Derivation and characterization of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells in clinical grade conditions

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Content DERIVATION AND CHARACTERIZATION OF HUMAN EMBRYONIC STEM (HES) CELLS AND HUMAN INDUCED PLU RIPOTENT STEM (HIPS) CELLS IN CLINICAL GRADE CONDITIONS by Jordan Elliott Pomeroy A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (SYSTEMS BIOLOGY AND DISEASE) May 2012 Copyright 2012 Jordan Elliott Pomeroy ii Epigragph “Science is a way of thinking much more than it is a body of knowledge” – Carl Sagan “Somewhere, something incredible is waiting to be known” – Carl Sagan iii Dedication I would like to dedicate this dissertation to my family and friends. Without their continuing love, encouragement, and support I would not have reached the conclusion of this wonderful goal. I look forward to sharing the next great moments with all the special people in my life at present and those who I have not yet had the pleasure to meet. iv Acknowledgements I would like to acknowledge the shapers of my scientific thought: Dr. Angel Amores, Dr. John Postlethwait, Dr. Robert Petroski, Dr. Martin Pera, Dr. Kouichi Hasegawa, and Dr. Jun Wu. Assumption has no value in the presence of experience. v Table of Contents Epigraph ii Dedication iii Acknowledgements iv List of Tables vii List of Figures viii Abstract x Introduction 1 Chapter One: A Xenobiotic‐Free (Xeno‐Free) Cell Culture System for the Derivation and Maintenance of Human Pluripotent Stem Cells 3 Introduction 3 Materials and Methods 9 Results 13 Conclusions 20 Chapter Two: Derivation of Xeno‐Free Human Induced Pluripotent Stem Cells 24 Introduction 24 Materials and Methods 33 Results 38 Conclusions 49 Chapter Three: Derivation of Xeno‐Free Human Embryonic Stem Cells 57 Introduction 57 Materials and Methods 70 Results 75 Conclusions 86 Chapter Four: Molecular Characterization of the Reprogramming Process Improves Selection of High Fidelity Human Induced Pluripotent Stem Cells 92 Introduction 92 Materials and Methods 98 vi Results 102 Conclusions 123 Conclusion 134 Bibliography 135 Appendices 144 Appendix A: Egg/Sperm Donor Consent Form 144 Appendix B: Embryo Donor Consent Form 151 vii List of Tables Table 1: Derived XF‐hiPSC Lines 45 Table 2: Embryo Derivation Results 79 Table 3: Live‐Cell ICC Selection Based Derivation Efficiency 123 viii List of Figures Figure 1: HES3 Maintained On Human Vitronectin/Fibronectin/hDFf v. Gelatin/mEF 16 Figure 2: HES3 Maintained With Standard KSR vs. KSR‐XF 17 Figure 3: 50X vs. 100X Xeno‐Free Growth Factor Cocktail 18 Figure 4: ICC of HES3 Maintained in KSR‐XF 19 Figure 5: PiggyBac Transposon and Transfection Efficiency 29 Figure 6: Transposon Reprogramming Protocol 31 Figure 7: Image of Cellular Focus Undergoing Reprogramming at Day 5 40 Figure 8: Images of Foci Undergoing Reprogramming at Weeks 2/3 41 Figure 9: Images of Foci at Weeks 4/5 and Subsequent Outgrowths 42 Figure 10: Images of hiPSCs 44 Figure 11: ICC Images of Full and Partial hiPSCs 46 Figure 12: Embryoid Body ICC Images of hiPSCs 47 Figure 13: Embryoid Body Quantification 50 Figure 14: Quantitative PCR for hiPSC Lines J1 and K1 54 Figure 15: Derivation of hESCS 62 Figure 16: Representative Human Embryos 77 Figure 17: Successful Derivation of mESCs by Laser Isolation 78 Figure 18: Embryo SC006‐C Outgrowth Images 80 Figure 19: Embryo SC011‐F Outgrowth Images 81 Figure 20: Embryo SC010‐A Outgrowth Images 82 Figure 21: Images of the XF‐hESC Line USC‐01 84 ix Figure 22: ICC Panel for the XF‐hESC Line USC‐01 85 Figure 23: Embryoid Body Images of USC‐01 86 Figure 24: ICC Reprogramming Timeline 93 Figure 25: Live‐Cell ICC Selection Methods 96 Figure 26: Genes Queried by Quantitative RNA Expression 97 Figure 27: TRA‐1‐60/GDF‐3 ICC Timeline 104 Figure 28: GCTM‐2/EpCAM ICC Timeline 105 Figure 29: OCT4/DNMT3b ICC Timeline 106 Figure 30: Nanog/E‐Cadherin ICC Timeline 107 Figure 31: EpCAM/DNMT3b ICC Timeline 108 Figure 32: EpCAM/E‐Cadherin ICC Timeline 109 Figure 33: ICC Reprogramming Marker Timeline Graphs 111 Figure 34: ICC Timeline Concatenated data 113 Figure 35: Live‐Cell ICC Images 115 Figure 36: Fluidigm Concatenated Data 116 Figure 37: Fluidigm Upregulated Data 118 Figure 38: Live‐Cell Staining Intensity Correlates With Expression 121 Figure 39: ‘Fully’ and ‘Partially’ Reprogrammed hiPSCs Derived from Live‐ Cell Selection 124 Figure 40: ICC of Live‐Cell Selected hiPSCs 126 Figure 41: Embryoid Body Analysis of Live‐Cell Selected hiPSCs 128 x Abstract Future use of pluripotent cells for regenerative medicine will require the derivation and characterization of new cell lines in clinical grade conditions. Removing potential sources of contamination, such as cell culture components sourced from animals, will aid the regulatory approval for future stem cell based therapeutics. In this dissertation, I describe the development of a xenobiotic‐free cell culture system for the derivation and maintenance of human pluripotent cell lines. I have derived >40 hiPSC lines and one hESC line in the xeno‐free cell culture conditions with maintenance of pluripotency charcacterized by morphology and immunocytochemistry marker expression for >50 passages and >30 passages respectively. The hESC line, USC‐01 and several hiPSC lines derived for this study demonstrate highly similar gene expression patterns although slight differences are apparent. USC‐01 and several hiPSC lines demonstrate the ability to differentiate into cells displaying characteristics of all three germ layers in an embryoid body differentiation assay. Further examination of the process of reprogramming somatic cells to a pluripotent state at both the RNA and protein expression levels indicates several genes/markers selective for hiPSCs achieving a pluripotent state most similar to the “gold‐standard” of pluripotency possessed by hESCs. This study confirms the usefulness of the markers TRA‐1‐60, E‐Cadherin and EpCAM for live‐cell selection of the best hiPSC colonies and also demonstrates the usefulness of the marker GCTM‐2. Expression analysis of colonies undergoing reprogramming also indicates that the genes FOXD3, CDH3, LCK, EDNRB, EPHA1, SOX2, xi and HAS3 are active in only a small subset of colonies 30 days after transfection of the piggyBac transposon reprogramming cassette. Since these genes are active in all hESC and most hiPSC positive control lines tested, confirmation of their activation could be used to select reprogramming hiPSC colonies most likely to achieve a pluripotent state similar to hESCs. Increased selection and derivation efficiencies of hiPSC lines demonstrating high fidelity to the hESC pluripotent state will streamline the generation of hiPSC lines for future testing as a replacement for hESCs in regenerative medicine. 1 Introduction Mammals undergo complex developmental processes after sexual reproduction. The fertilized embryo must traverse critical developmental checkpoints to differentiate into all the cells necessary for a fully functional organism. The word “totipotent” describes a cell that can differentiate into cells necessary for gestational support and contribute to the organism itself. The first cells (blastomeres) in a cleavage stage embryo are totipotent. A “pluripotent” cell is one step down the development ladder such that it can only contribute to the developing organism and does not contribute to the formation of gestational tissue such as the placenta. The first mammalian pluripotent cells were isolated from the inner cell mass (ICM) of mouse embryos by Evans and Kaufman in 1981 (Evans and Kaufman 1981).These pluripotent cells are known as embryonic stem cells (ESCs). The derivation of mouse ESCs revolutionized the field of developmental biology and allowed the production of transgenic knockout mice (Thomas and Capecchi 1987) which greatly influenced both science and medicine since researchers could identify the function of genes and elucidate the genetic basis of disease. Researchers also began to see the potential for pluripotent stem cells to produce an unlimited source of differentiated cells that could be used in regenerative medicine. Efforts to derive human ESCs, however, were not successful until 1998 when James Thomson et al. derived the first lines (Thomson, Itskovitz‐Eldor et al. 1998). Demonstration that human ESCs could be differentiated into functional somatic cell types, such as neurons, in vitro quickly followed with the studies of Reubinoff et al. in 2 2000 (Reubinoff, Pera et al. 2000). The field of ESC research has grown dramatically since these early discoveries, but much work remains to translate the potential of these cells to clinically relevant therapeutics. The genesis of the work described in this dissertation stems from the advent of technology demonstrating the ability to reprogram somatic cells into a pluripotent state similar to that possessed by human ESCs (Takahashi and Yamanaka 2006). The resulting induced pluripotent stem cells (iPSCs), however, exhibit low efficiency of generation, regardless of methodology, (Stadtfeld, Nagaya et al. 2008; Yamanaka 2009; Yu, Hu et al. 2009) and a divergent molecular profile that is most likely the result of an incomplete epigenetic remodeling (Kim, Doi et al. 2010), or genetically unstable (Hussein, Batada et al. 2011) reprogramming process (Pera 2011). There is a need, consequently, to characterize human ESCs (hESCs) vs. human iPSCs (hiPSCs) to determine the extent of their equivalency. Specifically, if hiPSCs are to replace hESC in cell therapy, they must exhibit a high level of fidelity to the standard pluripotent profile (Smith, Luong et al. 2009) of hESCs to lend confidence for future clinical use of these cells. One major problem with such comparative studies is the variability introduced by the wide variety of cell culture conditions previously used for derivation and maintenance of the hESCs and hiPSCs. The aim of this project is thus to derive hESCs and hiPSCs in identical clinical grade conditions to provide a platform for determining equivalency while limiting variability. 3 Chapter One: A Xenobiotic-Free (Xeno-Free) Cell Culture System for the Derivation and Maintenance of Human Pluripotent Stem Cells 1. Introduction Derivation of hESCs and hiPSCs in clinical grade conditions requires the development of a cell culture system that meets regulatory guidelines for clinical application of these cells. Standard cell culture systems for derivation and maintenance of human pluripotent stem cells typically utilize components sourced from animal products, such as fetal bovine serum (FBS) in the growth medium and mouse embryonic fibroblasts (mEFs) that serve as a supporting feeder layer for the pluripotent cells. Future clinical approval of hESCs or hiPSCs will most likely hinge on the creation of defined cell culture systems (Ludwig, Levenstein et al. 2006) and the replacement of components sourced from animal products (Ellerstrom, Strehl et al. 2006), for the derivation and maintenance of pluripotent cell lines. While efforts have focused on creating more defined systems for culture of human pluripotent cell lines, such as Knockout Serum Replacement (KSR – Life Technologies), such components still contain products sourced from animals. Many companies are now actively testing cell culture systems without animal products “xeno‐free (XF)”. In this study, I undertook beta testing the Knockout Serum Replacement – XenoFree (KSR‐XF) system from Life Technologies. Below I will detail the components of my xeno‐free cell culture system which include an extracellular matrix (ECM) coating of human vitronectin/fibronectin, 4 XenoFree human fibroblast feeders, and the KSR‐XF growth medium. The xeno‐free hESC cell culture system demonstrated an equivalent ability to maintain pluripotency of the HES3 line, measured by markers and maintenance of an undifferentiated state, as to that shown by HES3 grown in standard culture conditions. The successful establishment of this xeno‐free cell culture system enabled future derivations of XF‐hESCs and XF‐ hiPSCs. Specific Aim #1: Optimize the Use of Human Plasma Vitronectin/ Fibronectin As a XF-ECM Growth Matrix Most pluripotent cell culture systems begin with a base coating of ECM proteins on the growth surface of the plastic cell culture dishes. The ECM coating aids the attachment and proliferation of the pluripotent cells in addition to adhesion factors present in the liquid phase of the growth medium. Many standard systems supporting embryonic stem cells incorporate a base of 0.1% gelatin (denatured collagen), usually sourced from pig, that facilitates attachment of the fibroblast feeders and hESCs. However, gelatin alone does not support attachment and spreading of hESC, which requires integrin ligands present in serum or as additives. More defined systems use of a medley of ECM proteins such as fibronectin, vitronectin, and laminin. The use of multiple ECM proteins aids the culture of hESCs by providing binding sites for specific integrins (transmembrane binding proteins) expressed on the surface of the pluripotent cells. Integrins are important in cell biology because of their bi‐potential ability to provide structural contact with the external environment while also being linked to 5 internal second messenger systems influencing proliferation signals within the cell. In particular, the RGD binding domain of vitronectin serves as a binding substrate for Integrin α5 βv which has been shown to support and aid the proliferation of hESCs (Braam, Zeinstra et al. 2008). Some groups have demonstrated the use of synthetic vitronectin RGD domains for hESC support (Elefanty and Stanley 2010; Chen, Gulbranson et al. 2011), but we decided to use native human vitronectin for this study since there was not a ready source of the synthetic peptides. Fibronectin is important for both fibroblast feeders and hESCs. The addition of laminin is mainly indicated when using “feeder‐free” culture conditions (Braam, Zeinstra et al. 2008) not investigated in this study. Therefore, I decided to focus on optimizing vitronectin and fibronectin for the extracellular matrix coating. Previous work detailing the importance of the ECM proteins mentioned above generally used peptides from animal sources due to their low cost and ready supply. Obtaining human vitronectin and fibronectin is thus critical to establishing the extracellular matrix component of the xeno‐free cell culture system. Human fibronectin and vitronectin are commercially available, but the vitronectin is prohibitively expensive at $2,000.00 per milligram. Derivation, maintenance and characterization of new pluripotent cell lines require several milligrams for long term culture such that the commercial sourcing of vitronectin would be cost prohibitive. I thus isolated milligram quantities of human vitronectin from unused human Fresh Frozen Plasma (FFP). Commercial human fibronectin, however, is a viable source at $60/mg. The 6 combination of human vitronection and fibronectin provided a suitable XF‐ECM substrate for the attachment and proliferation of human pluripotent cells. Specific Aim #2: Establish a Xeno-Free Cell Culture System for Fetal Human Dermal Fibroblast Feeders Many cell culture systems require the use of an additional cell source that secretes factors vital to the health and proliferation of the study cells. Such supporting cells are termed “feeders.” Feeders are typically seeded on the culture dish at a density that provides a monolayer while allowing ample room for the attachment and proliferation of the study cells. The correct feeder density will provide a sufficient concentration of growth factors to support proliferation of pluripotent cells. A variety of cell types are used as feeders, but standard ESC cell culture systems employ mouse embryonic fibroblasts (mEFs) (Thomson, Itskovitz‐Eldor et al. 1998). mEFs are prepared by the enzymatic digestion of whole embryonic mice except for the head and visceral organs. The harvested mEFs must be mitotically inactivated before use as feeders to prevent continued proliferation while still providing viable, robust growth factor secreting cells. The two main methods for inactivation are irradiation and exposure to Mitomycin C. Irradiation is a very cost effective mechanism compared to the expensive protein Mitomycin C, but many institutions do not have an irradiator. This makes Mitomycin C a more appropriate inactivator for small batches of cells. Since I need to replace the animal sourced mEFs for a xeno‐free system, I substituted fetal human dermal fibroblasts (hDFfs) prepared in a similar methodology as described above since 7 such cells have been successfully used as human feeders in previous studies (Richards, Tan et al. 2003). It was also necessary to replace the FBS containing mEF medium with a xeno‐ free medium capable of supporting the expansion of hDFfs. Many companies advertise Low‐Serum mediums, but only American Type Culture Collection (ATCC) had a “serum‐ free” fibroblast medium containing recombinant human growth supplements suitable for use in this project. The hDFfs grown in the xeno‐free, humanized fibroblast medium supported proliferation and maintenance of pluripotency in the hESC line HES3. Specific Aim #3: Establish a Xeno-Free Cell Culture Medium for the Derivation and Maintenance of Human Pluripotent Cells The final component of the xeno‐free cell culture system is the xeno‐free culture medium. Standard hESC culture medium used in our lab comprises a base of DMEM supplemented with 20% FBS. This medium was used to derive some of the first hESC lines (Thomson, Itskovitz‐Eldor et al. 1998; Reubinoff, Pera et al. 2000), but the FBS negated its use in this project. In the years since these original lines were derived in the late 1990s, many companies have sought to produce more defined cell culture mediums to limit experimental/culture variability. The main source of variability in this cell culture medium is the FBS. As such, the biotechnology products company Life Technologies produced Knockout Serum Replacement (KSR) to replace the FBS component. KSR is a proprietary serum supplement with the chief ingredients remaining undefined, but the main idea was to remove components within FBS until 8 only the factors necessary to support the maintenance of hES pluripotency remained. While KSR represents a technological advancement, many components within this supplement are still sourced from animals. Consequently, it was essential to find a cell culture medium that was xeno‐free to move forward with “clinical grade” pluripotent cell line derivation. Few xeno‐free cell culture supplements were available when I began this project in 2009, but two mediums were in late stage development. Stem cell products companies were actively seeking to develop “clinical grade” products in anticipation of future clinical trials with stem cell derived therapeutics and the necessity to adhere to potential regulatory guidelines set forth by the Food and Drug Administration (FDA). One of these products was a xeno‐free version of KSR being developed by Life Technologies. The other was mTeSR‐2 from StemCell Technologies. I approached both companies to test these mediums for use in my xeno‐free cell culture system. Of the two, only KSR‐XF had published data supporting the derivation of hiPSCs (Rodriguez‐ Piza, Richaud‐Patin et al. 2010; Ross, Suhr et al. 2010) so I mainly focused on developing this medium. Since it was a xeno‐free version of the KSR that we already used on a regular basis in the lab, direct comparison between xeno‐free and standard cell culture systems was more ideal. I continued to experiment with both media at the start of the project, but I later found that I could not derive hiPSCs using mTeSR‐2. This failure added more impetus to focus on Xeno‐Free KSR. The KSR‐XF cell culture medium supported the maintenance of pluripotency of the HES3 line through repeated passages. 9 2. Materials and Methods Standard hESC Culture Conditions Culture dishes were coated with a 0.1% porcine gelatin (Sigma #G9136) solution for >1 hr at room temperature (RT). Irradiated mouse embryonic fibroblasts (mEFs) were freshly prepared by the USC stem cell core facility according to the WiCell‐2003 protocol “Derivation of Mouse Embryonic Fibroblasts, “ and plated at 25% confluency in mEF medium (DMEM [Gibco #11960], 10% fetal bovine serum [Gibco #16000‐044], 2 mM L‐Glutamine [Gibco #25030‐081], and 100 U/mL Pen/Strep [Gibco #15140‐122]) the night before passaging. The day of passaging, mEF medium was replaced with KSR hESC medium (DMEM‐F12 [Gibco #11330], 20% Knockout Serum Replacement [Gibco #10828‐028], 4ng/mL Human FGF‐b [Milipore #GF003], .01 uM 2‐Mercaptoethanol [Sigma #M3148], 0.1 mM MEM‐NEAA [Gibco #11140‐050], 2 mM L‐Glutamine, and 100 U/mL Pen/Strep) which was allowed to equilibrate in a 37 o C, 5% CO 2 incubator for 1 hr. The hES cell lines HES2 and HES3 were passaged into small clumps by manual dissection with fine needles and transferred to the equilibrated cell culture dishes. This process was repeated every 7 days. Xeno-Free hESC Culture Conditions Culture dishes were coated with native human vitronectin (0.5 ug/cm 2 – isolated in house, method below) and human fibronectin (0.5 ug/cm2‐ Gibco #33016‐015) diluted into sterile PBS at 2.5 ug/mL with a volume sufficient to effectively cover the cell culture surface. Dishes were incubated at RT for >3 hours. Native human vitronectin 10 was isolated from bags of unused Fresh Frozen Plasma (FFP) obtained from the Los Angeles County Blood Bank (IRB #HS‐10‐00134) using a heparin bead protein isolation column as described in (Yatohgo, Izumi et al. 1988). Briefly, thawed FFP was pre‐cleared on a Protein A Sepharose CL‐4B (GE Life Sciences #17‐0780‐01) column to remove proteins non‐specifically binding to the sepharose beads. 8M urea was added to the pre‐cleared human FFP to activate the heparin binding domain of vitronectin and the activated FFP was passed through a Heparin‐Sepharose 6 Fast Flow (GE Life Sciences # 17‐0998‐01) column. After several washes, the vitronectin was fractionally eluted and fractions with an A 280 of >0.05 were pooled for dialysis. The dialyzed, purified vitronectin was then quantified and lyophilized for long term storage @‐20 o C. The average recovery from one 250 mL bag of FFP was about 5 mg. XF‐hDFf feeders were prepared by expansion of an early passage cell stock obtained from ScienCell (#2300). Stock hDFfs were thawed into 15 mL of Serum Free fibroblast growth medium from ATCC which contains a Fibroblast Basal Medium (#PCS‐ 201‐030) and a Serum‐Free Fibroblast Growth Kit (#PCS‐201‐040) with the following human or recombinant growth factors: HSA 500 ug/mL, linoleic acid 0.6 mM, lecithin 0.6 ug/mL, 7.5 mM L‐Glutamine, rhFGF 5n ng/mL, rhEGF 5 ng/mL, rhTGF‐1 30 pg/mL, rhInsulin 5 ug/mL, hydrocoritisone 1 ug/mL, and ascorbic acid 50 ug/mL. The thawed cell suspension was transferred to a T‐150 cell culture flask coated with 1 ug/cm 2 fibronectin and allowed to expand to ~90% confluency, replacing medium every other day, before freezing for working stocks. Once reaching the desired confluency, the cells 11 were washed once with 10 mL sterile PBS before exposure to 2 mL TrypLE Select (Gibco #12563‐011). After detaching, cells were diluted 1:10 with XF‐Fibroblast Medium, counted, and centrifuged. The cell pellet was resuspended in the appropriate volume of XF‐Fibroblast Freezing Medium (10% DMSO in XF‐Fibroblast Medium) to give 1 x 10 6 cells/mL aliquots and slow frozen (~1 °C/min) in a Mister Frosty device to ‐80 o C for long term storage. For preparation of mitotically inactivated XF‐hDFfs for plating, frozen working stocks were thawed into XF‐Fibroblast Medium and transferred to 2 X T‐150 flasks coated with 1 ug/cm 2 native human fibronectin. Upon reaching 90% confluency, the XF‐ hDFfs were split 1:4. Cells were washed once with10 mL PBS followed by exposure to 2 mL of TrypLE Select. After detaching, 10 mL of XF‐Fibroblast Medium was added to each T‐150 and the entire volume is centrifuged. The cell pellet was resuspended in 120 mL of XF‐Fibroblast Medium and transferred to 8 x T150 fibronectin coated flasks. Cells are fed every other day until reaching 100% confluency wherein they are inactivated. Mitotic inactivation of the cells required >2 hour exposure to 20 ng/mL Mitomycin‐C (Milipore #47589) diluted in sterile PBS. Cells were then washed twice with 10 mL PBS before detaching with TrypLE Select. Cells were resuspended in XF‐Fibroblast Medium, counted, and centrifuged. The cell pellet was resuspended in XF‐Fibroblast Freezing Medium in plating aliquots of 3 x 10 6 cells/mL and slow frozen to ‐80 o C. XF‐hDFfs are viable using this methodology until P6. For plating, aliquots were thawed in XF‐ 12 Fibroblast Medium and plated onto vitronectin/ fibronectin coated culture dishes at a density of 5 x 10 4 cells/cm 2 and allowed to attach overnight before hESC passage. Xeno‐free hESC medium (DMEM‐F12, 20% XF‐Knockout Serum Replacement CTS [Gibco #12618‐013], 100 X XF‐Growth Factor Cocktail CTS [Gibco #A13560SA], 10 ng/mL Animal Free bFGF [Milipore #GF003‐AF], .01uM 2‐Mercaptoethanol, 0.1 mM MEM‐ NEAA, 2 mM L‐Glutamine, and 100 U/mL Pen/Strep) was added to fresh XF‐hDFf culture dishes on the day of hESC passage and allowed to equilibrate > 1 hour at 37 o C/5% CO 2 . Pluripotent cell colonies were passaged by manual cutting with a fine needle into small clumps of 50‐500 cells and transferred to equilibrated xeno‐free cell culture dishes. KSR‐XF hESC Medium was replaced daily with new passage occurring every 7 days. Immunocytochemistry of Pluripotent Cells Cultured in Xeno-Free Conditions After several passages in the KSR‐XF cell culture system, the HES3 cell line was transferred to 4‐well chamber slides coated with 0.5 ug/cm2 vitronectin/fibronectin and seeded with hDFf feeders. Colonies were allowed to expand for 5 days followed by removal of KSR‐XF hESC Medium and fixation with a 4% paraformaldehyde (PFA, Sigma #158127) solution in 1X PBS for 10 min at room temp. Fixed cells were then washed 3 times with 1X PBS and permeabilized with 0.1% Triton X‐100 for 5 min followed by washing three times with 1X PBS. Primary antibodies were diluted to working concentrations in PBS containing 2% animal serum (matched to secondary antibodies) and incubated for 1 hour at room temp followed by a 3X wash with PBS. Secondary 13 antibodies were diluted in PBS plus 2% animal serum and incubated for 1 hour followed by 3X wash with PBS. ProLong Gold anti‐fade reagent plus DAPI (Life Technologies #P36931) was utilized to mount the slides which were allowed to cure overnight. Images were taken with a Zeiss AxioImager Z1 microscope. Marker pairs for staining were GCTM‐2/SOX2, TRA‐1‐60/OCT4, and SSEA‐ 4/Nanog. Primary and secondary antibody combinations with dilutions were as follows: [GCTM‐2 neat (Mouse IgM, in house hybridoma) / AlexaFluor 488 goat anti‐mouse IgM (u chain) 1:1000 (Life Technologies #A21042)] [SOX2 1:1000 (Rabbit IgG polyclonal, Milipore #AB5603) / AlexaFluor 568 goat anti‐rabbit IgG (H+L) 1:1000 (Life Technologies #A11036)] [TRA‐1‐60 1:100 (Mouse IgM, Santa Cruz Biotechnology #sc‐21705) / AlexaFluor 594 goat anti‐mouse IgM (u chain) 1:1000 (Life Technologies #A21044)] [OCT4 1:200 (Mouse IgG 2b , Santa Cruz Biotechnologies #sc‐5279) / AlexaFluor 488 goat anti‐mouse IgG 2b 1:1000 (Life Technologies #A21141)] [SSEA‐4 1:50 (clone MC‐813‐70, Mouse IgG3, Milipore #MAB4304) / AlexaFluor 488 rabbit anti‐mouse IgG (H+L) 1:1000 (Life Technologies #A11059)] [Nanog 1:20 (Goat IgG polyclonal, R&D Systems #AF1997) / AlexaFluor 568 rabbit anti‐goat IgG (H+L) 1:1000 (Life Technologies #A11079)] 3. Results Human Vitronectin/Fibronectin Extracellular Matrix Supports hESC Maintenance Human fibronectin was commercially obtained from Life Technologies. I isolated human vitronectin from unused fresh frozen plasma (FFP) acquired from the LAC+USC Blood Bank after IRB approval. Receiving approval for the FFP, even though it was 14 otherwise destined for disposal, took 6 months from the start of contact to final signatures. Following the protocol in Yatohgo et al. (Yatohgo, Izumi et al. 1988) vitronectin was isolated from the FFP by employing a heparin bead column, followed by lyophilization and freezing at ‐20 o C for future use. With one week of labor, each 250 mL bag of FFP yielded ~5mg of vitronectin. The identity of the FFP isolated vitronectin was validated by Western Blot with commercially available vitronectin positive control. I isolated 20.5 mg human vitronectin from five bags of FFP: 0.6, 3.5, 6.2, 5.3 and 4.9 mg respectively. Subsequent testing of the human vitronectin and fibronectin under standard hES cell culture conditions indicated an optimal surface coating of 0.5ug/cm 2 for each ECM protein. A 0.1ug/ cm 2 coating was not sufficient for robust attachment and proliferation while 1 ug/cm 2 did not show improved culture of the hESC line HES2. Adequate coating requires exposing the culture surface to a 2.5 ug/mL solution of vitronectin/fibronectin in PBS for >2 hours. The vitronectin/fibronectin coating demonstrated hESC culture support equivalent to that of a 0.1% porcine gelatin extracellular matrix coating. I was able to demonstrate long term maintenance of the hESC line HES3 for >6 passages on this matrix combination (0.5ug/cm 2 VF) with the retention of several pluripotency markers and limited cellular differentiation. XF-hDFf Human Feeders Support hESC Maintenance I obtained commercially available, low‐passage hDFfs from ScienCell. The commercial stock of hDFfs was thawed in Serum Free Fibroblast Medium from ATCC and 15 transferred to T‐150 cell culture flasks coated with 0.5 ug/cm2 fibronectin. Under these conditions, thawed hDFfs exhibit rapid proliferation. Initial attempts to enzymatically passage the XF‐hDFfs with TrypLE Select and Defined Trypsin Inhibitor failed because the inhibitor caused dramatic cell lysis. Those cells that survived the exposure to Defined Trypsin Inhibitor exhibited marked reduction in proliferative capacity with an enlarged, flattened morphology. Further testing indicated that TrypLE could be effectively removed by dilution in Serum Free Fibroblast Medium followed by centrifugation, resuspension and transfer to fresh culture flasks. Using these refined techniques, I demonstrated the ability to expand XF‐hDFfs through P6 for feeder production. Cells allowed to go beyond P6 exhibit reduced proliferative capacity and shift from a small compact morphology to a large, flat cell type. Frozen P4‐P6 aliquots remain viable for >3 months at ‐80 o C. The final, effective feeder cell density at plating is 5 x 10 4 cells/cm 2 and the feeder dishes are ready to receive passaged pluripotent cells the next day. Waiting longer than 24 hours reduced pluripotent cell attachment and expansion. This density provides 80‐90% confluency the day after plating. Higher density inhibited the expansion of pluripotent colonies while lower density led to increased differentiation. The established cell lines HES2/HES3 were successfully maintained on these feeder dishes with standard KSR hESC medium for >6 passages validating their use in future experiments [Figure 1]. HES3 colonies continue to be compact with tight borders. The interface between the HES3 16 Figure 1: HES3 Maintained On Human Vitronectin/Fibronectin/hDFf vs. Gelatin/mEF These images illustrate the appearance of the HES3 cell line cultured under “standard” laboratory conditions which utilize 0.1% gelatin/mEFs/KSR (A) and “Xeno‐Free” conditions 0.5 ug/cm2 fibronectin‐ vitronectin/hDFfs/XF‐KSR (B). Grossly, both colonies possess similar morphology: compact, angular cells forming a polygonal colony with tight borders at the feeder interface. colonies and hDFfs (B), however, shows much closer apposition than as seen with mEFs (A). KSR-XF CTS hESC Medium Supports Maintenance of HES3 I tested two xeno‐free, commercially available hESC media. Initial tests with the KSR‐XF medium demonstrated short term maintenance of HES3 while the mTeSR‐2 medium exhibited more robust maintenance and had the ability to support short term feeder‐free maintenance. I had difficulty, however, using mTeSR‐2 for derivation experiments leading me to discontinue its testing. During my beta testing of KSR‐XF medium, Life Technologies informed me that they had produced a XF‐Growth Factor Cocktail (XF‐GFC) as they were also having issues with long term maintenance of hESCs. After supplementing the XF‐GFC to the KSR‐XF medium, I demonstrated long term maintenance of HES3 on defined matrices with 17 Figure 2: HES3 Maintained With Standard KSR vs. KSR‐XF These images illustrate the appearance of the HES3 cell line on human fibronectin/vitronectin/hDFf in normal KSR (A) and XF‐KSR (B). The HES3 cell line was transferred to the humanized KSR‐XF system at P50 and maintained for 8 passages in KSR‐XF. The colony in panel (A) represents cells returned to normal KSR for 2 passages and panel (B) are the same cells maintained in KSR‐XF for 2 more passages. The colonies maintain gross morphological similarities in both conditions supporting the use of the KSR‐XF system. results similar to those seen with standard KSR medium [Figure 2]. In Figure 2, the HES3 cell line was transferred from standard conditions at P50 and maintained for eight passages in KSR‐XF+GFC before being switched back to regular KSR for two passages (A). Image (B) shows the appearance of HES3 maintained in KSR‐XF for 10 passages. HES3 colonies possess a round, flat, compact morphology under both conditions. Unfortunately, the original XF‐GFC prototype was discontinued in Spring 2010 for a reformulation leading to a 6 month delay in this project. Life Technologies did not add or remove any components in the reformulation. Instead, they doubled the stock concentration (50X to 100X) for each component for increased long term stability. Final diluted working concentrations, however, remained the same. Reformulated 100X XF‐ GFC exhibited equivalent long term maintenance capacity for the HES3 line [Figure 3]. Colony morphology remained similar although colonies exposed to KSR‐XF with 100X 18 Figure 3: 50X vs. 100X Xeno‐Free Growth Factor Cocktail These images illustrate the appearance of the HES3 cell line in XF‐KSR cell culture conditions. During beta testing of the XF‐KSR growth factor cocktail, Life Technologies reformulated the components from a 50X stock solution to a 100X stock solution. The HES3 cell line maintains similar morphological characteristics after 5 passages in both 50X (A) and 100X (B) stock solutions demonstrating equivalence. GFC (B) commonly appeared more dense and compact than those maintained in 50X GFC (A). In response to this delay and constant “backorder” issues of the prototype products, I forged a direct relationship with the product development team at Life Technologies to ensure adequate supply. One major difference between the use of standard KSR and KSR‐XF is the method of passaging the hESCs. Under standard KSR culture, ‘’enzymatic” passaging is accomplished by adding a digestive enzyme cocktail including collagenase/trypsin/20%KSR (CTK) to the cell s. The cells are allowed to digest until the mEFs detach from the surface. The mEF containing CTK is aspirated and a small volume is left to continue digestion of the hESC colonies. After the colonies have adequately lifted from the culture surface, KSR medium is added to the dish and the colonies are triturated until they break into clumps of >200 cells wherein they are gravity sedimented and resuspended in fresh KSR then transferred to new cell culture dishes. I 19 Figure 4: ICC of HES3 Maintained in KSR‐XF These images show immunocytochemistry staining results for markers associated with the pluripotent state. HES3 P50+P10XF were fixed in 4% PFA and stained according to the methods presented in this section. Panel (A) shows the results for GCTM2 (green), SOX2 (red), DAPI (blue). Panel (B) shows the results for TRA‐1‐60 (red), OCT4 (green), and DAPI (blue). Panel (C) shows the results for SSEA‐4 (green), Nanog (red), and DAPI (blue). Strong staining for each of these markers suggests robust maintenance of pluripotency of the HES3 line after 10 passages in the KSR‐XF cell culture system. attempted to use the “enzymatic” passaging technique on my XF culture system, substituting KSR‐XF into the CTK digestive cocktail, but the hDFfs did not respond well to this technique. Instead of detaching as single cells, the feeders would detach as a sheet effectively trapping the hESC colonies. The light trituration necessary to break the hESC colonies apart without dissociation to single cells was insufficient to release the hESC colonies from the tight “wadding” of undissociated hDFfs. Increasing digestion time with XF‐CTK did not break up the sheets of hDFfs without fully dissociating the hESC colonies to single cells. Single cell passaging is notoriously difficult with hESCs due to the fact that they rapidly differentiate or die when dissociated. After the failure of XF‐enzymatic passage, I resorted to manual passage of the hESC colonies in the KSR‐XF culture system. Expanded colonies at Day 7 were dissected into small pieces containing >100 cells. The strong bonds developed between the hESC colony and hDFf feeders required close dissection to ensure transfer of the outermost edges of the colony. Manual passaging provided robust attachment post‐passage and 20 the HES3 cell line exhibited strong pluripotency marker expression by immuno‐ cytochemistry (ICC) after 10 passages [Figure 4]. The HES3 cell line demonstrates the expected patterns of surface staining for TRA‐1‐60, GCTM‐2, and SSEA‐4 noted by the intense fluorescence separated by visible cell borders. Nuclear staining for OCT4, SOX2, and Nanog was strong throughout the e colonies. Limited spontaneous differentiation was noted during the initial maintenance testing. 4. Conclusions The xeno‐free hESC cell culture system is composed of a human vitronectin/fibronectin ECM coating, XF‐hDFf feeders, and KSR‐XF hESC Medium. The use of defined culture systems often presents practical challenges in terms of costs alone. Commercially available human plasma fibronectin is within the research budget of most laboratories desiring to derive multiple human ES and IPS cell lines, but human plasma vitronectin is not. After demonstrating the ability of commercial vitronectin to support the maintenance of pluripotency in the HES2 line, I initiated an IRB to obtain unused FFP from the LAC+USC Blood Bank. I was able to isolate over 20 mg of vitronectin from the FFP which has a market price of $40,000. This amount was sufficient for commencement of the derivation project and saved valuable research dollars for other expensive xeno‐free cell culture components. Initiating this IRB protocol, moreover, provided some valuable experience in terms of human subjects approval. Receiving approval for the FFP, even though it was destined for disposal, took 6 months from initiation to final approval. 21 Optimizing the production of XF‐hDFfs required more than a year of testing. I first demonstrated that hDFfs work as a replacement for mEFs under standard KSR hESC cell culture conditions. I then tested the only commercially available XF‐Fibroblast medium and showed it supported the expansion of hDFfs. There were many low‐serum fibroblast media available, but ATCC had the only serum‐free medium, which also came with a high price of >$200 per 500 mL bottle. Successful feeder production starts with low‐passage (P2) hDFf stocks which are expanded in fibronectin coated cell culture flasks. It was highly important to passage the XF‐hDFfs before they reached 100% confluency because the proliferative capacity was greatly reduced either as a result of overcrowding or the extended exposure to TrypLE necessary to dissociate the densely packed cells. With careful monitoring, the XF‐hDFfs provided viable Mitomycin‐C mitotically inactivated feeders out to P6. Passaging the XF‐hDFs beyond P6 resulted in a remarkably reduced proliferative capacity and dramatic enlargement of the individual fibroblasts rendering them less useful as feeder stocks. These low‐passage XF‐hDFf feeders provided long‐term maintenance of pluripotency when they were plated at a density of 5x10 4 cells/cm 2 on top of the human vitronectin/fibronectin ECM. The choice to move forward with beta testing of the KSR‐XF medium components resulted from its similarity to the standard KSR system used in the Pera Lab and due to its eventual support for the derivation of XF‐hiPSCs. The published hiPSC derivation data with early versions of KSR‐XF also provided impetus to move forward with the Life Technology product (Rodriguez‐Piza, Richaud‐Patin et al. 2010; Ross, Suhr 22 et al. 2010). The XF medium, mTeSR‐2, initially demonstrated better long‐term maintenance of pluripotency than the KSR‐XF system, but its failure to facilitate XF‐ hiPSC derivation limited its usefulness in this study. mTeSR‐2 has a very high FGF‐b concentration (>80 ng/mL) which helps stabilize the pluripotent state. This high concentration of FGF‐b, however, induced rapid proliferation of untransfected fibroblasts in the hiPSC reprogramming experiments described below which overcrowded the cells undergoing reprogramming. The eventual addition of the XF‐GFC from Life Technologies to the KSR‐XF medium raised the pluripotency maintenance support level close to that demonstrated by mTeSR‐2. Nevertheless, problems with availability of the prototype KSR‐XF components from Life Technologies hampered early progress with this system. I finally arranged a direct line of communication with the Stem Cell Products Development group at Life Technologies to ensure a steady supply of KSR‐XF and the 100X XF‐GFC. Life Technologies invited me to speak about the development of this medium at the International Society for Stem Cell Research annual meeting in June 2011. With all three xeno‐free cell culture system components validated, I next perfected serial passage techniques to ensure the transfer of high quality, pluripotent cells from generation to generation. This required manual passage of individual colonies dissected using a fine hypodermic needle. Unfortunately, manual passage is a highly labor intensive technique that limits the ability to expand pluripotent cell lines into large surface area culture flasks. Experiments requiring large numbers of XF‐hESCs would 23 thus require a full‐time skilled technical assistant, automation, or optimization of enzymatic passage techniques. Nevertheless, manual passage eliminated exposure to trypsin which has been shown to increase susceptibility to chromosomal abnormalities, possibly leading to a state of “adaptation” (Mitalipova, Rao et al. 2005). Even though the manual passaging process is time consuming, the pluripotent cell lines cultured under these xeno‐free conditions exhibited strong ICC staining for pluripotency markers and limited spontaneous differentiation after long‐term passage (P8). Establishing a xeno‐free hESC cell culture system was extremely challenging. Starting with proof‐of‐concept studies to validate the effectiveness of the humanized ECM, hDFf feeders and KSR‐XF hESC Medium followed by scaling up to quantities necessary for derivation and long term maintenance of pluripotent cells required foresight and patience. Major roadblocks to progress were the inhibitory price of commercial human vitronectin and the inconsistent product availability of prototype xeno‐free medium components from Life Technologies. Despite the obstacles present during optimization, the XF‐hESC cell culture system provides stable, reliable maintenance of pluripotent cells. 24 Chapter Two: Derivation of Xeno-Free Human Induced Pluripotent Stem Cells 1. Introduction Cellular reprogramming to pluripotency is the process of taking a fully differentiated cell and returning it to a pluripotent state, present in the early human embryo, defined by the ability of a cell to differentiate into all cells present in an organism. The field of cellular reprogramming has existed for greater than 60 years. The first experiments, performed by Briggs and King in the 1950s with later experiments by Sir John Gurdon, involved somatic cell nuclear transfer (SCNT) wherein the nucleus of a somatic cell was inserted into an enucleated Xenopus egg cell (Briggs and King 1952; Gurdon 1962). The resulting chimeric cell was able to proceed through development into a fully functional organism. Amazingly, the “cloned” individual began as a fully differentiated “programmed nucleus” that was able to completely revert to the naïve epigenetic state capable of differentiating into all the cell types necessary in the organism. The source of the reprogramming factors in SCNT is the cytoplasm of the egg cell. The totality of factors involved in this reprogramming process remain elusive, but continued experimentation has started to narrow down the key genes involved in reprogramming to the pluripotent state. One key observation was the speed of the reprogramming process. Fully programmed nuclei were able to convert to a pluripotent state rapidly in SCNT, but the process took longer, about 10 days (Cowan, Atienza et al. 25 2005; Yu, Vodyanik et al. 2006; Hasegawa, Zhang et al. 2010) in cellular fusion experiments with ESCs and somatic cells. The experiments in the Yamanaka lab in 2006 elegantly identified four nuclear transcription factors (OCT4, SOX2, KLF4 and CMYC) capable of driving a somatic cell to a pluripotent‐like state, but this required three weeks of transgene expression (Takahashi and Yamanaka 2006) for full reprogramming. These transgene mediated reprogrammed cells are called induced pluripotent stem cells (iPSCs). The differences in reprogramming time between these three reprogramming methodologies point to a dynamic reprogramming system dependent on the combination and concentration of specific reprogramming factors. Understanding the differences in the length of time to fully reprogram a cell will undoubtedly unlock additional factors responsible in the reprogramming process and illustrate the correct stoichiometric values for these factors which allow for the rapid reprogramming observed in SCNT. While the biology of the reprogramming process is highly important, the potential of iPSCs to provide an unlimited source of donor matched cells and tissue is an equally relevant clinical topic. Contemporary research on iPSCs indicates a long road ahead toward full understanding of the possible pitfalls created by driving a somatic cell to a pluripotent state. Some of these problems include the role of leftover epigenetic memory not reset during the reprogramming process (Kim, Doi et al. 2010), genetic stability (Hussein, Batada et al. 2011), immunogenicity of donor matched iPSCs (Zhao, Zhang et al. 2011), and the true fidelity of the pluripotent‐like state presented by iPSCs 26 in relation to the “gold standard” pluripotent state observed in ESCs (Smith, Luong et al. 2009). One piece of the clinical puzzle is defining the reprogramming conditions that would pass muster when scrutinized by regulatory bodies such as the Food and Drug Administration (FDA). In this chapter, I will discuss the derivation of “fully” reprogrammed human iPSCs in the clinical grade xeno‐free cell culture system described in Chapter One. This study will help pave the way to eventual therapeutic use of this cellular technology. Specific Aim #1: Optimize the Transfection Protocol for the Delivery of Reprogramming Factors Via the PiggyBac Vector Various methods exist to reprogram somatic cells to a pluripotent state. Yamanaka illustrated proof‐of‐concept by introducing four transcription factors: OCT4, SOX2, KLF4 and CMYC into fibroblasts. The four factors were introduced to the fibroblasts via individual viral vectors that facilitated integration into the host genome and reprogramming transgene expression. Our lab utilized the four vector reprogramming technique to successfully derive hiPSCs in a paper describing relative reprogramming efficiencies between cytoplast fusion and iPSCs (Hasegawa, Zhang et al. 2010). The experiments done in this paper also delivered valuable lessons for characterization and maintenance of hiPSCs. As the field of iPSC research has advanced, new techniques have been utilized to address the issues of reprogramming efficiency and the production of integration‐free iPSCs. Integration of the reprogramming transgenes into the host DNA has the potential to alter gene expression by direct 27 insertion into a gene or disruption of its critical regulatory regions, so reprogramming without integration is paramount to future clinical use of hiPSCs. One method to increase efficiency was to reduce the number of vectors necessary from four to one. This was accomplished by creating a polycistronic reprogramming cassette containing the four factors carried by a single virion (Carey, Markoulaki et al. 2009). The polycistronic reprogramming cassette eliminated the necessity that one individual cell be independently transduced by all four vectors. While the polycistronic cassette increased the efficiency of reprogramming, reduced the amount of labor necessary to produce fresh virus for each transduction and reduced the number of host genome integrations, the viral vector still had the major issue of genomic integration. Once the virally delivered transgenes integrated into the host genome, they could not be removed. Such integration would be problematic for clinical use of such cells, because the cassette may integrate into an important region of DNA thus disabling a necessary gene or potentially inducing neoplastic growth, similar to what has been observed during some trials of gene therapy (Hacein‐Bey‐Abina, von Kalle et al. 2003; Howe, Mansour et al. 2008). Further research using non‐integrating adenoviral vectors (Stadtfeld, Nagaya et al. 2008) and recombinant protein transfection techniques (Zhou, Wu et al. 2009) led to the first integration free iPSCs. The non‐ integrating techniques, however, were plagued by extremely low derivation efficiencies. To solve the problems of genome integration and low efficiency, several groups inserted the reprogramming cassette into the piggyBac transposon (Kaji, Norrby et al. 28 2009; Woltjen, Michael et al. 2009; Yusa, Rad et al. 2009) which has the notable characteristic of being able to integrate and be subsequently removed without leaving a trace in the host genome. This means that the reprogramming factors can be delivered for long enough to achieve reprogramming, and seamlessly removed when utilizing the “fully” reprogrammed cells for further experimentation. Interestingly, the piggyBac transposon was isolated from the Cabbage Looper Moth and it is unique among transposons in that it removes seamlessly (Fraser, Ciszczon et al. 1996). Many transposons, such as Sleeping Beauty, leave behind some kind of footprint even if it is as small as 1 or 2 nucleotide changes (Liu, Aronovich et al. 2004). Such small changes could still be enough to cause damage via mutation and compromise regulatory approval. The use of piggyBac transposons represents a unique intersection in science where two seemingly unrelated fields merge to produce amazing technical capability. Since my study focused on creating clinical grade iPSCs it was necessary to produce integration‐free reprogrammed cells. I thus chose to use the four “Yamanaka” genes OCT4, SOX2, KLF4 and CMYC carried on the piggyBac transposon (pPB:CAG.OSKM‐ pu Δtk) synthesized by Yusa et al. in 2009 (Yusa, Rad et al. 2009). This reprogramming system utilizes a two plasmid system with the transposon mediated cassette contained on one plasmid and the transposase enzyme necessary for integration and removal located on a second plasmid. The transposon cassette consists of TTAA flanking repeat regions, necessary for interacting with the piggyBac transposase, surrounding a polycistronic reprogramming cassette with expression driven by the CAG synthetic 29 Figure 5: PiggyBac Transposon and Transfection Efficiency This schemetic (A) represents the relevant regions of the piggyBac reprogramming transposon. The four reprogrogramming transcription factors (OCT4, SOX2, KLF4, and CMYC) are linked by self cleaving 2A peptide sequences. The synthetic, constitutively active CAG promoter drives expression of this polycistronic repgrogramming cassette which is trailed by a positive/negative selection element. Puromycin exposure positively selects cells with successful transposon integration while gancyclovir will eliminate cells possessing the transposon thus facilitating negative selection for eventual transposon removal. PiggyBac specific terminal repeats flank the reprogramming/selection cassette. A second plasmid containing the piggyBac transposase (pCyL43) is co‐transfected with this transposon plasmid to initiate integration and removal. Transfection methodologies were optimized (B) for the two‐plasmid piggyBac transposon reprogramming system and hDFfs. The AMAXA Nucleofector system provided the highest reproducible transfection efficiency (5‐10%): AMAXA Keratinocyte Kit #VPD‐1002, NHDF High Viability Program #P‐022, 4 mg PB:CAG.OSKM‐puDtk transposon, 2 mg pCyL43 piggyBac transposase, and 2 x 10 6 hDFfs @P5. Efficiency was queried by the percentage of nuclei positive for OCT4 after 72 hrs (C). Scale bar = 100 um. promoter. The reprogramming factors are linked by 2A self‐cleaving linker peptide sequences followed by a positive/negative selection cassette employing puromycin positive selection and tyrosine kinase mediated negative selection [Figure 5]. The transposon is coded by the name pPB:CAG.OSKM‐pu Δtk and I obtained it from the Wellcome Trust Sanger Institute plasmid database. The tranposase plasmid, pCyL43:PB was also obtained from the Sanger Institute. Optimal transfection conditions will 30 generate sufficient fibroblasts with integrated reprogramming transposons for efficient generation of hiPSCs. Specific Aim #2: Reprogram Fetal Human Dermal Fibroblasts to Induced Pluripotent Stem Cells Once the reprogramming transgenes have been delivered to the target somatic cell, one must observe the culture dishes for 3‐5 weeks until viable “ESC‐like” colonies appear [Figure 6]. Several somatic cell types have been used to generate hiPSCs, but donor fibroblast cell lines are easily accessible and expandable for large scale experiments. I used the same hDFfs for feeder preparation for the reprogramming experiments in this study. Observations from previous reprogramming studies indicate that reprogramming fibroblasts undergo a process of mesenchymal to epithelial transition (MET) (Li, Liang et al. 2010). This is exemplified by fibroblasts changing from an elongated cell morphology to a round, flat epithelioid morphology. These changes are visible early during reprogramming, but a diverse population of non‐fibroblast cellular foci appear during the reprogramming process. The heterogenous nature of each of the foci undergoing reprogramming presents a challenge when deciding to select colonies for hiPSC derivation, but one can use the morphological phenotype of hESCs as a guide. hESCs usually form flat, round, compact colonies with tight borders. Identifying foci displaying the morphological appearance of hESCs is thus helpful for initial triaging for further propagation and a few studies have confirmed this observation (Meissner, Wernig et al. 2007). Using 31 Figure 6: Transposon Reprogramming Protocol This diagram references all the key points of the reprogramming process. Low‐passage hDFfs are expanded in an FBS containing medium before harvest and transfection with the AMAXA Nucleofector. The piggyBac transposon reprogramming system is composed of two plasmids, one which contains the OSKM reprogramming cassette and the second containing the piggyBac transposase enzyme. Five days post‐transfection, the medium is replaced with KSR‐XF hESC Medium and the cells are allowed to proliferate for >30 days until hESC‐like reprogramming foci are isolated for propagation and characterization. morphological characteristics alone, I isolated several foci from weeks 3‐5 and demonstrated establishment and long‐term passage of hiPSCs with strong hESC‐like characteristics. Specific Aim #3: Validate Pluripotentcy of XF-hiPSCs by ICC, EB Assay, and Teratoma Formation Morphology similar to hESCs alone is not sufficient for determining the pluripotential nature of hiPSCs. hiPSC lines with varying post‐isolation morphological characteristics resulted from propagation of foci undergoing reprogramming. Some of these hiPSC lines were very hESC‐like but others gave rise to colonies in which the individual cells and overall morphology diverged greatly from hESCs. Previous literature has suggested that the colonies most similar to hESCs are “fully” reprogrammed while 32 the divergent cell lines are “partially” reprogrammed (Masaki, Ishikawa et al. 2007; Chan, Ratanasirintrawoot et al. 2009). These studies looked at reprogramming foci prior to isolation and post‐isolation to describe the “full” vs. “partial” characteristics. However, it would be very useful to identify selectable criteria that enabled prospective isolation of “fully” and “partially” reprogrammed hiPSCs. Standard characterization protocols for pluripotent cells require a diverse panel of pluripotency markers queried by ICC, gene expression analysis as well as embryoid body (EB) analysis and teratoma formation, both of which indicate the ability of the pluripotent cell line to maintain self‐renewal and to differentiate into cells possessing characteristics of all three germ layers: ectoderm, endoderm and mesoderm. ICC is the first and simplest characterization step necessary to segregate “partially” and “fully” reprogrammed hiPSCs. I have utilized a panel of pluripotency markers shown to be expressed on hESCs to characterize several hiPSC lines. My standard panel contains the following markers shown to be involved in maintenance or descriptive of the pluripotent state: transcription factors – OCT4, SOX2, NANOG (Chambers and Smith 2004; Jaenisch and Young 2008), cell surface markers – Alkaline Phospatase, TRA‐1‐60, SSEA‐4 (Thomson, Itskovitz‐Eldor et al. 1998), GCTM‐2 (Mason and Pera 1991; Schopperle, Kershaw et al. 2003), intercellular signaling molecules ‐ EpCAM (Lu, Lu et al. 2010), E‐ Cadherin (D'Amour, Agulnick et al. 2005), and GDF3 (Levine and Brivanlou 2006). Based on marker expression, a hiPSC line is considered “fully” reprogrammed if the majority of colonies stain strongly positive for all the markers with a pattern similar to hESCs. 33 In other mammalian systems such as mouse and rat, it is possible to take the characterization a step further by creating animals completely sourced from the pluripotent cells. This is achieved by injecting pluripotent cells into blastocysts where they integrate into the developing embryo and form a chimeric animal. The resulting animal is comprised of cells sourced from the host blastocyst and the injected pluripotent cells. If the pluripotent cells contribute to the formation chimeric animal’s germ tissue, particularly the gametes, F1 generation chimeric animals may be mated to produce an animal completely sourced from the pluripotent cells. Since such testing is impossible in human due to ethical and technical roadblocks, EB assays and teratoma formation are the final characterization steps. hiPSC cell lines exhibiting hESC‐like morphology, similar pluripotent marker expression patterns and the ability to differentiate into all three germ layers will be validated as “fully” reprogrammed while “partially” reprogrammed lines will fail to demonstate hESC‐like characteristics in these assays. Karyotype analysis demonstrating long‐term chromosomal stability is also important to ensure a normal genetic makeup prior to utilization. 2. Materials and Methods PiggyBac Transposon Transfection Conditions The piggyBac transfection system used in this study contains two plasmids. The transposon plasmid pPB‐CAG.OSKM‐pu Δtk containing the reprogramming cassette and the transposase plasmid pCyL43:PB which facilitates seamless integration/excision of the transposon were obtained from the Wellcome Trust Sanger Institute (Yusa, Rad et 34 al. 2009). Bacterial stabs were re‐plated followed by clone selection and expansion for Maxi‐Prep (Qiagen EndoFree Maxi Kit #12363). The identity of each plasmid was confirmed with a customized restriction enzyme digest created by plasmid DNA sequence analysis. Low passage (<P4) hDFf fibroblasts stocks used for feeder preparation were also utilized as the starting somatic cells for reprogramming to hiPSCs. Several transfection systems were tested for optimal integration efficiency of the two plasmid piggyBac transfection system: Lipofectamine 2000 (Life Technologies #11668), Lipofectamine LTX (Life Technologies #15338), Neon Transfection System + Starter Pack (Life Technologies #MPK5000, #MPK5000S), and the Amaxa Nucleofector II System with the NHDF Nucleofector Kit and the Human Keratinocyte Nucleofector Kit (Lonza #VPD‐1001, #VPD‐ 1002). Protocols included with each system were followed, with programs, cell numbers, reagent concentrations, and plasmid concentrations varied to achieve optimal transfection. Each system was tested on the hDFfs cultured under xeno‐free conditions and in a serum containing fibroblast medium (MEM‐alpha [Gibco #32561], 10% FBS, 0.1 mM MEM‐NEAA, 2mM L‐Glutamine, and 100U/mL Pen/Strep). Transfection efficiency was measured by ICC staining for the percentage of OCT4 positive cells after plating transfected cells onto 0.1% gelatin coated 4‐Well glass chamber slides and allowing 72 hours for transgene expression. Optimal transfection conditions providing a reproducible 5‐10% transfection efficiency were as follows. 2 x 10 6 hDFfs (P2/3) were cultured in serum containing 35 fibroblast medium and harvested at 90% confluency. Following the protocol contained in the Amaxa Human Keratinocyte Nucleofector Kit, the cells were pelleted and resuspended in 100 uL transfection solution containing 4 ug pPB‐CAG.OSKM‐pu Δtk transposon plasmid and 2 ug pCyL43:PB transposase plasmid. Cells were transferred to the transfection cuvette and nucleofected with the NHDF High Viabilty (P‐022) transfection program on the Amaxa Nucleofector platform. Transfected cells were immediately diluted into equilibrated serum containing fibroblast medium and plated for derivation and characterization. hiPSC Derivation Following the optimal transfection protocol described above, the cells were transferred to 3 x 10 cm culture dishes coated with 0.5 ug/cm2 vitronectin/fibronectin. Serum containing fibroblast medium was replaced the following day and every other day after until reprogramming Day 5, when the medium was switched to KSR‐XF on an alternate daily feeding schedule. Reprogramming dishes were observed daily for the appearance of non‐fibroblastic foci. Mature foci possessing hESC‐like colony morphology were isolated by microscope assisted manual dissection starting on Day 20 and continuing until the reprogramming plate became unusable due to overcrowding and detachment of the cellular layer from the culture surface (~Day 35). Isolated foci were transferred to xeno‐free cell culture plates and observed for attachment/outgrowth. Proliferating isolates were continually passaged for characterization. 36 hiPSC Characterization by ICC, Embryoid Body, and Teratoma Formation Isolated reprogramming colonies demonstrating long‐term, stable passage were characterized by ICC for markers of pluripotency, embryoid body differentiation, and teratoma formation. ICC marker combinations were GCTM‐2/SOX2, TRA‐1‐60/OCT4, and SSEA‐4/Nanog with staining following the procedures described in Chapter One. Embryoid bodies were created from a modified “Spin‐EB” protocol (Ng, Davis et al. 2008). Briefly, hiPSC colonies were isolated by microscope assisted manual dissection and pelleted. Colony pellets were enzymatically digested to single cells with TrypLE containing the Rho Kinase Inhibitor Y‐27632 (10 ng/mL, Sigma #Y 0503). Digested cells were diluted in KSR‐XF medium + ROCK Inhibitor and 5 x 10 3 cells/tube were transferred to low‐binding PCR tubes and centrifuged at 400g for 2 minutes. After two days of re‐ aggregation, small EBs were transferred to a 96‐well low‐binding plate containing STEMdiff APEL medium (StemCell Technologies #05210). APEL medium was changed every other day until Day 8 when expanding EBs were transferred to 48‐well plates coated with 0.1% porcine gelatin in HES medium (DMEM [Gibco #11960], 20% FBS, 1X Insulin‐Transferrin‐Selenium (ITS) [Gibco #41400‐045], 0.1 mM 2‐Mercaptoethanol [Gibco #21985‐023], 0.1 mM MEM‐NEAA, 2 mM L‐Glutamine, and 100 U/mL Pen/Strep). HES medium was changed every other day and EBs were observed for attachment and differentiated cellular outgrowths for two more weeks at which time they were fixed in 4% PFA for ICC. Several markers of differentiation were used to characterize the EB outgrowths: Nestin‐1:40 (Ectoderm, Milipore #MAB5326, Clone 10C2, mIgG1), 37 Alphafetoprotein‐1:500 (Endoderm, Sigma #A8452, Clone C3, mIgG2a), and Smooth Muscle Actin‐neat (Mesoderm, DAKO #M0851, Clone 1A4, mIgG2a). Secondary antibodies were as follows: Nestin / AlexaFluor 594 goat anti‐mouse IgG1 1:1000 (Life Technologies #A21125), Alphafetoprotein / AlexaFluor 488 goat anti‐mouse IgG2a 1:1000 (Life Technologies #A21131), and Smooth Muscle Actin / AlexaFluor 488 goat anti mouse IgG2a 1:1000 (Life Technologies #A21131). Fixed cells were permeabilized with 0.2% Triton X‐100 followed by three washes with PBS. Fixed cultures were incubated with primary antibodies for 4 hours followed by three washes with PBS. Secondary antibodies were incubated for one hour followed by three washes in PBS. Hoescht 33342 1:1000 (Molecular Probes #H3570) was added during the secondary antibody incubation for nuclear counterstaining. EB outgrowths were fluorescently imaged on a Zeiss Axiovert A1. For assessment of teratoma formation, Over 40 colonies from each hiPSC line were manually isolated and digested with TrypLE + ROCK Inhibitor as described above. The digestion solution was diluted 1:50 with KSR‐XF followed by pelleting. The cell pellet was resuspended in 50 uL of a 5% Matrigel (BD #356234) solution in mEF medium and kept on ice until testicular capsule injection. Adult male NOD‐SCID mice were anesthetized with Phenobarbital before surgical exposure of both testes. Approximately 2 x 10 4 cells in 10uL of 5% Matrigel solution were injected underneath the testicular capsule. Mice were allowed to recover and teratoma development was monitored for >6 weeks by palpation of the lower abdominal area. Mature teratomas were isolated 38 from the mice and fixed in 4% PFA followed by cryosectioning and H&E staining. Standard histological assessments of fully differentiated tissue were applied to identify tissue arising from all three germ layers. 3. Results Optimal Transfection Conditions for the piggyBac Reprogramming Plasmids Both piggyBac plasmids were validated by restriction enzyme digestion. DNA from bacterial colonies bearing the transposon plasmid pPB:CAG.OSKM‐pu Δtk showed the expected restriction fragment pattern, and DNA isolation was scaled up via the Qiagen Endofree Maxi Kit with a high yield (>1 mg at 1.4 ug/uL). The pCyL43:PB transposase plasmid, however, was found to be defective on initial testing. The Sanger Institute did not provide sequence validation information for pCyL43:PB, but I was able to identify the sequence of the piggyBac transposase with the help of a colleague in the Ying Lab. The sequence was obtained for the atub‐pBac‐K10 construct from the Drosophila Genomics Resource Center at Indiana University (https://dgrc.cgb.indiana.edu/files/repository/1155.txt?id=74880bce8bf559aaaea4bb89 4c8e316a). Restriction enzyme digestion based on the published piggyBac transposase sequence confirmed the identity of a new pCyL43 stock from Sanger, and the plasmid DNA production was scaled up (> 500 ug at 750 ng/uL). The activity of the piggyBac plasmids was confirmed by the presence of OCT4 staining in the transfected hDFfs 7 days post‐transfection. 39 Initial transfection efficiencies using the transposon system and Lipofectamine 2000 were extremely low (<0.01%). The transfection reaction contained 1 x 10 6 hDFfs, 2 ug transposon and 1ug transposase with average reagent concentrations as listed in the Lipofectamine 2000 protocol. I used the same xeno‐free hDFf fibroblast line for reprogramming that I used for production of human fibroblast feeders. It was immediately apparent, however, that these cells exhibit extremely low survival post‐ transfection (>95% cell death). I thus obtained a new aliquot of hDFfs from ScienCell and expanded the cells in a standard fibroblast medium containing a MEM‐alpha base supplemented with 10% FBS. I ran an empirical transfection assay using several transfection methodologies in combination with varying transfection reagent, plasmid DNA, and hDFf cell numbers [Figure 6b]. The chemical transfection methods Lipofectamine 2000 and Lipofectamine LTX displayed the lowest maximum transfection efficiency (< 0.1% and < 1% respectively). The Neon Electroporator from Life Technologies demonstrated improved transfection efficiency (3‐5%). The highest efficiency (5‐10%) was achieved with the AMAXA nucleofector system using the Human Keratinocyte Nucleofector Kit and the NHDF High Viability (P‐022) electroporation protocol with 4ug transposon, 2 ug transposase and 2 x 10 6 hDFfs. I estimated transfection efficiency by determining the percentage of fibroblasts expressing OCT4 three days post‐transfection. 40 Figure 7: Image of Cellular Focus Undergoing Reprogramming at Day 5 This image illustrates several small foci undergoing reprogramming appearing five days post‐transfection with PB:CAG‐OSKM‐puDtk and the pCyL43 transposase plasmid. Notably, transfected hDFfs become more compact and aggregate into round colonies. These foci are surrounded by normal, untransfected fibroblasts that will begin acting as autologous feeders during reprogramming. Previous studies indicate a process of mesenchymal‐to‐epithelial transition (MET) underlying the changes observed here. Derivation of Xeno-Free hiPSCs Following transfection with the optimized AMAXA protocol described above, the first signs of reprogramming were noticeable on Day 2/3 as some cells lost the elongated fibroblast phenotype and begin to form small aggregates with a compact, round cellular phenotype. By Day 4/5 larger aggregates of these compact cells appear surrounded by normal looking fibroblasts [Figure 7]. I term these aggregates “reprogramming foci” since each focus is most likely the product of one fibroblast with 41 Figure 8: Images of Foci Undergoing Reprogramming at Weeks 2/3 Reprogramming foci number and shape continue to evolve during the reprogramming process. By Day 12 (A), most foci form dense, elongated colonies that track with the fibroblast bands. By Day 18 (B), many foci begin enlarging, no longer tracking with the fibroblasts, and several small foci become visible. By Day 20 (C, D, E) large round colonies appear with many foci displaying heterogeneous morphologies indicating multiple emerging cell types. By Day 24 (F), round ESC‐like colonies are large enough for mechanical isolation. Colonies resembling hESCs are rare during Weeks 2/3 and most fail to develop into “fully” reprogrammed hiPSCs after isolation. transposon integration(s). Evidence of MET is noticeable in the reprogramming dishes during the first week as the foci display morphological characteristics of epithelial cells , such as compact, closely associated cells, while the surrounding non‐transfected fibroblasts retain an elongated, mesenchymal morphology. Week 1 was defined by changes visible in single cells, but weeks 2/3 were defined by the wide variety of reprogramming foci [Figure 8]. Each 10 cm dish contained, on average, >1000 visible foci undergoing reprogramming with varying morphological characteristics. Some foci exhibit rapid proliferation to a large size, while others remained small or only first appeared during this phase. Some foci remained round (C, F) with many exhibiting an elongate phenotype (A, B, D, E). Some foci stayed 42 Figure 9: Images of Foci at Weeks 4/5 and Subsequent Outgrowths Reprogramming weeks 4/5 are characterized by increasing numbers of colonies with hESC‐like morphology. Colony C4 (A), isolated at Day 24, exhibited attachment, but had non‐hESC like outgrowths (B) at passage three. Most colonies isolated by morphological criteria alone have a “partially” reprogrammed phenotype as seen with colony C4. The proliferating cells tend to be dark, diffuse, and form palisading outgrowths without tight colony borders. K1 (C), isolated on Day 32, initially showed robust “partially” reprogrammed cellular outgrowths, but soon began to show a dimorphic phenotype by passage five with cells exhibiting “partially” and “fully” reprogrammed characteristics (D). With further passages, I was able to split K1 into separate “partially” and “fully” reprogrammed hiPSC lines. Colony J1 (E), also isolated on Day 32, exhibited the most hESC‐like morphology in the entire reprogramming dish. Its outgrowths (F) formed colonies extremely similar to hESCs by passage two. These images are a small sampling of the diverse colony populations arising during reprogramming. Morphological criteria can aid hiPSC selection, as illustrated by colony J1, but the process remains highly inefficient with most isolated colonies forming “partially” reprogrammed hiPSCs. flat (C, E, F) while others began expanding vertically (B, D). The edges of some foci formed a tight border with others being more diffuse. While each reprogramming focus had a unique morphology, there were some trends present that helped create classifications among the reprogramming foci. One classification consisted of round, compact foci that resembled hESC colonies. I used the hESC‐like morphological criteria to select foci from Weeks 3‐5. My earliest successful derivation attempt came at Day 19 and my latest success came at Day 43 35. Waiting longer than Week 5 was not an option because the cellular layer in the over‐confluent dishes rapidly begins detaching from the growth surface. Many reprogramming foci selected by hESC‐like morphology failed to attach. Of those selected foci that attached and proliferated, most did not display hESC characteristics [Figure 9]. The cell lines C4, K1 and J1 illustrate three typical phenotypes for isolated foci (Transformed, Partial, and Full). Transformed phenotypes are characterized by rapidly proliferating cells that remain loosely associated with rough colony borders. Partial phenotypes exhibit dense colonies with rough border regions composed of small dark cells. Lines with a Full phenotype have characteristic dense, round colonies with smooth colony edges. The hiPSC line C4 (A) attached and proliferated for a few passages, but the cells were dark and the colonies were diffuse (B). hiPSC C4 is a typical example of the “transformed” group which does not survive long term passage. The “partial” group was characterized by the hiPSC line K1 (C) that continued to proliferate for several passages while maintaining its unique morphological traits of small, dark cells in dense, fast‐proliferating colonies (D). Interestingly, I was able to split the K1 line into two cell lines that possessed “partial” and “full” characteristics (K1, K1ENR). The “full” hiPSC line J1 (E) was isolated from a reprogramming focus on Day 32. Of all the reprogramming foci in the 10 cm dish, J1 had the most hESC‐like appearance. J1 immediately demonstrated cellular outgrowths with highly similar characteristics to hESCs such as small cells in compact, round, flat colonies (F). I used morphological criteria to derive several hiPSC lines [Figure 10] that fall into the “full” (A‐F) and “partial” 44 Figure 10: Images of hiPSCs I have derived several hiPSC lines from three different donor hDFf stocks. The first six panels represent “fully” reprogrammed lines based on their morphological similarities to hESCs: J1 (A), K1 ENR (B), U1 (C), Y4 ENR (D), AA4 (E), and AE4 (F). Several lines exhibiting a similar, but non hESC‐like morphology are shown in the bottom three panels. These “partially” reprogrammed lines continue to stably proliferate after several passages: K1 (G), W4 (H), Y4 (I). Interestingly, the image for the Y4 “partially” reprogrammed line illustrates the point at which a dimorphic cell population exists during early passaging. These colonies were passaged into separate culture dishes to form the Y4 and Y4 ENR cell lines. During subsequent passages, the two Y4 lines maintain their “fully” and “partially” reprogrammed morphological traits. While these two types of hiPSCs are commonly derived from the transfection dishes, several other intermediate cell types also grow out of isolated reprogramming foci. Unfortunately, these intermediates fail to proliferate through several passages for full characterization. (G‐I) morphological descriptive categories. These morphologically divergent lines originate from several independent transfection experiments and from three different 45 Table 1: Derived XF‐hiPSC Lines This table lists all XF‐hiPSC lines derived and propagated for study. Three different hDFf donor lines were successfully used for reprogramming experiments. Each date corresponds to the initiation of a new transposon transfection experiment. “Fully” reprogrammed cell lines are noted in black font while “partially” reprogrammed cell lines are noted in red font. The hiPSC line J1 has been continually passaged since initial isolation >P50. The hiPSC lines K1/K1E and Y4/Y4E represent lines isolated from the same reprogramming foci, but they have been split into “fully” and “partially” reprogrammed cell lines that stably maintain divergent morphological characteristics. hDFf donor cell lines [Table 1]. Conservatively, 1 out of 50 reprogramming foci isolated by morphological characteristics gave rise to a “fully” reprogrammed hiPSC line. These “full” hiPSCs exhibited morphology similar to hESCs at the cellular and colony level and some lines have retained this morphology with limited spontaneous differentiation for >50 passages. “Partial” hiPSC lines demonstrating long term maintenance for >50 passages while maintaining a stable, unique morphological identity were also isolated. XF-hiPSCs Demonstrate Pluripotency Via ICC and Embryoid Body Analysis “Fully” and “Partially” reprogrammed lines were stained by ICC for the markers GCTM‐2/SOX2, TRA‐1‐60/OCT4 and SSEA‐4/Nanog [Figure 11]. SOX2 and OCT4 stain strongly positive on both hiPSC lines, but it is not possible to decipher endogenous or 46 Figure 11: ICC Images of Full and Partial hiPSCs These images illustrate pluripotent marker expression on the J1 “fully” reprogrammed and K1 “partially” reprogrammed XF‐hiPSC cell lines. Representative colonies are stained with the cell surface marker GCTM‐2 (green) and nuclear marker SOX2 (red) (A, D). J1 has a marker distribution similar to hESCs while K1 shows patchy GCTM‐2 staining and diffuse colony borders. SOX2 expression is strong on both cell lines which is expected due to its inclusion on the integrated reprogramming transposon. Colonies were also stained with the nuclear marker OCT4 (green) and cell surface marker TRA‐1‐60 (red) (B, E). J1 exhibits an hESC‐like staining pattern while K1 has a central area positive for TRA‐1‐60 and palisading diffuse edges positive for OCT4. OCT4 is also part of the reprogramming transposon so strong expression is expected. The cell surface marker SSEA‐4 (green) and nuclear marker Nanog (red) stain strongly on both cell lines (C, F), but the pattern is widely divergent. J1 again possesses hESC‐like staining characteristics while K1 illustrates non‐compact borders and patchy staining for SSEA‐4. The nuclear marker Nanog is not included on the transposon so its strong staining is indicative of reprogrammed pluripotency transcription factor networks. Both XF‐hiPSC cell lines J1 and K1 show pluripotency marker expression, but J1 exhibits a more hESC‐like staining pattern than K1. transgene expression due to the inclusion of these two genes on the piggyBac transposon reprogramming cassette (A, B, D, E). The nuclear marker Nanog, originating from endogenous activation of this pluripotency gene, stains brightly on both hiPSC lines (C, F), but the staining is limited to colony borders in the J1 “fully” reprogrammed line while Nanog positivity in the K1 “partially” reprogrammed line exhibits a diffuse, 47 Figure 12: Embryoid Body ICC Images of hiPSCs This embryoid body assay was performed to investigate the pluripotent nature of the hiPSCs. The top three panels correspond to the “fully” reprogrammed line AA4 while the bottom three panels correspond to the “partially” reprogrammed line Y4. After three weeks of differentiation, both lines showed strong expression for markers of all three germ layers. Nestin (red) is a marker for neural tissue of the ectoderm lineage (A, D). Alphafetoprotein (green) is a marker of liver progenitors of the endoderm lineage (B, E) and Smooth Muscle Actin (green) is a marker of smooth muscle contractile tissue of the mesoderm lineage (C, F). Hoescht dye (blue) was used as a nuclear counterstain in all of the images. The Y4 “partially” reprogrammed line predominantly formed Nestin positive cells. This was expected since the Y4 line, along with other “partially” reprogrammed lines, commonly spontaneously differentiate into cells with long axonal‐like processes. It was unexpected, however, that the Y4 line also differentiated into cells of endodermal and mesodermal lineage. Y4, much like the K1 line, was split into “partially” and “fully” reprogrammed lines early on during passaging. It is possible that some “fully” reprogrammed cells remained in the Y4 line enriched for its “partially” reprogrammed characteristics, or the continued presence of the reprogramming transposon leads to a dynamic state with some cells switching from “partially” to “fully” reprogrammed. Scale bars = 50 um. palisading pattern at the colony border. GCTM‐2 and TRA‐1‐60 are strongly positive on the surface and borders of J1 (A, B), but positivity is limited to a few cells at the center 48 of the K1 colony (D, E). SSEA‐4 positivity spans the entire surface of J1 (C) and K1 (F), but the finger‐like projections at the edge of K1 are highlighted compared to the tight colony borders of J1. All markers stain positive on the “fully” and “partially” reprogrammed cell lines. “Partially” reprogrammed lines, however, exhibit a spotty staining pattern with some colonies having limited positivity or being marker negative entirely. All hiPSC lines tested in the Spin‐EB assay demonstrated robust EB formation. An interesting observation was that “full” hiPSCs developed multiple, small EBs after aggregation while “partial” hiPSCs usually developed a single, large EB despite equal cell number input. After attachment on gelatin coated plates, the EBs exhibited robust cellular outgrowth consisting of multiple cell types. “Partial” EBs predominantly produced long, axon‐like cellular projections emanating from the central EB mass. “Full” EBs produced a variety of morphologically nondescript cells. Interestingly, large central masses in the “full” EBs appeared to retain pluripotent morphology while it was hard to decipher cellular identity in the center of “partial” EB outgrowths due to their high density and three dimensional shapes. Both “full” and “partial” EBs [Figure 12] showed a majority of Nestin (Ectoderm) (A, D) positive cells with small islands of Alphafetoprotein (Endoderm) positive cells interspersed (B, E). Few cells positive for Smooth Muscle Actin (Mesoderm) were noted in “full” (C) and “partial” (F) hiPSCs, but both hiPSC morphological subtypes displayed the ability to differentiate into all three germ layers as measured by expression of the selected markers. Interestingly, the 49 axonal‐like projections noted under phase microscopy stained brightly for Nestin in the “partially” reprogrammed EB outgrowths (F). Such outgrowths were not seen in the “fully” reprogrammed EBs. After staining the EBs in this assay, I quantified the proportion of EB wells staining positive for the various markers [Figure 13]. Overall, when comparing EBs produced from the hESC line USC01, “Full” lines G4E, G4F, AA4 and “Partial” lines Y4, G5D (A), there is no statistical difference between the ability of each line to produce cells with characteristics of all three germ layers. G5D was the lone exception as it did not have SMA positive cells, but only 2 wells were stained. Qualitatively, however, the “Partial” lines exhibited a propensity greater than “Full” lines to form Nestin positive outgrowths, and they were the only lines to produce long, axon‐like projections (B). I am currently performing teratoma assays on two of my hiPSC cell lines: G4E and G4F. The teratoma data will be included in a future manuscript describing XF‐hiPSC derivation. 4. Conclusions I have successfully created large stocks of the piggyBac reprogramming plasmids pPB:CAG.OSKM‐pu Δtk and pCyL43:PB. The plasmids were validated by restriction enzyme digestion. Empirical testing of transfection conditions indicate that the AMAXA Nucleofector system provides the highest efficiency with 5‐10% of the cells surviving nucleofection demonstrating integration of the reprogramming transposon. A 5‐10% transfection efficiency is the minimum necessary to have enough integrated cells to 50 Figure 13: Embryoid Body Quantification These graphs depict the staining results for the embryoid body assay. hESCs, “full” and “partial” hiPSCs were dissociated and allowed to aggregate as embryoid bodies before plating. EB outgrowths were stained for the markers Nestin (Ectoderm), Alphafetoprotein (Endoderm), and Smooth Muscle Actin (Mesoderm). Wells with positive staining were counted and displayed as a percentage of total EB wells (A). All cell lines differentiated into cells expressing markers characteristic of all three germ layers except G5D which failed to show SMA positivity. Interestingly, only the “partial” hiPSCs produced outgrowths with structures highly characteristic of axonal projections (B). This is in line with previous expression data suggesting that the “partial” lines possess a more neuronal profile. Even with the predominantly nestin positive outgrowths, the “partial” lines were able to differentiate into cells characteristic of all three germ layers. 51 proceed with experimentation. With this efficiency, ~1 x 10 5 cells have integrated piggyBac transposon(s) assuming that half of the cells do not survive the transfection. With an estimated hiPSC generation efficiency of 0.1%, transposon integrated fibroblasts progressing to a “Fully” reprogrammed state, I can expect to maximally generate 100 new hiPSC lines with each reprogramming experiment. This efficiency was more than enough to generate several unique XF‐hiPSC lines for further characterization and equivalency testing with XF‐hESCs. Observing the number of foci undergoing reprogramming in each 10 cm dish provides an initial indication of the transfection efficiency and lends support for continuing the experiment. As mentioned previously, the fibroblast medium is replaced with KSR‐XF at Day 5 and the reprogramming cells begin to flatten and spread out upon exposure to the KSR‐XF. Therefore, it is important for the dishes to be highly confluent before switching to the KSR‐XF since the high number of surrounding normal fibroblasts help keep the foci closely associated as well as providing an endogenous feeder layer to support the reprogramming cells. The hiPSCs generated in these experiments are not xeno‐free throughout the entire process since the fibroblasts are expanded in FBS containing medium prior to transfection and for the first 5 days post‐transfection. It is possible that further refinement of the transfection conditions would allow for greater transfection efficiency on cells grown entirely in xeno‐free conditions, but my initial testing indicates that the fragile XF‐hDFfs do not survive the transfection in sufficient numbers to proceed with reprogramming. 52 To date, I have derived more than 40 hiPSC lines and several of these lines have been maintained for >20 passages without loss of pluripotency markers. The morphology of the hiPSC colonies closely mimics the morphology of either HES2 or HES3 in the same culture system although the “full” hiPSCs appear more compact and three‐ dimensional. ICC, however, indicates a similar profile of pluripotency marker expression (GCTM‐2, TRA‐1‐60, SSEA‐4, Nanog, SOX2, OCT4). hiPSCs and the hESC line HES3 demonstrate a similar RNA expression profile of pluripotency markers by qPCR . Spontaneous differentiation is also evident in the hiPSC lines derived in this study, but few colonies will spontaneously differentiate suggesting a stable reprogrammed state. I have also tested several lines for embryoid body formation and the majority possess the ability to differentiate into cells expressing markers characteristic of all three germ layers: Nestin (ectoderm), AFP (endoderm) and SMA (mesoderm). Interestingly, I derived several “partially” reprogrammed hiPSC lines that possess dramatically different colony morphological characteristics. The colonies proliferate more rapidly and have diffuse borders with dark, palisading cells. ICC staining for pluripotency markers indicates that the majority of the cells in the “partially” reprogrammed hiPSCs lose or never had expression of the surface markers GCTM‐2 and TRA‐1‐60. Further examination of the “partially” reprogrammed lines indicates a propensity toward a neural phenotype. This is suggested by the spontaneous differentiation of these lines into cells with axonal‐like elongated projections emanating from the EB and staining predominantly positive for the neural protein nestin. qPCR 53 analysis of the K1 line before EB formation shows dramatically decreased expression of pluripotency associated genes (OCT4, Nanog, DNMT3b, Nodal, GDF3, CRIPTO1) while showing increased expression of SOX2 and PAX6, both strongly expressed in neural cells [Figure 14]. It is possible that the neural phenotype is a default pathway in this reprogramming system or the “partially” reprogrammed hiPSCs represent cells that have not reached a stable reprogrammed state. “Partially” reprogrammed hiPSC lines like K1, however, continue to exhibit the same cellular and colony characteristics for >50 passages indicating that the cells have reached a stable but incompletely reprogrammed phenotype. In fact, I was able to split the K1 hiPSC line into two clonogenic populations, one which remained in a “partial” state (K1) and another which exhibited a stable “fully” reprogrammed state (K1ENR). Whether these two lines originate from a single fibroblast with integrated transgenes or from two separate, independently integrated fibroblasts that then blended together to form one heterogenous focus during the reprogramming process remains to be tested. One could do Southern Blot analysis to see if the two K1 lines exhibit a different transposon integration profile. If the integration profile is the same, then the two lines originated from a single cell whereas a different profile would indicate multiple cells of origin. The first scenario would indicate a more intriguing phenomenon wherein the “partially” reprogrammed line possesses a mix of cells with the same transposon integrations each of which is subject to an independent, dynamic reprogramming 54 Figure 14: Quantitative PCR for hiPSC Lines J1 and K1 This graph illustrates the relative expression of several pluripotency and lineage related genes. The HES3 line is used as a reference for relative expression for the J1 (red, green) and the K1 (purple) hiPSC lines. J1 exhibits similar expression compared to HES3 except for decreased PAX6 and elevated GATA4. K1, however, exhibits marked decreases in pluripotency gene expression except for slightly elevated SOX2. K1 also exhibits dramatically elevated PAX6 (ectoderm) expression with strong decreases inGATA4 (endoderm) and MIXL1 (mesoderm). With elevated SOX2 and PAX6, the “partial” K1 line possesses characteristics of a neural phenotype while the “full” J1 line has similar expression to the pluripotent control HES3. process. This phenomenon would account for the late appearance of K1 ENR after several passages (P3‐5) wherein only a small subset of cells in the original selected focus achieve a “fully” reprogrammed state. Several passages would then be necessary for the small “fully” reprogrammed population to expand enough for visual segregation. Accordingly, even “fully” reprogrammed lines displaying early morphology similar to hESCs require a few passages before all the colonies exhibit a stable “fully” 55 reprogrammed state. Since I am manually passaging these hiPSCs, I select for the best looking colonies to propagate. Consequently, I am adding a level of selection bias that accounts for this observation. Such stability issues also point to a reprogramming process that may require more than 3‐5 weeks for completion. The heterogeneity displayed by the isolated foci undergoing reprogramming is a gross sign illustrating the amazingly complex biology underlying the reprogramming process. As the transgenes begin to reprogram the cell, epigenetic imprinting must be removed and the pluripotency gene networks must be restarted if the cell is to reach a naïve, pluripotent ESC state. The very low reprogramming efficiency reported by hiPSC studies indicates that successful removal of epigenetic imprinting and pluripotency network remodeling is a difficult process. As a result, most cells will assume some intermediate phenotype between a somatic cell and a pluripotent cell. Heterogeneous reprogramming foci are glaring evidence of this complicated process. Deriving hiPSCs by selecting foci based solely on their morphological resemblance to hESCs remains inefficient with only 1‐2% reaching a stable, “fully” reprogrammed identity. The majority of morphologically selected foci fail to attach or attach without proliferating. This failure could be due to mechanical damage received by manual fine needle dissection or it could be due to the functional proliferative capacity of the reprogramming focus itself. Technological advancements are thus necessary to improve selection of the best “fully” reprogrammed hiPSCs. Live‐cell staining with cell surface pluripotency markers, such as GCTM‐2 and TRA‐1‐60, or the 56 inclusion of selective reporter genes on the reprogramming cassette will help select hiPSCs possessing the most hESC‐like characteristics. 57 Chapter Three: Derivation of Xeno-Free Human Embryonic Stem Cells 1. Introduction The political and ethical climate surrounding the derivation and use of hESCs continues to evolve. Currently, the Dickey‐Wicker Congressional Amendment of 1995 prohibits the use of federal funds to derive new hESCs. Dr. Martin Pera, as a result of federal funding impediments to new hESC derivation, obtained a $1.4 million grant from the California Institute of Regenerative Medicine (CIRM) to support the derivation of new pluripotent cell lines at the Keck School of Medicine of the University of Southern California [CIRM RL1-00667-1]. The state funds distributed through CIRM do not have restrictions related to the use of human embryos for hESC derivation and research. With strong financial support for new hESC derivation, we began developing the methodology to derive new, clinically relevant hESC lines. The following commentary describing the justification for new hESC derivation and current derivation technology is excerpted from a review I co‐authored with Dr. Pera and Dr. Kouichi Hasegawa (Hasegawa, Pomeroy et al. 2010). Scientific Justification and Ethical Criteria For New Derivations Any efforts to derive new hESC lines should be justified scientifically. A recent analysis of the literature indicated that there are now over 1000 hESC lines in existence and that roughly 70% of these have been characterized to some degree in peer‐ reviewed publications (Loser, Schirm et al. 2010). However, much of the scientific 58 literature on hESCs is based on only a handful of cell lines (McCormick, Owen‐Smith et al. 2009; Scott, McCormick et al. 2009). Given that there are over a thousand hESC lines in existence, there is certainly a case for more work on a wider range of these established cell lines (assuming that the majority are in fact available for distribution). However, what scientific rationale justify generation of additional cell lines? First, genetic or epigenetic variation in the ability of hESC lines to undergo differentiation into particular lineages remains a largely unknown factor. Thus it is uncertain how many cell lines might be required to provide a panel with, for example, high competency for beta islet cell formation. Similarly, little is known about how genetic or epigenetic stability varies between different hESC lines. Second, estimates of the number of hESC lines that will be required to provide adequate coverage for tissue matching in transplantation vary. Limited studies suggest that existing cell lines are not representative of a wide range of ethnic diversity (Laurent, Nievergelt et al. 2010; Mosher, Pemberton et al. 2010), and the problem of matching populations of mixed racial origin has not really been addressed. Third, only a small minority of the published cell lines have been derived under conditions that are optimal for future clinical use. Technology for hESC derivation, maintenance, and expansion are constantly evolving and there is a strong argument for deriving cell lines using improved methodology. While the advent of iPSC technology may circumvent ethical roadblocks around the use of embryos in research, there are still questions over the biological equivalence and safety of iPSC lines compared to hESC (Lee, Park et al. 2009). Some concerns 59 include limitations or reductions in developmental potential (Hu, Weick et al. 2010; Kim, Lee et al. 2010), variability relating to the starting cell type used for reprogramming (Kulkeaw, Horio et al. 2010), and an epigenetic / gene expression profile which may suggest and independent pluripotent state for iPSCs when compared to ESCs (Chin, Mason et al. 2009; Doi, Park et al. 2009). hESCs, as such, remain the standard by which all other pluripotent cell lines are judged (Hyun, Hochedlinger et al. 2007; Smith, Luong et al. 2009). The research community has been moving towards uniform international ethical standards for the derivation of new cell lines. The European Union started the process in 2004 with Directive 2004/23/EC followed by the International Society for Stem Cell Research (ISSCR) in 2007 and the National Institutes of Health (NIH) in 2009. Each of these sets of guidelines, subjected to public comment and extensive refinement, adheres to some very basic principles to ensure the highest level of safety and compassion in the study of hESCs: 1) Only unused embryos created for the purpose of in vitro fertilization (IVF) should be used for the derivation of ESCs. 2) Donors should voluntarily consent to the donation of embryos for research without influence from those participating in the study. 3) The standard of IVF care should be unaffected by the decision to donate. 4) No financial compensation was made for the donation. 60 5) Donors should be informed of alternatives to donation, that embryos would be used for the derivation of ESCs, that no direct medical benefit was intended, that the ESCs may have commercial potential to which they would not be entitled, that identifying information would remain confidential, and that they may withdraw from the study until the embryos are actually used. The push to implement these standards is reflected in the European Human Embryonic Stem Cell Registry (hESCreg) and the NIH Embryonic Stem Cell Registry. The NIH registry currently comprises 64 lines that adhere to US federal guidelines with 12 more in submission as of May 2010 (150 with 43 more in submission as of March 2012). While hESCreg has over 650 lines listed, the provenance of only a small fraction has been validated to meet European/International guidelines. As hESC technology moves into the translational and clinical stage, standards for derivation of cell lines will become more stringent. The first step toward the clinic involves the derivation and characterization of Good Manufacturing Practice (GMP) quality cell lines (Ahrlund‐Richter, De Luca et al. 2009). In order to qualify for GMP, the cell lines must be derived and cultured in: 1) Defined and 2) controlled conditions 3) by trained staff 4) with full documentation. Some proprietary lines have been derived under GMP conditions (ESI, WiCell), and only one cell line, H1, has been maintained under GMP conditions and approved for clinical trials (Geron Corporation). It should be noted that GMP does not preclude the use of products derived from animal sources, such as fetal calf serum, so long as the product meets the GMP standards defined above 61 and no other suitable products are available. Future clinical acceptance of any new hESC line, however, can only be improved by the combination of xeno‐free GMP methods for derivation and maintenance. Human Embryo Culture, Assessment, and Establishment of hESCs Embryo Culture and Assessment Figure 15 illustrates some key phases in the life history of a hESC line, beginning with embryo culture. Human embryo culture has been refined over the thirty plus years since the first successful in vitro fertilization (IVF) procedure. In general, procedures for culture of the fertilized egg from the two pronuclear (2PN) stage of fertilization through initial cleavage and transfer to the uterus for implantation have sought to mimic the conditions that a zygote would experience while traveling through the fallopian tube. Human tubal fluid nearest the ovary is high in pyruvate and lactate, while the concentration of glucose increases as the zygote nears the uterus. Therefore, it is common to culture human embryos in sequential media wherein a human tubal fluid analogue medium is used from fertilization to the 8‐cell stage at day 3, and this is followed by a switch to a high glucose, complete medium for compaction and blastocyst formation on days 5‐6 (Bongso and Tan 2005; Mercader, Valbuena et al. 2006; Ilic, Genbacev et al. 2007; Sathananthan and Osianlis 2010). However, some workers argue that a single media system yields equivalent results (Biggers and Summers 2008). Many embryo culture protocols have employed co‐culture systems using fibroblasts, endometrial cells, or other cell types to support development, and a recent meta‐ 62 Figure 15: Derivation of hESCS 63 analysis indicated that co‐culture does indeed improve embryo quality (Kattal, Cohen et al. 2008). However, co‐culture of embryos has the same drawbacks as the use of feeder cells during establishment and maintenance. Embryo quality is a critical factor in hESC derivation. For the most part, assessment of embryo quality continues to rely on morphological criteria (Bongso and Tan 2005), though metabolomics (Botros, Sakkas et al. 2008), and proteomics (Katz‐ Jaffe, McReynolds et al. 2009) may ultimately provide more objective and accurate evaluation. The highest level of success comes from implantation of high‐quality, expanded blastocysts on Day 5/6, and reported success in hESC derivation is also greatest under these parameters. Since the best available embryos are of course used for transfer to the uterus, surplus embryos for hESC derivation may not always be of the highest quality. Several groups (Mitalipova, Calhoun et al. 2003) have reported derivation of hESC lines from poor quality embryos, and a recent study confirmed the potential of this approach(Lerou, Yabuuchi et al. 2008) , though success rates were quite low unless the embryos were able to reach the blastocyst stage. Timing of inner cell mass (ICM) isolation is another critical factor determining the outcome of derivation. Most of hESC lines have been derived from blastocysts at day 5/6 of culture. A recent study indicated up to 50% efficiency when blastocysts were allowed to develop until day 6 (Chen, Egli et al. 2009). At this stage, isolated ICM attaches to the feeder layer with relative ease and starts proliferating. These results are reflective of the higher rates of successful implantation during IVF treatment when blastocyst stage embryos are used. 64 Avoiding Immunosurgery and Exposure to Animal Products Although some workers have derived hESC from explanted blastocysts, most have relied on immunosurgery for isolation of the inner cell mass (ICM) (Solter and Knowles 1975; Bongso, Fong et al. 1994). Because trophectoderm cells show rapid rates of growth and may inhibit the expansion of the ICM in culture, their early removal is considered beneficial by most workers. Immunosurgery requires the use of xenomaterials in the form of animal‐sourced antibodies and complement. Whole and partial‐embryo culture methods (Kim, Oh et al. 2005) can eliminate the need for immunosurgery, but do not enrich for ICM during initial derivation. Two alternate approaches avoid the pitfalls of immunosurgery with animal components and whole embryo culture. First, the inner cell mass can be isolated mechanically by dissection with sharpened metal needles (Strom, Inzunza et al. 2007). The second possibility is to perform the isolation with infrared lasers, which are widely used for drilling holes in the zona pelucida of eggs and early embryos for preimplantation genetic diagnosis (PGD) testing. The infrared laser can be used to isolate the inner cell mass through ablation of the zona pelucida and trophectoderm (Turetsky, Aizenman et al. 2008). Proof of concept of this technique was demonstrated on genetically abnormal embryos identified during PGD, with 3 out of 8 inner cell masses producing disease‐specific hESC lines. A more recent study using laser‐assisted derivation in human embryos reported derivation efficiency as high as 52% when isolating ICM from day 6 blastocysts (Chen, Egli et al. 2009). 65 Blastomere Culture Several groups have examined the potential for hESC derivation from single blastomeres. Given that preimplantation genetic diagnosis entails biopsy of a single blastomere and allows for the normal development of the remaining cleavage stage embryo, blastomere biopsies were undertaken to create new hESCs without the destruction of embryos, to avoid ethical concerns. The technique was first reported in the mouse in 2006 (Chung, Klimanskaya et al. 2006), and this report was closely followed by the derivation of hESCs from human blastomeres (Klimanskaya, Chung et al. 2006). Co‐culture of the blastomere with existing hESC lines was a necessity in these studies, a potential limitation to the use of the technique for deriving clinical grade lines. Another study employed co‐culture with the parent embryo with some success (Chung, Klimanskaya et al. 2008). This method of co‐culture with the parent embryo also produced blastomere derived hESCs in the presence of human feeders and minimal xenomaterials (Ilic, Giritharan et al. 2009). It is possible that maintaining the parent embryo in culture with the biopsied blastomere may restrict its future use in IVF treatment, so elimination of this step is important. In 2009 a group used 4‐cell stage embryos and isolated individual blastomeres (Geens, Mateizel et al. 2009). Each blastomere was allowed to develop in sequential medium until day 3 or 4 when they were transferred to inactivated MEFs. Two new cell lines were established, only one of which was karyotypically normal. 66 Derivation of hESCs from blastomeres represents an interesting technical achievement, and the technique has the potential to provide insight into possible differences in cell lines derived from presumed totipotent blastomeres of early stages versus pluripotent cells derived from the later stage ICM of the blastocyst. Whilst it has been argued that such an approach can leave a viable embryo intact (on the basis of experience with preimplantation genetic diagnosis) and thus circumvent ethical issues around embryo destruction, it seems unlikely that clinicians would chose to implant an embryo that had undergone biopsy in preference to one that had not (unless there were a clinical indication for carrying out the biopsy). Therefore, it is unclear whether such procedures would ultimately impact on the long term viability of the embryos, which would likely be discarded anyway. Specific Aim #1: Obtain Fully Consented Human Embryos Based on the scientific justification described in the previous passages, we decided to obtain human embryos for hESC derivation in “clinical grade” conditions. Obtaining surplus human embryos has been the most demanding and difficult project so far, but my experience in coordination and execution of a human subjects study has gained tremendously. The most difficult part of deriving new hESCs is obtaining high quality human embryos that are in limited supply. Dr. Martin Pera began this process in 2008 by initiating an IRB and getting stem cell research oversight (SCRO) committee approval for consenting donors and obtaining embryos from our collaborator, Dr. Richard Paulson, of USC Fertility. While the ethical review documents were in 67 preparation, support for hESC research at the federal government level was dramatically shifting. In 2001, Federal funding support for hESC research was limited to the 76 hESC lines reported to be in existence (the actual number of available cell lines turned out to be much lower). Such restrictions, only a few years after the first successful derivations of hESCs, severely hampered progress in this field since many of these lines were unusable or not readily available for distribution. With the change of presidential administrations in January 2009, President Obama directed the National Institutes of Health (NIH) to draft new guidelines for the federally funded/sanctioned research on new hESC lines. It was imperative that the IRB and embryo consenting process matched the new guidelines which were released on July 7 th , 2009. Chiefly important among these guidelines is the necessity that all donated embryos be surplus to clinical requirement and no longer needed for future fertility treatment. Surplus embryos are thus only available after donors have voluntarily decided to end fertility treatment regardless of success. Another main guideline is the need to obtain consent from all parties involved in the fertility treatment. This means that up to four individuals would need to give consent: Patient/Partner and Sperm/Egg donor. Other guidelines address a patient’s release of rights to future commercial gain, that medically trained consenting personnel must not be directly involved in the research, and that fertility care should not be affected by the decision to donate. After vetting our documents against the new NIH guidelines, we submitted them for final approval in late 2009 at which point we 68 were cleared to begin contacting potential donors (Appendix A ‐ Egg/Sperm Donor and Appendix B ‐ Embryo Donor Consent Forms). Specific Aim #2: Optimize Laser Assisted Inner Cell Mass Enrichment Protocol While the consenting process was underway, I was busy establishing the xeno‐ free cell culture system above and had successfully derived hiPSCs. These results lent confidence for the first hESC derivation attempts starting in December 2010. Standard derivation procedures, as described above, utilize the immunosurgery technique to selectively lyse the trophectoderm of the developing blastocyst thus leaving the inner cell mass (ICM) enriched for plating onto the feeders/extracellular matrix (Solter and Knowles 1975; Bongso, Fong et al. 1994). I was unable to use this technology since immunosurgery requires antibodies isolated from animal serum rendering this process not xeno‐free. Therefore, I procured the XY Clone 1420 um infrared laser from Hamilton Thorne to mechanically dissect the ICM from the fully expanded blastocysts. Infrared lasers are used clinically to drill a hole in the zona pellucida to facilitate removal of single cells in early cleavage embryos for the purpose of preimplantation genetic diagnosis. Recently, researchers have used such lasers to artificially hatch blastocyst stage embryos and remove the bulk of trophectoderm leaving an enriched ICM (Turetsky, Aizenman et al. 2008). This laser‐assisted ICM enrichment technique allows for XF derivation of hESCs. It is imperative that the laser‐assisted ICM enrichment protocol be fully optimized 69 before beginning derivation attempts with scarce, fully‐consented human embryos. To this end, I utilized mouse blastocysts on which to hone my laser derivation skills. I demonstrated derivation of a mouse ESC line with the laser derivation rig and this success provided confidence moving forward with human embryos. Specific Aim #3: Derive and Characterize XenoFree Human Embryonic Stem Cells The majority of fully‐consented human embryos received for derivation of hESCs were low quality. Of the 45 embryos received to date, most did not survive the thawing process or failed to develop into expanded blastocysts necessary for laser aided isolation of ICMs. Several embryos did not progress past the the blastomere stage and failed to compact to form morula stage embryos. Amongst those that made it to a blastocyst stage, the ICM was frequently fragmented. The initial derivation attempts were unsuccessful in large part due to the low embryo quality. Despite the low quality, many embryos exhibited attachment and proliferation. Most of the outgrowths did not possess hESC‐like characteristics. A few fully expanded blastocysts yielded dimorphic cell proliferations following isolation, but I was unable to manually dissect a hESC‐like population of cells from these outgrowths. Further refinement of the protocol led me to enzymatically dissociate the entire proliferative population followed by re‐plating. Subsequent outgrowths demonstrated hESC‐like colonies which became the first new XF‐hESC line at the Keck School of Medicine, USC01. USC01 continues to possess the pluripotent characteristics of hESCs after >30 70 passages. The new, karyotypically normal (46XY) XF‐hESC line stably expresses several markers of pluripotency, and differentiates into cells exhibiting characteristics of all three germ layers in embryoid body assays. 2. Materials and Methods Human Embryo Donor Consent IRB (HS‐08‐00576) and SCRO (2008‐6‐1) approval for obtaining consent from human embryo donors for the purpose of deriving new hESC lines was granted in 2009. The consent process adheres to the 2009 NIH guidelines for the use of new hESC lines. Surplus human embryos resulting from the completion or discontinuance fertility treatment at USC Fertility were identified and approached for consent for donation. Consent was obtained from the patients receiving fertility treatment as well as gamete donors if the donated embryo was the result of a donated gamete(s). Unidentifiable sperm donors contributing to the fertilization of a donated embryo were allowed for embryos fertilized and stored before November 2007, but all sperm donors were consented past this date. Donor contact was undertaken by fertility specialists trained in human subjects research before sending consent documents for signatures. After receipt of all signed consent forms, donors were contacted a final time for any questions and informed of a 30 day waiting period after which the fully consented human embryo became available for hESC derivation. Fully consented human embryos were re‐coded for this study with any identifiable patient information removed from the record. 71 Human Embryo Culture to Blastocyst and Transfer for Derivation Fully consented human embryos were stored at USC Fertility, a fully licensed fertility clinic associated with the University of Southern California, until used for derivation. Embryos were cryogenically stored at the 2PN, Day 3 and Blastocyst stage. Upon thaw, embryos were cultured under standard protocols at USC Fertility with a two stage blastocyst culture protocol until the equivalent of Day 5/6 embryonic development stage. Day 5/6 embryos were then transferred to a HEPES buffered blastocyst medium (In Vitro Care #2002) supplemented with Serum Substitute Supplement (Irvine Scientific #99193), and transported by car to the Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research with a 30 minute average travel time. Transported embryos were kept near 37 o C by placing the vial close to my skin while covering myself with a jacket. Upon arrival at the derivation facility, embryos were immediately transferred to equilibrated Quinn’s Advantage Protein Plus Blastocyst medium (SAGE In‐Vitro Fertilization Inc. #1529) in a 37 o C incubator. Embryo quality was scored according to the Gardner Blastocyst Grading System (Gardner 1998). Briefly, the embryo is scored from 0 (undeveloped) to 6 (hatched) with two letter grades for the quality of the ICM (A‐C) and the trophectoderm (A‐C). Laser Assisted Enrichment of the Inner Cell Mass and XF-hESC Derivation Isolation of the ICM from Day 5/6 human embryos was accomplished by laser dissection of the zona pellucida and removal of the bulk of the trophectoderm. Each embryo was transferred to a 35 uL drop of XF‐KSR culture medium covered with embryo 72 tested mineral oil (Sigma #M5310) to protect it from contamination while on the derivation system. A 1420 um infrared laser (XY Clone – Hamilton Thorne) attached to a 40X objective on a Zeiss Axiovert A1 microscope provided a fixed, pulsatile, (100% Power, 400 us pulse) and ablative beam for embryo dissection. The Day 5/6 embryo was moved into the path of the laser through a bi‐manual manipulation system consisting of the following components: 2 X joystick fine micromanipulators (Narishige #MO‐302), 2 X coarse manipulators (Narishige #MN‐4), 1 X air operated coarse microinjector (Narishige #IM‐5A), 1 X oil operated fine microinjector (Narishige #IM‐6), and glass embryo holding pipettes: 18‐25 um ID, 60 mm L, 30 o angle (Swemed #14308). Embryos were always oriented with the ICM on the side of the fine microinjector to prevent mechanical damage. The glass holding pipettes formed the direct interface with the embryos with positive/negative pressure from the screw drive microinjectors producing fine control. ICM enriched embryonic material was transferred to xeno‐free cell culture dishes and allowed to attach for 48 hours before observation for cellular outgrowths. Fresh KSR‐XF medium was added daily. Cellular outgrowths were observed twice daily for the appearance of hESC‐like cells and to monitor proliferation of non‐hESC‐like cells. Once proliferating cells began to demonstrate aggregation/ detachment, the entire outgrowth was mechanically isolated and enzymatically digested with TrypLE + ROCK Inhibitor. Dissociated cells were re‐plated onto fresh xeno‐free cell culture dishes in XF‐KSR + ROCK Inhibitor. New hESC‐ 73 like colonies were allowed to expand before mechanical passage onto fresh culture dishes every 7 days. Frozen stocks of new XF‐hESC lines were prepared by the vitrification method (ESI Methodology Manual Embryonic Stem Cell Culture, www.escellinternational.com). Briefly, 10 whole colonies were manually isolated and transferred to XF‐Freezing solutions of increasing sucrose concentration (0%, 10%, 20%): XF‐KSR supplemented with sucrose, 10% DMSO and 10% propylene glycol. Colonies were then transferred to freezing straws by capillary action and immediately immersed in liquid nitrogen. Straws were placed in vials for long‐term liquid nitrogen storage. Thawing requires the reverse procedure wherein colonies are transferred to thawing medium with decreasing sucrose concentrations (20%, 10%, 0%) before transferring to fresh XF cell culture dishes. Characterization of XF-hESC Lines New XF‐hESC lines were characterized by ICC, embryoid body assay and teratoma formation. ICC was performed with the following pluripotency markers after 4% PFA fixation: GCTM‐2, TRA‐1‐60, GDF‐3 (rabbit IgG polyclonal, Abcam #AB38547), EpCAM (mIgG1, DAKO #M0804), E‐Cadherin (mIgG2 b , R&D Systems #MAB18381), SSEA‐ 4, OCT4, SOX2, DNMT3b (Goat IgG, Santa Cruz Biotechnology #sc‐10235), Nanog , and Alkaline Phosphatase (Vector Labs #SK‐5300). The marker combinations were: TRA‐1‐ 60/GDF‐3, GCTM‐2/EpCAM, OCT4/DNMT3b, E‐Cadherin/Nanog, EpCAM/DNMT3b, EpCAM/E‐Cadherin, SSEA‐4/SOX2, and Alkaline Phospatase. Primary and secondary antibody combinations with dilutions were as follows: [TRA‐1‐60 1:100 / AlexaFluor 594 74 goat anti‐mouse IgM ( μ chain) 1:1000 (Santa Cruz Biotechnologies #A21044)] [GDF‐3 1:50 / AlexaFluor 488 donkey anti‐rabbit IgG 1:1000 (Santa Cruz Biotechnologies #A21206)] [ GCTM‐2 neat / AlexaFluor 488 goat anti‐mouse IgM ( μ chain) 1:1000 (Santa Cruz Biotechnologies #A21042)] [EpCAM 1:50 / AlexaFluor 568 goat anti‐mouse IgG 1:1000 (Santa Cruz Biotechnologies #A21124)] [OCT4 1:200 / AlexaFluor 488 rabbit anti‐ mouse IgG (H+L) 1:1000 (Santa Cruz Biotechnologies #A11059)] [DNMT3b 1:300 / AlexaFluor 568 rabbit anti‐goat IgG 1:1000 (Santa Cruz Biotechnologies #A11079)] [E‐ Cadherin 1:20 / AlexaFluor 488 rabbit anti‐goat IgG (H+L) 1:1000 (Santa Cruz Biotechnologies #A11078)] [Nanog 1:20 / AlexaFluor 594 F(Ab’)2 fragment of rabbit anti‐ mouse IgG (H+L) 1:1000 (Santa Cruz Biotechnologies #A21205)] [EpCAM 1:50 / AlexaFluor 594 F(Ab’)2 fragment of rabbit anti‐mouse IgG (H+L) 1:1000 (Santa Cruz Biotechnologies #A21205)] [DNMT3b 1:300 / AlexaFluor 488 rabbit anti‐goat IgG (H+L) 1:1000 (Santa Cruz Biotechnologies #A11078)] [EpCAM 1:50 / AlexaFluor AlexaFluor 568 goat anti‐mouse IgG 1:1000 (Santa Cruz Biotechnologies #A21124)] [E‐Cadherin 1:20 / AlexaFluor 488 goat anti‐mouse IgG2 b 1:1000 (Santa Cruz Biotechnologies #A21141)] [SSEA‐4 1:100 / AlexaFluor 488 goat anti‐mouse IgG3 1:1000 (Santa Cruz Biotechnologies #A21151)] [SOX2 1:1000 / AlexaFluor 568 goat anti‐rabbit IgG (H+L) 1:1000 (Santa Cruz Biotechnologies #A11036)]. Cells were plated in 8‐well chamber slides coated with 0.5 ug/cm2 vitronectin/fibronectin and 5 x 10 4 cells/cm 2 hDFfs. All cells were permeabilized with 0.2% Triton X‐100 after fixation except for the TRA‐1‐ 60/GDF‐3 well. All primary and secondary antibodies were prepared in a 2% serum 75 solution matching the species of secondary antibody except for TRA‐1‐60/GDF‐3 which was prepared in a 1% BSA solution. Primary antibodies were incubated for 2 hours followed by 3X wash with PBS and secondary antibodies were incubated for 1 hour followed by 3X wash with PBS. Slides were wet mounted with ProLong Gold Antifade Reagent and allowed to cure overnight before imaging on a Zeiss AxioImager Z1 microscope. Embryoid body assay and teratoma formation were performed under the same conditions detailed in the materials and methods section for Chapter Two. Karyotyping was performed by the USC DNA core utilizing the GTW banding technique. 3. Results Obtain Fully Consented Human Embryos The consenting team first began contacting donors representing a pool of 150 embryos previously consented for another fertility research project. The embryos in this pool were frozen from 1989 to 2005, but each embryo needed new consent in order to adhere to the 2009 NIH guidelines. The consenting team immediately ran into difficulty tracking down current contact information for the individuals responsible for each embryo. This difficulty dramatically reduced the number of available embryos from this pool, but 36 fully‐consented embryos were obtained from this group. After exhausting this initial pool of embryos, the consenting team began contacting recent patients who had concluded fertility treatment but had embryos remaining in frozen storage. Another 9 fully‐consented embryos came from this secondary group. The 76 consenting team is currently approaching new donors at the beginning of fertility treatment, but it may be several years before such patients conclude fertility treatment resulting in surplus embryos. The extensive effort put forth by the consenting team has yielded ~60 embryos for use in the derivation project, 45 of which I have received for derivation experiments. Quality of the fully consented embryos was very low [Figure 16]. 8/45 embryos failed to survive the thawing process. Based on the Gardner Blastocyst Grading System, 21 embryos were scored as Grade 0 blastocysts since they failed to show signs of development. 17 of the Grade 0 embryos were highly fragmented and arrested in the cleavage stage (A‐C). Four of the Grade 0 embryos exhibited initial signs of compaction to the morula stage but did not advance further (D‐F). Four embryos showed signs of initial cavitation present in early blastocysts (Grade 2/3, G‐I), but the cells present in these embryos showed obvious signs of fragmentation. 12/45 embryos developed to the expanded blastocyst stage, Grade 4+ (J‐L), with one starting to hatch (Grade 5) and another fully hatching (Grade 6). Despite advancing to an expanded blastocyst stage, only 5/12 Grade 4+ embryos had a discernible inner cell mass. Only one embryo, SC011‐ F (K), exhibited the highest quality score of Grade 4AA with a discernible, healthy ICM and surrounding trophectoderm without fragmentation. The majority of the high quality blastocysts came from a single donor pool, SC011. As stated above, all living embryos were transferred for derivation attempts at developmental day 5/6 regardless of quality. 77 Figure 16: Representative Human Embryos These images show the various stages of development and quality of embryos at time of transfer for hESC derivation. The Gardner blastyocyst scoring system was used to describe the embryo quality. The majority of the embryos failed to reach the desired expanded blastocyst stage (Grade 4) occuring at Day 5/6 post‐fertilization. Most embryos did not progress farther than the early cell division stages and exhibited severe cellular fragmentation: Grade 0 Fragmented (A, B, C). Some embryos began to show early proliferation toward the compacting morula stage, but did not progress further: Grade 0 Compacting (D, E, F). A small subset of embryos began to show cavitation leading to the blastocyst stage, but the quality remained low: Grade 2 Early Blast (G, H, I). Nine out of 45 embryos developed to fully expanded blastocysts ideal for laser assisted derivation of hESCs: Grade 4+ Expanded Blastocyst (J, K, L). Most of the expanded blastocysts came from one donor pool (SC011) suggesting that embryo quality is strongly linked to the health of original donor tissue. Overall, the quality of the embryos in this study was very poor. All embryos were dissected for derivation, regardless of quality. 78 Figure 17: Successful Derivation of mESCs by Laser Isolation Optimizing laser‐assisted dissection of the ICM from blastocyst stage embryos required proof‐of‐concept studies using mouse embryos. Such practice streamlined the derivation process and provided evidence that the laser didnot cause damage to the ICM preventing derivation of ESCs. The first image (A) illustrates the appearance of a fullyexpanded mouse blastocyst at Day 3.5. The second image (B) shows representative muESC colonies appearing after twopassages. This successful derivation and practice with >50 mouse embryos facilitated the first attempts at laser‐assisted derivation of new hESC lines. Optimize Laser Assisted Inner Cell Mass Enrichment Protocol I assembled the derivation rig and familiarized myself with the relevant parts/software. I practiced on more than 50 mouse blastocysts to refine the laser‐ assisted derivation technique. Mouse blastocysts obtained from frozen stocks at USC Fertility demonstrated remarkably lower quality than those isolated fresh in the USC Transgenic core facility. The mouse embryo pictured in Figure 17 illustrates the appearance of a high quality blastocyst (A) at Day 3.5 of development. The zona pellucida is relatively thin and opaque. The trophectoderm forms a thin external cellular layer underneath the zona while the ICM is compact and large. Fully optimized laser‐ assisted ICM isolation required less than 10 minutes for complete dissection and transfer of the isolated ICM to equilibrated cell culture dishes. I successfully 79 Table 2: Embryo Derivation Results This table describes the XF‐hESC derivation results. I received 45 fully‐consented, surplus human embryos from USC Fertility. The embryos in this study were frozen from 1989 to 2007 at different stages of development: 2PN, Day 3, and Blastocyst. Eight embryos did not survive the thawing process. Of the 37 embryos transferred for derivation, 14 exhibited attachment and outgrowth post‐isolation. Only one embryo, SC010‐A, demonstrated successful derivation. This embryo gave rise to the XF‐hESC line USC01. 80 Figure 18: Embryo SC006‐C Outgrowth Images These images illustrate the attachment and outgrowth of embryo SC006‐C after laser‐assisted derivation in XF‐KSR conditions. The development of SC006‐C arrested at the morula stage (A), but the overall health of the cells appeared good with mild fragmentation. Two days after plating (B), the embryo had attached without any outgrowths. By Day 4 (C) a robust proliferative outgrowth appeared with most cells exhibiting a “trophectodermal” phenotype. At Day 6 (D) the cellular outgtrowth had reached full expansion with cells starting to show aggregation. The aggregation had become severe at Day 9 (E) and much of the outgrowth started to detach. The aggregation and detachment was fully complete by Day 11 (F). These images detail the common fate of embryos exhibited attachment and proliferation when derived under XF‐KSR conditions. Notably, only one non‐hESC like cell type was observed preventing mechanical isolation and propagation of pluripotent cells. demonstrated derivation of mESCs using the laser‐assisted protocol with the new mESC line surviving several passages (B). Derive and Characterize Xeno-Free Human Embryonic Stem Cells 5/45 embryos (11.1%) developed into fully expanded blastocysts with discernible ICMs. I attempted laser assisted derivation on 35 embryos and transferred the isolates to fresh KSR‐XF cell culture dishes. 14 embryos or ICM isolates attached and exhibited outgrowths with the morphology of most outgrowths resembling trophectoderm [Table 2]. These “trophectodermal” outgrowths proliferate rapidly before aggregating and 81 Figure 19: Embryo SC011‐F Outgrowth Images These images illustrate the attachment and outgrowth of embryo SC011‐F post laser‐assisted derivation. This embryo successfully developed into a fully expanded blastocyst (A) as did most of the embryos from this donor. Laser‐assisted dissection produced two clumps of cells (B) with the left clump comprised of trophectodermal cells and the right clump consisting of a trophectoderm/ICM mix. On Day 2 after plating, the dissected ICM exhibited attachement and initial outgrowth (C). By Day 3 (D), a robust proliferative cell mass is present. Interestingly, Day 4 (E) a dimorphic cell population appeared with a central cell mass exhibiting hESC‐like characteristics. Day 6 (F) clearly shows two cell types with a central, dense cell mass suspected to contain pluripotent cells and a surrounding proliferative outgrowth beginning to show aggregation. I chose to mechanically isolate the central cell mass on Day 7, and I transferred it to a new cell culture dish. Unfortunately, the isolated cells did not attach. This embryo, however, evidenced the viability of healthy embryos and supported the expansion of the ICM post dissection in KSR‐XF cell culture conditions. detaching two weeks after isolation. Embryo SC006‐C [Figure 18] was a poor quality embryo that developed to the morula stage without progressing to the blastocyst stage by embryo Day 6 (A). I hatched and plated the entire embryo (B) since I could not discern a solitary ICM. After 4 days, a large “trophectodermal” proliferation was noticeable (D). On the sixth day (E), the cellular outgrowth began to demonstrate aggregation followed by complete detachment eleven days (F) after isolation. Only one 82 Figure 20: Embryo SC010‐A Outgrowth Images These images illustrate the attachment and outgrowth of the embryo SC010‐A. Based on previous unsuccessful derivation attempts, I allowed this embryo to develop as long as possible before derivation. In this case, the embryo developed into a fully expanded blastocyst and hatched from the zona pellucida. This made laser‐assisted dissection impossible so I plated the entire hatched blastocyst with (A) showing its appearance two days after plating. By Day 3 (B) rapid proliferation and initial signs of trophectodermal aggregation were noted. The embryo continued to expand at Day 4 (C), but the signs of aggregation increased. The first signs of detachment were evident at Day 5 (D) and this led me to mechanically disect the entire outgrowth followed by TrypLE dissociation in the presence of Rho Kinase Inhibitor. I pursued enzyme digestion in order to release pluripotent cells from the aggregating trophectoderm thus allowing them to interface with the vitronectin/fibronectin/hDFf cell culture substrate. Six days after digestion (E) a small, proliferating mass with hESC‐like characteristics appeared. Two other hESC‐like outgrowths were discovered at the edge of the cell culture dish on Day 13 with one of these colonies pictured (F). I mechanically passaged these outgrowths for future propagation and characterization. cell type was observed proliferating from embryo SC006‐C during is derivation course. Embryo SC006‐C did not result in a new XF‐hESC line. Embryo SC011‐F exemplifies the highest quality blastocyst (Grade 4AA) obtained in this project [Figure 19]. The well‐developed ICM (A) allowed for removal of a majority of the trophectoderm (B). Four days after isolation (D), SC011‐F showed two distinct cell populations, one which resembled the “trophectodermal” outgrowths seen 83 in previous derivation attempts and a central, dense, hESC‐like outgrowth. Manual isolation of this secondary cell type on Day 7 (F) was unsuccessful because the central cell mass did not reattach. Embryo SC011‐F failed to produce a new XF‐hESC line. I allowed my last set of embryos to develop as long as possible at the fertility clinic prior to transferring them to the lab for isolation. One of the embryos (SC010‐B) developed into a fully expanded blastocyst and I performed laser isolation. This embryo attached, but did not show proliferation. The second and last consented embryo (SC010‐A) developed into a fully expanded blastocyst, but hatched on its own prior to transfer from the clinic [Figure 20]. Since I could not perform laser isolation on this embryo, I plated it directly onto an equilibrated xeno‐free cell culture dish (A). Marked proliferation and aggregation of the isolated embryo was observed by Day 3 (B). Detachment of the outgrowth was already apparent by Day 4 (C) so I dissociated the entire cellular outgrowth on Day 5 (D) in TrypLE plus ROCK Inhibitor. The dissociated cells were transferred to a fresh xeno‐free cell culture dish. Surprisingly, six days after dissociation (E), I observed a proliferating outgrowth from the trypsinized proliferative cell mass that had hESC‐like morphological characteristics at the cellular level. I attempted to manually isolate this interesting colony nine days after plating, but the cell mass was lost during transfer. Nevertheless, I continued to observe the plate for more outgrowths and discovered two proliferating colonies at the very edge of the dish 13 days after plating (F). I transferred these larger outgrowths to fresh xeno‐free culture dishes and both produced colonies with strong resemblance to hESCs [Figure 21]. The 84 Figure 21: Images of the XF‐hESC Line USC‐01 These images depict the first Xeno‐Free hESC line derived at USC. The first four panels (A, B, C, D) depict a single P2 colony expanding on Days 1, 2, 3, and 5. Colonies continue to possess hESC‐like morphology at P3 and P5 (E, F). first four panels of this figure (A‐D) depict a single colony proliferating from Day 2‐5. The last two panels (E, F) show colonies exhibiting tight borders with small, round, hESC‐ like cells after repeated passage (P3, P5 respectively). These are the first images of USC‐ 01, the first XF‐hESC line derived at USC. USC‐01 is currently at 34 passages in the KSR‐XF hESC medium and remains strongly positive for a variety of pluripotency markers by ICC: Surface (TRA‐1‐60, GCTM‐ 2, GDF‐3, EpCAM, E‐Cadherin, SSEA‐4 and Alkaline Phosphatase) and Nuclear (OCT4, SOX2, Nanog, and DNMT3b) [Figure 22]. The quality of staining based on intensity and cellular localization for each marker matches that observed for previously derived, non‐ XF hESCs. USC01 also exhibits strong expression of pluripotency genes by qPCR (see Chapter Four). Spontaneous differentiation is common in the central regions of large 85 Figure 22: ICC Panel for the XF‐hESC Line USC‐01 These fluorescent images depict several markers of pluripotency expressed on the XenoFree hESC line USC01 cultured in XF‐KSR+CX at P5. (A) Cell surface markers TRA‐1‐60 (red) and GDF‐3 (green). (B) Cell surface markers GCTM‐2 (green) and EpCAM (red). (C) Nuclear markers OCT4 (green) and DNMT3b (red). (D) Cell surface marker E‐Cadhering (red) and nuclear marker Nanog (green). (E) Cell surface marker EpCAM (red) and nuclear marker DNMT3b (green). (F) Cell surface markers EpCAM (red) and E‐Cadherin (green). (G) Cell surface marker SSEA‐4 (green) and nuclear marker SOX2 (red). All of the preceding images have nuclear counterstaining with DAPI (blue). Alkaline Phosphatase (H) staining is strong (dark contrast). Strong positivity for all markers in this ICC staining panel support the pluripotent identity of the Xeno‐Free hESC line USC01. Scale bars = 50 um. colonies, but it is rare for entire colonies to fully differentiate during the 7 days between passaging. EB analysis indicates robust differentiation into cells with characteristics of all three germ layers [Figure 23]. Nestin (A) remained the most prevalent marker among differentiated cells mirroring what was seen with hiPSC EBs, but strongly staining Smooth Muscle Actin (C) cells were more numerous in USC‐01 EBs. AFP (B) positive cells were the least prevalent in these differentiation experiments. The intensity of staining for all markers appeared greater for hESC EBs than seen with hiPSC EBs with USC01 differentiated cells possessing more developed cellular architectures. Teratoma analysis is currently underway and I expect results very soon. Karyotyping by the Giemsa‐ trypsin‐Wrights banding technique indicated that USC01 is 46 XY at P12. Out of 20 cells 86 Figure 23: Embryoid Body Images of USC‐01 analyzed, 14 had a full 46XY chromosomal content while the other 6 demonstrated random loss of one autosome (compatible with a normal diploid karyotype). 4. Conclusions The development of the KSR‐XF cell culture system described in Chapter One paved the way for implementing xeno‐free techniques starting with the first derivation steps. Manual dissection replaced immunosurgery for ICM isolation since the antibodies and complement proteins are sourced from animal serum. The XY Clone infrared laser combined with micromanipulation techniques added precision to the manual dissection that would not be afforded with dissection by hand alone. The value of laser dissection for ICM enrichment was further evidenced by the derivation of mESCs. The newly derived mESC line demonstrated that the XY Clone laser was not damaging the ICM and that viable cells resulted from the process. With the derivation techniques and culture system solidified, I received my first thawed embryos from the fertility clinic. I immediately realized that the success of the project would not hinge upon the XF‐hESC medium or laser isolation techniques alone. Instead, the quality of the starting 87 material would dictate the probability of deriving new hESC lines. Unfortunately, the quality of the donated human embryos was very low in this study. There are a variety of reasons for the high number of sub standard embryos. In general, the best embryos generated for fertility treatment are transferred for treatment. Patients with infertility may also not produce the best embryos. Many embryos had been frozen several years ago, possibly under freezing conditions not meeting contemporary protocol. Regardless of the reasons, >80% of the donated embryos possessed marginal or substandard quality. It would be wise to collaborate with a large number of fertility clinics in future experiments to mitigate the problems associated with embryo quality. Despite quality issues, 14 embryo isolates attached and demonstrated early proliferation. The proliferative outgrowths, however, had a similar pattern of rapid expansion followed by cellular aggregation and detachment. The appearance of these outgrowths suggested a “trophectodermal” identity. The better looking embryos typically produced a larger proliferative outgrowth indicating the presence of more viable starting cells, but only one embryo, SC011‐F demonstrated a dimorphic cell population at the center of the outgrowth. The speed of aggregation and detachment of the trophectodermal cells, however, impaired the expansion of the secondary cell type believed to represent an ICM outgrowth. Manual dissection of the secondary cell type did not yield continuous cultures of hESCs . The experience I had above was most definitely frustrating, but it led me to conclude that there was room for technical improvement in the derivation procedure. 88 First, of those embryos that develop to a fully expanded blastocyst it is imperative to allow them to develop as long as possible to increase the size of the ICM and allow for easier laser isolation. Second, residual presumptive trophectoderm from the isolation proliferates rapidly in this culture system and impedes the successful interaction of the ICM with the feeders/ECM. It may be possible that there are some hESCs dispersed within the trophectoderm, but they cannot expand within the dominant, over‐ proliferating trophectodermal cells. The hyperproliferating cellular explant from manual dissection should thus be enzymatically dissociated by Day 7 to release the pluripotent cells. This will provide greater opportunity for a direct interface between the pluripotent cells and the vitronectin/fibronectin /hDFf feeders. Adding ROCK Inhibitor also provides stability for the dissociated single or small clumps of hESCs. The new line, USC‐01, survived derivation after implementation of these technical changes. Moreover, previously unsuccessful embryos most likely would have yielded viable pluripotent cells had I performed the dissociation prior to full aggregation/detachment of the outgrowth. I will employ this technique in all future derivations. The successful derivation of the XF‐hESC line USC01 validates the thorough preparation and I anticipate the arrival of more embryos as the consenting process continues. When comparing my derivation efficiency of 0% (0/30) from low quality embryos and 20% (1/5) from high quality embryos, it is clear that embryo quality directly affects the success of derivation attempts. Lerou et al. describe a 0.6% derivation efficiency from low quality Day 3 embryos which increased to 4.1% for Day 5 low quality embryos, 89 and 8.5% for low‐quality Day 5 embryos that progressed to the blastocyst stage (Lerou, Yabuuchi et al. 2008). My data reflect the results obtained in the Lerou paper and indicate that high quality embryos are necessary for the derivation of a large library of hESCs. Further characterization of USC‐01 by ICC indicates strong expression for a large panel of pluripotency markers. Staining intensity and cellular localization of the markers is comparable to the established cell lines HES2 and HES3. Embryoid body assays also demonstrate the ability of USC‐01 to produce differentiated cells possessing characteristics of all three germ layers. The staining intensity and underlying architecture of positively staining structures appears more developed in the USC‐01 generated EBs compared to the staining seen with hiPSCs. This may indicate a lack of equivalency between the newly derived hESCs and hiPSCs. It is important to remember, however, that the hiPSCs in this study still have integrated transposons actively expressing transgenes which could limit the differentiation potential of hiPSCs in an EB assay. Teratoma analysis is currently underway, but I expect that the assay will produce highly differentiated tissues on pathological examination. At P12, karyotype analysis confirmed the 46XY stable chromosomal status of this XF‐hESC line. I will perform the karyotype analysis again on older cells, greater than P30, to demonstrate long‐term chromosomal stability in xeno‐free cell culture conditions. Early passage stocks have also been cryogenically banked for future distribution with successful thawing for distribution. 90 The derivation of USC‐01 and several hiPSC lines under identical xeno‐free cell culture conditions described in this study provides a platform for future equivalency comparisons. True comparative studies require several pluripotent cell lines for broad characterization and identification of similar/dissimilar traits. To that end, more XF‐ hESC lines are required to complete such analysis. Also, it would be informative to derive iPSC from trophectoderm outgrowths of embryos that gave rise to hESC, for comparing the two cell types on identical genetic backgrounds. It is also worth noting that the origin of these cell lines is not entirely xeno‐free. For the hiPSCs, the donor tissue for the hDFf stocks was passaged in FBS containing medium and high efficiency transfection required the cells to be grown in FBS containing fibroblast medium until 5 days post‐transfection. USC‐01 is almost entirely xeno‐free except for that the commercial hDFf feeder stocks were expanded in FBS containing medium before being prepared in xeno‐free conditions. A 100% xeno‐free cell culture system will require the in‐house preparation of xeno‐free primary human fibroblast cell lines for both feeders and hiPSC production which was beyond the scope of this study. Nevertheless, the xeno‐free methodology described here will easily translate to a 100% xeno‐free system. Derivation of new hESC lines under defined conditions will generate valuable data less influenced by variability introduced through earlier cell culture systems. One step toward more defined systems is the removal of animal products from the derivation process. From the initial isolation of the ICM all the way to the media and growth factors used to direct differentiation, implementing xeno‐free techniques 91 demonstrates the feasibility of “clinical grade” hESC derivation. The successful derivation of the new XF‐hESC line USC‐01 provides proof‐of‐concept for future “clinical grade” pluripotent cell lines. 92 Chapter Four: Molecular Characterization of the Reprogramming Process Improves Selection of High Fidelity Human Induced Pluripotent Stem Cells 1. Introduction I developed this project as a secondary research topic since the hESC derivation process was beleaguered by delays and very limited availability of good quality embryos. The second half of my characterization project, XF‐hiPSC derivation, was streamlined and reproducible from a technical standpoint which allowed me to ask questions about the molecular basis of the reprogramming process. The reprogramming process, by nature, is very inefficient. Several groups showed improvement in reprogramming efficiency by incorporating additional nuclear transcription factors into the reprogramming mix (Liao, Wu et al. 2008; Mali, Ye et al. 2008), inclusion of small molecules into the growth media like the histone deacetylase inhibitor butyrate (Mali, Chou et al. 2010), or lowering the oxygen concentrations in the incubator to more closely mimic physiological conditions (Utikal, Polo et al. 2009). These improvements in reprogramming efficiency added impetus to development of selection protocols for the best “fully” reprogrammed hiPSCs for further characterization and study. One lab identified a surface marker of pluripotency (TRA‐1‐60) that appeared late during reprogramming; selection of colonies positive for this marker by live cell staining increased hiPSC derivation efficiency (Chan, Ratanasirintrawoot et al. 2009). Another lab identified the markers EpCAM and E‐Cadherin as “late” markers suitable for selection in mouse iPSCs (Chen, Chuang et al. 2011). In October of 2010, I began a 93 Figure 24: ICC Reprogramming Timeline To produce a timeline illustrating the appearance of pluripotency related markers, transfected fibroblasts were transferred to glass chamber slides and fixed/stained every three days with the listed marker combinations (Day 3‐24). study to produce a timeline of pluripotency marker appearance during the reprogramming process to find late markers selective for “fully” reprogrammed hiPSCs. Selection of foci undergoing reprogramming by cell surface pluripotency markers provides the opportunity to isolate the most hESC‐like hiPSCs, and isolate foci at progressive timepoints for molecular characterization through quantitative gene expression analysis Specific Aim #1: A Timeline of Pluripotency Marker Appearance During the Reprogramming Process Using ICC to Determine the Order of Activation for Key Pluripotency Regulator Genes The first phase of this project centered on determining the temporal activation of genes involved in pluripotency during the reprogramming process. I began by preparing chamber slides containing fibroblasts transfected with the piggyBac reprogramming cassette. Every 3 days, I fixed the slides and stained for a combination 94 of pluripotency markers: TRA‐1‐60/GDF‐3, GCTM‐2/EpCAM, OCT4/DNMT3b, Nanog/E‐ Cadherin, EpCAM/DNMT3b, and EpCAM/E‐Cadherin [Figure 24]. TRA‐1‐60 and GCTM‐2 are cell surface glycoproteins expressed on pluripotent cells, whose functional significance is not known. The secreted marker GDF‐3, a BMP inhibitor, is implicated in the regulation of cell fate in stem cells (Levine and Brivanlou 2006). Theintercellular adhesion molecule EpCAM is involved in the maintenance of pluripotency (Ng, Ang et al. 2010) and has been shown to influence proliferation through its intracellular domain EpICD (Maetzel, Denzel et al. 2009) and subsequent interactions with beta‐catenin. The intercellular adhesion molecule E‐Cadherin is also expressed on pluripotent cells and has the ability to influence pluripotent cell fate through the stabilization of beta‐catenin (Orsulic, Huber et al. 1999; Sato, Meijer et al. 2004). Their appearance during reprogramming indicates a transition from a fibroblastic state to a state more associated with pluripotency. The nuclear transcription factors OCT4, DNMT3b and Nanog are master regulators of pluripotency so their appearance indicates the re‐activation of endogenous pluripotency networks. Understanding the timing of expression for these pluripotency markers during reprogramming provides a framework for assaying the molecular changes underlying the reprogramming process. 95 Specific Aim #2: “Late” Markers of Pluripotency Used to Select Reprogramming Colonies at Days 10, 20, and 30 to Analyze Reactivation of Pluripotency Networks Via Quantitative Expression Profiles With the Fluidigm Biomark HD Platform Reprogramming a cell from a fibroblastic state to a pluripotent state requires the reactivation of pluripotency gene networks. The low efficiency of cellular reprogramming is most likely caused by the failure to completely re‐activate the necessary gene networks to maintain pluripotency. To test the above hypothesis, I performed live‐cell staining with a combination of TRA‐1‐60/E‐Cad and GCTM2/EpCAM at Day 10, 20 and 30 during reprogramming [Figure 25]. Live‐cell staining can only be performed on surface antigens since visualization of nuclear antigens would require the permeabilization of the cell membranes effectively killing the target cells. Selection by live‐cell staining presents an advantage since production of reporter cell lines is not feasible for mid or high throughput derivation of iPSC. The cell surface markers in this assay are highly expressed on pluripotent cells and achieve elevated expression on reprogramming foci after 2‐3 weeks of reprogramming. Intense staining for these markers may indicate the re‐activation of pluripotency associated transcriptional networks which can be assayed by quantitative expression analysis. After isolating colonies for intense vs. negative marker expression by live cell staining, total RNA was extracted from whole reprogramming foci. Quantitative RNA expression analysis was then performed on >40 genes chosen for their involvement in regulating pluripotency and early lineage commitment [Figure 26]. 96 Figure 25: Live‐Cell ICC Selection Methods Following the same transfection procedures as previously described, transfected cells were plated into 35mm dishes. The dishes were live‐cell stained with the marker combinations GCTM‐2/EpCAM and TRA‐ 1‐60/E‐Cadherin at Day 10, 20 and 30. Double positive/negative reprogramming foci were manually isolated and lysed for RNA isolation. Quantitative expression analysis was performed on the isolated foci to determine the underlying expression profile of reprogramming foci during the reprogramming process. The Fluidigm Biomark HD quantitative expression platform is a new technology that facilitates this study. The Fluidigm system allows one to query the expression of 48 genes from sample sizes as small as one cell. Standard RNA expression quantification methodologies require large RNA inputs from several thousand cells to query expression of only a few genes. Single‐cell and small colony analysis is possible with standard expression studies, but the labor necessary to query large sample numbers against multiple genes is inhibitory. Looking at the expression of several genes from small reprogramming foci would thus not be possible without the powerful Fluidigm microfluidics platform. After running the 48 gene expression panel on >150 samples selected by pluripotency marker positivity/negativity at increasing intervals during reprogramming, the expression data supports the value of live‐cell ICC for selection of 97 Figure 26: Genes Queried by Quantitative RNA Expression Expression levels of 49 genes were quantitatively analyzed by the Fluidigm Biomark HD system. The genes selected for this study were chosen based on their involvement in maintenance of the pluripotent state, early lineage commitment (ectoderm, endoderm, mesoderm, neural crest), or expression on hDFfs. The housekeeping genes PPIA and B2M were used as controls for relative expression quantification. “fully” reprogrammed hiPSCs while also providing a unique look at the activation of pluripotency transcriptional networks that could provide an increased level of selection. Specific Aim #3: Utilize “Late” Markers of Pluripotency to Select for “Fully” Reprogrammed hiPSCs and Validate these Cell Lines by ICC, qPCR, Embryoid Bodies, and Teratoma Formation The data generated by the Fluidigm Biomark HD expression analysis experiments indicated that reprogramming foci with intense marker positivity possess an expression profile similar to hESCs and “fully” reprogrammed hiPSCs. The final aim of this project is to verify that live‐cell staining selection with “late” markers of pluripotency increases 98 the derivation efficiency of “fully” reprogrammed hiPSCs. To accomplish this experiment, I isolated a large sample of dual positive/dual negative foci undergoing reprogramming at Day 30 and observed them for several passages. The majority of foci selected by live‐cell staining and demonstrating attachment/outgrowths produced “fully” reprogrammed hiPSCs while marker negative foci generated “partially” reprogrammed hiPSCs. Foci selection by live‐cell staining provides a significant advantage to selection by foci morphology alone. 2. Materials and Methods ICC Reprogramming Timeline Low passage hDFfs were nucleofected with the pPB.OSKM‐pu Δtk transposon and pCyL43:PB transposase plasmids as described in Chapter Two. After resuspension in equilibrated MEM‐alpha fibroblast medium, nucleofected cells were plated in 2‐well chamber slides coated with 0.5 ug/cm 2 vitronectin/fibronectin at a density of 1 x 10 3 cells/cm 2 . MEM‐alpha fibroblast medium was changed the day after transfection and every other day until Day 5 when the medium was replaced with XF‐KSR hESC medium with feeding continuing every other day. Non‐transfected hDFfs were also plated at the same density as negative controls for ICC staining. Beginning on Day 3 and ending on Day 24, slides were fixed with 4% PFA at three day intervals. Fixed slides were stained with the following pluripotency marker combinations on Day 3, Day 6, Day 9, Day 12, Day 15, Day 18, Day 21, and Day 24: TRA‐1‐60/GDF‐3, GCTM‐2/EpCAM, OCT4/DNMT3b, 99 Nanog/E‐Cadherin, EpCAM/DNMT3b, and EpCAM/E‐Cadherin. Staining conditions were identical to those described in Chapter Three. Positivity for the pluripotency markers was determined by comparison to the “fully” reprogrammed hiPSC line J1. Three categories were utilized to score the ICC slides: Negative (matches negative control/background), Positive (stains above background with correct cellular localization), and Intense (staining intensity resembles positive control staining). Quantification by integration of fluorescence above individual nuclei using Image J software supported the three category scoring for marker positivity although assessment of marker staining was performed visually. All colonies present on the slides were scored according to the above criteria and noted to be Double Negative, Single Positive, or Double Positive. All intense staining colonies were also scored/included in the positive category. Fluidigm Biomark HD Expression Profiling of Single Foci Undergoing Reprogramming Low passage hDFfs were nucleofected with the pPB.OSKM‐pu Δtk transposon and pCyL43:PB transposase plasmids as described in Chapter Two. After resuspension in equilibrated MEM‐alpha fibroblast medium, nucleofected cells were plated into 35 mm cell culture dishes coated with 0.5 ug/cm 2 vitronectin/fibronectin at a density of 1 x 10 3 cells/cm 2 . MEM‐alpha fibroblast medium was changed the day after transfection and every other day until Day 5 when the medium was replaced with XF‐KSR hESC medium with feeding continuing every other day. 100 On Days 10, 20, and 30 reprogramming dishes were stained by live‐cell ICC with the marker combinations: GCTM‐2/EpCAM and TRA‐160/E‐Cadherin. Primary and secondary antibodies were diluted into XF‐KSR hESC medium and allowed to consecutively incubate for 20 min at 37 o C with 3X sterile PBS wash following each incubation. 1 mL sterile PBS was added to each 35 mm cell culture dish for imaging with a Zeiss Axiovert A1. Hoescht dye (1:1000) was added during the secondary antibody incubation for nuclear counterstaining. Primary and secondary antibody pairings with dilutions were as follows: [GCTM‐2 (neat) / AlexaFluor 488 – A21042 (1:1000)] [EpCAM (1:50) / AlexaFluor 568 – A21124 (1:1000)] [TRA‐1‐60 (1:100) / AlexaFluor 594 – A21044 (1:1000)] [E‐Cadherin (1:20) / AlexaFluor 488 – A21141 (1:1000)]. Double negative and double positive colonies were imaged and isolated by manual dissection for expression analysis. Non‐transfected hDFfs (negative control) and hiPSCs/hESCs (positive controls) were also live‐cell stained and isolated. Isolated colonies were immediately lysed in 250 uL RLT lysis buffer contained in the Qiagen RNeasy Micro Kit (Qiagen #74004) and RNA was extracted following the Qiagen protocol. The small amount of extracted RNA prohibited quantification prior to analysis, so colonies of similar size were isolated to reduce input variability in future expression studies. The entire extracted RNA sample was reverse transcribed to cDNA according to the Qiagen QuantiTect Rev. Transcription Kit (Qiagen #205313). The cDNA samples were pre‐amplified with TaqMan PreAmp Master Mix (AB Biostystems #4391128) and PCR primers specific for several pluripotency and lineage specific genes. 101 An 18 cycle pre‐amplification protocol was constructed with the following parameters: 10 min @ 95 o C, 18X (15 sec @95 o C, 4 min @ 60 o C). Pre‐amplified samples were diluted 1:5 with TE for storage. Expression analysis was performed with the Fluidigm Biomark HD microfluidics platform with a 48 x 48 IFC chip. Samples and controls were run in duplicate such that 20 test samples and four controls (positive, negative, RT‐, and no template) can be run on each chip with 48 genes being tested by real time PCR. All samples were prepared according to the Fluidigm protocol. Briefly, pre‐amplified samples are mixed with the provided 20X GE Sample Loading Reagent and TaqMan Universal PCR Master Mix (Life Technologies #4304437) and transferred to a 96‐well PCR plate for loading. TaqMan Gene Expression Assay probes are mixed 1:1 with DA Assay Loading Reagent and transferred to a 96‐well PCR plate for loading. Samples and assays are loaded into the 48 x 48 well chip and run on the Fluidigm Biomark HD system for 40 cycles. Delta Ct values for analysis were calculated by subtracting the Ct value for cyclophilin from the test Ct value. A cutoff Ct value of 40 was given for samples that did not show amplification for a target gene. Delta Ct values were uploaded to the MultiExperiment Viewer software for hierarchical clustering analysis. Genes of interest were then compared between selection groups by 2x2 contigency tables and the Fisher’s Exact Test. 102 Live-Cell Selection and Characterization of hiPSCs Low‐passage hDFfs were nucleofected and plated as described in the previous section. After 30 days, reprogramming dishes were live‐cell stained with the marker pairs GCTM‐2/EpCAM and TRA‐1‐60/E‐Cadherin. Staining conditions were identical to the previous section except that Hoescht Dye was not added due to possible toxicity. Double positive and double negative foci were imaged, isolated by manual dissection, and transferred to fresh XenoFree culture dishes. hiPSCs exhibiting attachment and outgrowth were continually passaged as long as they were viable and observed for “fully” and “partially” reprogrammed morphological characteristics. “Fully” and “Partially” reprogrammed hiPSCs were plated in 8‐well chamber slides for ICC and stained for the following markers: TRA‐1‐60/GDF‐3, GCTM‐2/EpCAM, OCT4/DNMT3b, Nanog/E‐Cadherin, EpCAM/DNMT3b, EpCAM/E‐Cadherin, SSEA‐ 4/SOX2, and Alkaline Phosphatase. Staining conditions were previously described in Chapter Three. Live‐cell selected hiPSCs were also prepared for spin‐EB differentiation assays and teratoma formation as described in Chapter Two. 3. Results Pluripotency Associated Markers Demonstrate Progressive Positivity Between “Early” and “Late” Markers With Intensely Staining Foci Describing a Small Population. Every three days (3, 6, 9, 12, 15, 18, 21, 24), I fixed reprogramming slides with 4% PFA and performed ICC with a panel of pluripotency markers (Nuclear: OCT4, SOX2, Nanog, DNMT3b – Receptors: E‐Cadherin, EpCAM – Surface: SSEA4, TRA‐1‐60, GCTM‐2). 103 The following marker combinations were used: TRA‐1‐60/GDF‐3, GCTM‐2/EpCAM, OCT4/DNMT3b, Nanog/E‐Cadherin, EpCAM/DNMT3b, EpCAM/E‐Cadherin [Figure 27, Figure 28, Figure 29, Figure 30, Figure 31, Figure 32 respectively]. Each of the reprogramming timeline figures illustrates representative staining from marker combinations at each timepoint. The selected colonies represent the best images with the most intense staining. Colonies of varying staining positivity/intensity were present on each slide indicating non‐uniform reprogramming. As the reprogramming process progressed, the percentage of colonies staining for the markers increased [Figure 33]. GDF‐3 staining began to appear on Day 9 while TRA‐1‐60 first became positive on Day 12 (A), which is also the first time at which colonies stained double positive. Accounting for marker positivity (above background with proper cellular localization) alone, both GDF‐3 and TRA‐1‐60 appear on slightly less than 50% of colonies by Day 24 with about 35% staining double positive. When scoring the most intensely staining colonies, those that have staining similar to the positive control, such colonies do not appear until Day 18 for both markers with single and double intensely positive colonies representing about 10% on Day 24. GCTM‐2 shows positive staining by Day 12 with EpCAM predominantly appearing at Day 15 (B). Over half of the colonies become EpCAM positive by Day 24 while slightly over 40% show GCTM‐2 positivity. Only 25% of colonies show double positive staining for these markers. Day 18 produces the first intensely positive colonies for GCTM‐ 2/EpCAM with about 10% intensely positive for both markers on Day 24. The 104 Figure 27: TRA‐1‐60/GDF‐3 ICC Timeline These images depict the timeline of expression of the pluripotency markers TRA‐1‐60 (red) and GDF‐3 (green). Cells were fixed every three days post transfection and stained by ICC. DAPI (blue) was used as a nuclear counterstain in all panels. The hiPSC line J1 was used as a positive control for reference staining. GDF‐3 is a secreted protein that has an intercellular staining pattern. Initial cytoplasmic GDF‐3 staining was noted at Day 6 with strong intercellular staining first appearing at Day 12. TRA‐1‐60 marks a cell surface glycoprotein that is present on all pluripotent cell lines. TRA‐1‐60 exhibits stippled staining on Day 9 with strong cell surface staining arising by Day 15. Interestingly, both markers strongly stain a small subset of cells early on with full colony involvement not appearing until Day 21/24. The intensity of staining for both markers increases during the reprogramming timeline. Scale bars = 50 um. 105 Figure 28: GCTM‐2/EpCAM ICC Timeline These images depict the timeline of expression of the pluripotency markers GCTM‐2 (green) and EpCAM (red). Cells were fixed every three days post transfection and stained by ICC. DAPI (blue) was used as a nuclear counterstain in all panels. The hiPSC line J1 was used as a positive control for reference staining. GCTM‐2 stains a cell surface glycoprotein present on pluripotent cells. Light, stippled GCTM‐2 staining starts by Day 9 with strong staining appearing by Day 15/18. High intensity GCTM‐2 staining similar to the J1 control appears at Day 21/24 with whole colony involvement. EpCAM is an intracellular adhesion molecule that also has intracellular second messenger connectivity. EpCAM first appears by Day 12 with a strong intercellular staining pattern that gains intensity during the reprogramming process until it is similar to the J1 control at Day 21/24. Scale bars = 50 um. 106 Figure 29: OCT4/DNMT3b ICC Timeline These images depict the timeline of expression of the pluripotency markers OCT4 (green) and DNMT3b (red). Cells were fixed every three days post transfection and stained by ICC. DAPI (blue) was used as a nuclear counterstain in all panels. The hiPSC line J1 was used as a positive control for reference staining. The nuclear transcription factor OCT4 is a master regulator of the pluripotent state. OCT4 expression is immediately evident at Day 3 reflecting its activity on the reprogramming transposon. DNMT3b is another transcription factor implicated in regulating pluripotency. DNMT3b first demonstrates notable staining at Day 12 with strong nuclear staining appearing by Day 18. The colony shown at Day 24 exhibits staining very similar to the J1 positive control. OCT4 and DNMT3b exhibit overlapping staining with all DNMT3b positive cells also showing OCT4 positivity. Scale bars = 50 um. 107 Figure 30: Nanog/E‐Cadherin ICC Timeline These images depict the timeline of expression of the pluripotency markers Nanog (green) and E‐Cadherin (red). Cells were fixed every three days post transfection and stained by ICC. DAPI (blue) was used as a nuclear counterstain in all panels. The hiPSC line J1 was used as a positive control for reference staining. Nanog is a nuclear transcription factor that was one of the first genes found to regulate the pluripotent state. Nanog expression first appears by Day 3/6 of the reprogramming process with strong staining similar to the J1 positive control appearing at Day 21, especially in the colony pictured at Day 24. E‐ Cadherin is an intracellular adhesion molecule with intracellular signalling capabilities. E‐Cadherin expression appears by Day 12 of the reprogramming process with strong intercellular staining appearing by Day 15. The staining intensity of Nanog and E‐Cadherin increases during the course of the reprogramming process as evident in these selected colony images. Scale bars = 50 um. 108 Figure 31: EpCAM/DNMT3b ICC Timeline These images depict the timeline of expression of the pluripotency markers DNMT3b (green) and EpCAM (red). Cells were fixed every three days post transfection and stained by ICC. DAPI (blue) was used as a nuclear counterstain in all panels. The hiPSC line J1 was used as a positive control for reference staining. Both of these markers were previously used in combination with other markers, but I wanted to look for overlapping positivity between different combinations. DNMT3b exhibited light expression by Day 9 with strong expression appearing by Day 18. EpCAM began to show expression by Day 12 with increasing expression during the reprogramming process. Interestingly, the colony image for Day 18 illustrates a small subset of cells strongly positive for EpCAM, indicating a dynamic reprogramming process within one colony. Scale bars = 50 um. 109 Figure 32: EpCAM/E‐Cadherin ICC Timeline These images depict the timeline of expression of the pluripotency markers E‐Cadherin (green) and EpCAM (red). Cells were fixed every three days post transfection and stained by ICC. DAPI (blue) was used as a nuclear counterstain in all panels. The hiPSC line J1 was used as a positive control for reference staining. Both of these markers were previously used in combination with other markers of pluripotency. For the EpCAM and E‐Cadherin combination, I wanted to investigate the appearance of two intercellular adhesion markers. Both EpCAM and E‐Cadherin show intercellular staining patterns by Day 12 with intensity increasing during the process of reprogramming. By Day 21, both markers exhibit strong overlapping expression with some areas being stronger for one marker over another. Such overlapping and non‐overlapping staining is very similar to the J1 positive control. The colony shown in Day 24 looks remarkably similar to J1 possibly indicating a more “fully” reprogrammed state. Another interesting observation about the colony shown at Day 24 is its small size compared to most colonies appearing after 24 days of reprogramming. This could indicate that a high rate of proliferation is not necessary to achieve a “fully” reprogrammed state. In fact, many small colonies exhibited the strongest expression during the late stages of reprogramming. Scale bars = 50 um. 110 concordance between markers is greater for intensely staining colonies as seen with the previous marker pair. OCT4 is part of the integrated reprogramming cassette accounting for its immediate appearance and high rates of positivity (C). DNMT3b staining, however, first appears at Day 12 with more than 60% of colonies positive by Day 24. Interestingly, all colonies positive for DNMT3b are also positive for OCT4. Intensely staining DNMT3b starts at Day 18 with fewer than 10% of colonies possessing such characteristics by Day 24. Nanog staining first appears early at Day 6 with a rapid increase to 40% of colonies positive by Day 12 (D). E‐Cadherin positivity first begins on Day 12 with a steady increase to 40% of colonies positive by Day 24. Slightly more than 20% of colonies exhibit concordant staining with Nanog and E‐Cadherin after 24 days of reprogramming. A few intensely staining colonies appear at Day 15 for both markers, but concordant intense staining fails to cross 10% of total colonies at Day 24. The combination of EpCAM and DNMT3b gave slightly different results to the previously examined combinations of these markers with others. (E). Both markers appear at Day 12 and exhibit a lower saturation percentage on Day 24 (EpCAM – 30%, DNMT3b – 40%). Less than 25% of colonies on Day 24 are positive for both markers. As for intense staining, Day 18 shows the first numerous appearances with fewer than 10% intensely staining for both markers. 111 Figure 33: ICC Reprogramming Marker Timeline Graphs These graphs illustrate the appearance of pluripotency markers during the process of reprogramming. Reprogramming slides were fixed every three days and stained with the marker pairs TRA‐1‐60/GDF‐3 (A), GCTM‐2/EpCAM (B), OCT4/DNMT3b (C), Nanog/E‐Cadherin (D), DNMT3b/EpCAM (E), and EpCAM/E‐Cadherin (F). Reprogramming foci were scored as negative, positive, or intense for one or both markers. Intensely staining foci numbers are also included in the positive data. The results are listed as a percentage of total foci present in the well of the chamber slide. Markers appearing early are OCT4 at Day 3 and Nanog at Day 6. GDF‐3 expression starts by Day 9 while TRA‐1‐60, GCTM‐2, EpCAM, E‐Cadherin and DNMT3b do not appear on numerous foci until Day 12. Positivity increases after appearance, but appears to reach a maximum for most markers by Day 24. On the whole, intensely positive foci do not appear until Day 18 for all markers and demonstrate a higher concordance for double positive compared to single positive foci. The exception is EpCAM and E‐Cadherin which show high concordance in both the positive and intensely positive groups. Intensely staining, double‐positive foci may represent reprogramming foci most likely to achieve a “fully” reprogrammed state. 112 The combination of EpCAM and E‐Cadherin (F) is the first pair of markers both exhibiting intercellular localization. The two markers appear positive on Day 12 with increasing positivity towards 35% on Day 24. Interestingly, the percentage double positive is highly concordant with the single positive data for EpCAM/E‐Cadherin suggesting that most colonies are double positive. Intensely positive colonies appear on Day 18 with a plateau for both markers below 10% on Day 24. The intense staining data is also highly concordant between single and double staining. Compiling the data from all the marker pairs into a concatenated timeline illustrates some important relationships between the markers [Figure 34]. Nanog and GDF‐3 are early positive markers appearing on Day 6 and Day 9 respectively (A). GCTM‐ 2, TRA‐1‐60, EpCAM, E‐Cadherin, and DNMT3b are late positive markers appearing on Day 12. Regardless of time of appearance, the percentage of colonies staining positive for these markers increases to 30‐50% of reprogramming foci by Day 24. Intense staining foci (B), however, generally appear at reprogramming Day 18 with around 10% of colonies exhibiting intense staining by Day 24. As a general observation, marker positivity is not uniform on reprogramming foci. Staining was deemed positive even if a small proportion of cells within one focus exhibited expression so long as the staining was above background and in the correct cellular localization. As the reprogramming process progresses, the number of colonies with positive marker expression increased along with the number of cells within each 113 Figure 34: ICC Timeline Concatenated data These graphs depict the percentage of reprogramming foci positive (A) and intensely positive (B) for pluripotency markers during the process of reprogramming. The markers GCTM‐2, TRA‐1‐60, GDF‐3, EpCAM, E‐Cadherin, Nanog, DNMT3b and OCT4 were tested by ICC on reprogramming cells fixed every three days. Positivity was scored as staining above background with proper cellular localization while intense positivity was scored as staining highly similar to the J1 “fully” reprogrammed hiPSC line. Overt positivity for all markers ranged between 30‐50% at Day 24 while intense positivity hovered around 10%. Foci exhibiting intense staining also appear later during the reprogramming process indicating that time and staining intensity select a population of reprogramming foci more likely to progress to a “fully” reprogrammed state. The cell surface markers GCTM‐2, TRA‐1‐60, E‐Cadherin and EpCAM are thus useful for live‐cell selection of these late, intense foci undergoing reprogramming. 114 foci exhibiting marker positivity. Intense staining was also a function of the percentage of each foci exhibiting strong marker expression. “Late” Markers of Pluripotency Identify Reprogramming Foci With RNA Expression Profiles Closer to Pluripotent Cells Live‐cell staining with the “late” marker combinations GCTM‐2/EpCAM and TRA‐ 1‐60/E‐Cadherin enabled selection of double positive reprogramming foci for RNA expression analysis [Figure 35]. The positive contol XF‐hESC line USC01 exhibited strong positivity for all four markers (A, D). The positive control “full” XF‐hiPSC line AA4 (B) demonstrates strong GCTM‐2/EpCAM positivity. The positive control “full” XF‐hiPSC line AE1 (E) exhibits strong positivity for TRA‐1‐60/E‐Cadherin. Using the XF‐hESC line USC01 and “full” XF‐hiPSC lines as positive controls for comparison I selected several reprogramming foci with equivalent marker positivity. GD30F (C) is an example of intense GCTM‐2/EpCAM staining arising after 30 days of reprogramming and TD302B (F) is a reprogramming focus with intense TRA‐1‐60/E‐Cadherin staining after 30 days. Double negative colonies were also isolated for RNA expression analysis. All colonies isolated at Day 10 after live‐cell staining were marker negative which was expected from the reprogramming timeline study since most markers did not have intense staining until Day 18. By Day 20, several colonies exhibited intense staining with increasing number and size of strongly staining colonies by Day 30. I ran eight Fluidigm chips containing over 150 samples and controls [Figure 36]. Cluster analysis of the data provides two major clusters along the vertical (SAMPLE) axis. 115 Figure 35: Live‐Cell ICC Images These images depict characteristic live‐cell staining for the selection markers GCTM‐2 / EpCAM (Top) and TRA‐1‐60 / E‐Caderin (bottom). The first image in each section (A, D) demonstrates positive control staining on the Xeno‐Free hESC line USC01. The second image depicts positive control staining on the Xeno‐Free hiPSC lines AA4 (B) and AE1 (E). The last image in each section illustrates strong staining on reprogramming hiPSCs 30 days post‐transfection (C, F). While the staining intensity is similar between the controls and the reprogramming colonies, the reprogramming colonies are much more dense due to the over‐crowded nature of the cell culture dishes during late reprogramming stages. This high density does not allow for single‐cell resolution of the selection markers as seen in the positive controls. Reprogramming colonies with high intensity live‐cell staining for both marker combinations were selected for further analysis and propagation. Scale bars = 50 um. The top vertical cluster groups the positive control hESC, hiPSC cell lines with a majority of the Day 30 double positive samples and a few Day 20 double positive samples. I have deemed this the “Pluripotent” cluster. The bottom vertical cluster groups the negative control hDFf samples along with the majority of the Day 20 double positive, all Day 10 double negative, and all Day 20/30 double negative samples. I have deemed this the 116 Figure 36: Fluidigm Concatenated Data 117 “Fibroblastic” cluster. A few major clusters also appear along the horizontal (GENE) axis. In relation to the fibroblastic group, the pluripotent group exhibits decreased expression of several genes (Downregulated Cluster): BMP2, KLF4, DKK1, and EGFR. These genes can be seen on the left side of the cluster map. In an opposite relationship, the pluripotent group shows dramatically increased expression of several genes (Early Upregulated Cluster): ERBB3, DNMT3B, EPCAM, CDH1, LEFTY1, NODAL, NANOG, CRIPTO1, OCT3/4. This group of strongly upregulated genes includes several canonical pluripotency master regulators. Several of the samples in the “fibroblastic cluster, however, show increased expression of these genes. Another cluster of genes exhibits strong expression in the positive controls, but limited expression among some of the double positive staining samples from Day 20/30 in the pluripotent group (Late Upregulated Cluster): FOXD3, CDH3, EPHA1, HAS3, LCK, EDNRB, and SOX2. Some of these genes are known pluripotency regulators (FOXD3, CDH3, SOX2) while the functions of the remaining genes in the maintenance of the pluripotent state are largely unknown. I charted the proportion of reprogramming foci showing upregulation of the genes mentioned above [Figure 37]. The “early” upregulated genes are shown in the top graph (A), and the “late” upregulated genes are shown in the bottom graph (B). While it is apparent that both groups show an upregulation with time, the “early” group demonstrates upregulation by Day 10 with expression also noted in the Day 20N and Day 30N double negative samples. A few of these genes are significantly different 118 Figure 37: Fluidigm Upregulated Data These graphs illustrate the expression of pluripotency related genes in reprogramming foci selected for GCTM‐2/EpCAM, TRA‐1‐60/E‐Cadherin positivity/negativity every 10 days post‐transfection of the reprogramming transposon. Several genes are upregulated “early” and lack specificity for marker positivity while a select group of “late” genes appear in about half of the Day 30 foci and are restricted to those foci positive for markers after live‐cell staining selection. 119 between Day 30P double positive foci and “fully” reprogrammed hiPSC controls (DNMT3b, EpCAM and CDH1 – p < .05). Of these genes, only CDH1 is expressed in a significantly different proportion of foci between Day 20P and Day 30P double positive isolated foci. The “late” group has very limited expression in double negative foci (Day 10N, Day 20N, Day 30N) and double positive foci isolated at Day 20P. About half of the double positive foci isolated at Day 30P show expression of these “late” genes. The expression differences between the “fully” reprogrammed hiPSC controls and Day 30P foci are significant to very highly significant for all the genes. The expression differences between Day 20P and Day 30P double positive isolated foci are significant (p < .05) to very highly significant (p < .001) for all genes except SOX2. Furthermore, the expression differences between Day 30P double positive foci and Day 30N double negative foci are highly significant (p < .01) to very highly significant for all the “late” genes except HAS3. Another interesting observation is that mouse OCT4 (muOCT4) is expressed on all hiPSC and reprogramming foci samples tested indicating continued activity of the reprogramming transposon. Specificity of human and mouse OCT4 was tested by inclusion of transposon plasmid as a control for mouse OCT‐4. The mouse primer detects expression of muOCT4 on the transposon but not huOCT4 on the hESC positive controls. Conversely, the human OCT4 primer does not amplify muOCT4 on the transposon but does amplify endogenous human OCT4 on the hESC positive controls. It is clear from the data that Day 10 colonies continue to exhibit a profile similar to the control hDFfs. All Day 10 samples fall into the Fibroblastic cluster. Some Day 10 selected 120 foci exhibit increased expression for a few pluripotency related genes, but pan‐ activation of all the queried pluripotency genes was not observed. All marker negative foci selected at Day 20 group together in the Fibroblastic cluster. About half of the Day 20 double positive samples, however, group with the Pluripotent cluster (GD19D, GD20D, TD20E, TD202G, TD19A, TD19B, TD19C, TD20B, GD19F, GD19B, TD19D) with the remainder retaining a more fibroblastic expression profile. By Day 30, the expression profile of marker positive colonies exhibits stronger expression of pluripotency genes that more closely resembles that of the positive controls. All but one (TD30C) Day 30 double positive foci group in the Pluripotent Cluster. All Day 30 marker negative reprogramming foci group with the Fibroblastic Cluster. Intensely staining foci selected at Day 30 exhibit the most pluripotent expression profile [Figure 38] by clustering closest to the positive controls (A). Foci G30F and TD302B (B) are two samples with intense staining and strong clustering with the positive controls in the Pluripotent Cluster. Intensely staining Day 20 double positive foci (GD20D, TD19B) also cluster closer to the positive controls than a less intensely staining counterpart GD20B. Double negative Day 10 samples clearly cluster with the hDFf negative controls. 121 121 Figure 38: Live‐Cell Staining Intensity Correlates With Expression The top panel (A) shows clustering analysis for positive control hESCs and “fully” reprogrammed hiPSCs along with double positive foci selected at Day 20 and 30. Double negative foci selected at Day 10 and hDFf negative controls are also included. The clustering analysis shows strong segregation between the various groups with most double positive foci clustering with positive controls and double negative samples clustering with negative controls. The most intensely staining double positive foci (B) group with positive controls suggesting that intense marker positivity can be used to select for the most “fully” reprogrammed foci. 122 “Late” Markers of Pluripotency Increase “Fully” Reprogrammed hiPSC Derivation Efficiency Colonies exhibiting dual positivity showed dramatically higher derivation efficiency compared to negative colonies [Table 3]. None of the marker negative colonies gave rise to “fully” reprogrammed hiPSCs. Of 53 marker negative colonies, 30 attached and exhibited outgrowths resembling “transformed” and “partially” reprogrammed hiPSCs. I was only able to passage two of these lines long‐term for further characterization (T2Qn, T3Rn). Of the 73 marker positive colonies live‐cell selected, 19 attached and exhibited outgrowths. Fourteen of these newly derived hiPSCs exhibited a “fully” reprogrammed state observed by morphology with the other five showing a “partially” reprogrammed identity by morphology. With 14/73 live‐cell selected foci generating hiPSCs with morphology suggestive of a “fully” reprogrammed state, the selection aided derivation efficiency was 19.2%. The live‐cell selected hiPSC lines G4E, G4F, and T2G exhibit a “fully” reprogrammed morphology while the hiPSC lines G5C, G5D, and T3D exhibit a “partially” reprogrammed morphology [Figure 39]. Several of these live cell selected “fully” and “partially” reprogrammed hiPSCs were propagated long‐term and characterized by ICC, EB and teratoma assays [Figure 40]. The live‐cell selected, “fully” reprogrammed hiPSC line G4F demonstrates strong marker positivity for the pluripotency markers TRA‐1‐60, GCTM‐2, GDF‐3, EpCAM, E‐ Cadherin, SSEA‐4, Alkaline Phospatase, OCT4, DNMT3b, Nanog, and SOX2 (A‐H). The strong staining is restricted to round, compact colonies much like that seen with the XF‐ 123 Table 3: Live‐Cell ICC Selection Based Derivation Efficiency Live‐cell staining with the pluripotency marker combinations GCTM‐2/EpCAM and TRA‐1‐60/E‐Cadherin aids the selection of “fully” reprogrammed XF‐hiPSCs. I isolated 73 double positive reprogramming foci and 53 double negative foci and transferred them to xeno‐free culture dishes for derivation. 19 double positive foci exhibited attachment/outgrowths (25.3%) compared to 30 double negative foci (56.6%). 14/19 (73.7%) double positive outgrowths exhibited a “fully” reprogrammed morphology after several passages while 0/30 (0.0%) of the double negative outgrowths gave rise to “fully” reprogrammed hiPSCs. Overall, 14/73 double positive foci gave rise to “fully” reprogrammed hiPSCs by morphology. Several of these “full” hiPSCs were shown to possess characteristics similar to hESCs. Live‐cell selection greatly increases derivation efficiency compared to selecting by morphology alone in which ~2% of selected foci give rise to “fully” reprogrammed hiPSCs. hESC line USC01. The live‐cell selected, “partially” reprogrammed hiPSC line G5D also demonstrates positivity for each of the markers in this study (I‐P), but the surface markers show patchy staining across the colonies. The nuclear markers also show limited areas of positivity across each colony. Embryoid body analysis for the live‐cell selected, “fully” reprogrammed hiPSC line G4F shows positivity for Nestin, Alphafetoprotein, and Smooth Muscle Actin confirming the presence of differentiated cells expressing genes characteristic of all three germ layers [Figure 41]. Teratoma analysis is currently underway for G4F with the results expected shortly. 4. Conclusions The data presented in this chapter support the hypothesis that reprogramming colonies exhibiting intense staining for a combination of the late surface GCTM‐ 2/EpCAM and TRA‐1‐60/E‐Cadherin are more likely to give rise to “fully” reprogrammed 124 Figure 39: ‘Fully’ and ‘Partially’ Reprogrammed hiPSCs Derived from Live‐Cell Selection These images illustrate the fate of reprogramming colonies selected by live‐cell ICC with combinations of “late” pluripotency markers. GCTM‐2 (green) / EpCAM (red) and TRA‐1‐60 (red) / E‐Cadherin (green) were used in combination for selection. Colonies demonstrating marked positivity for both markers were isolated for propagation. After several passages, colony morphology indicated that live‐cell selected colonies gave rise to both “fully” and “partially” reprogrammed hiPSCs. Most live‐cell selected hiPSCs demonstrated a “fully” reprogrammed morphology. Marker negative colonies isolated at Day 30 only gave rise to “partially” reprogrammed hiPSCs. 125 iPSCs. Several genes (FOXD3, CDH3, LCK, EPHA1, EDNRB, HAS3 and SOX2) were also identified by quantitative expression analysis of foci undergoing reprogramming that may have value in the further refinement of selection for the best “fully” reprogrammed hiPSCs. The reprogramming marker timeline experiment confirmed the late appearance of TRA‐1‐60 (Day 12) and of EpCAM/E‐Cad (Day 12). TRA‐1‐60 has previously been identified as a selective pluripotency marker appearing “late” in the reprogramming process (Chan, Ratanasirintrawoot et al. 2009). EpCAM and E‐Cad were also recently shown to be selective, late markers in mouse iPSCs (Chen, Chuang et al. 2011). I also demonstrated that GCTM‐2 appears late during the reprogramming process (Day 12) with intense staining appearing by Day 18. This is the first study to indicate the late appearance of GCTM‐2, and it appears similar to the timeline for TRA‐1‐60. This was expected since GCTM‐2 and TRA‐1‐60 bind to the same antigen, but bind at different epitopes. The endogenous nuclear marker of pluripotency, Nanog, appears at Day 6 with widespread positivity by Day 9. The secreted pluripotency marker and BMP antagonist GDF‐3 also appears early at Day 9. The endogenous nuclear pluripotency marker, DNMT3b, demonstrated expression by Day 12. The early appearance of the endogenous nuclear markers makes them less selective for “fully” reprogrammed iPSCs and their use would not be possible in a live‐cell selection process due to their intracellular nature. The early appearance of GDF‐3 also precludes its use as a selective marker. This study also marks the first characterizations of DNMT3b and GDF‐3 during 126 Figure 40: ICC of Live‐Cell Selected hiPSCs These fluorescent images depict several markers of pluripotency expressed on two XF‐hiPSC lines that were selected on Day 33 by live‐cell staining with the marker combination GCTM‐2/EpCAM. The hiPSC line G4F (top, P12) exhibits a “fully” reprogrammed morphology while the line G5D (bottom, P12) exhibits a “partially” reprogrammed morphology. Both lines are positive for all markers in this panel except for SOX2 which was absent from all G5D colonies. G4F exhibits strong colony‐wide staining for the markers, similar to hESCs, while G5D shows non‐uniform positivity in small areas of each colony. ICC confirms a divergent phenotype between the “fully” and “partially” reprogrammed lines. (A, I) Cell surface markers TRA‐1‐60 (red) and GDF‐3 (green). (B,J) Cell surface markers GCTM‐2 (green) and EpCAM (red). (C, K) Nuclear markers OCT4 (green) and DNMT3b (red). (D,L) Cell surface marker E‐Cadherin (red) and nuclear marker Nanog (green). (E, M) Cell surface marker EpCAM (red) and nuclear marker DNMT3b (green). (F, N) Cell surface markers EpCAM (red) and E‐Cadherin (green). (G, O) Cell surface markerSSEA‐4 (green) and nuclear marker SOX2 (red). Alkaline Phosphatase (H, P) staining is faint, but positive (red). All of the preceding images have nuclear counterstaining with DAPI (blue). Scale Bars = 50 um. reprogramming. OCT4 staining does not provide a basis for selection because existing reagents do not discriminate between its expression from endogenous and transgene 127 sources. Increasing intensity of OCT4 staining during reprogramming, however, may point to the increased contribution of endogenously expressed OCT4, as this pluripotency network is re‐activated by reprogramming transgene expression. While the previous paragraph details the first appearance of positive staining for the examined pluripotency markers, intense staining resembling that expressed on positive control hiPSCs first appears around Day 18 for all markers. The percentage of reprogramming foci exhibiting intense staining plateaus around 10% for all markers by Day 24 whereas positive staining above background increases over the course of the timeline to 30‐50% of total colonies. The appearance of foci exhibiting intense staining may suggest a strong re‐activation of pluripotency regulatory networks. Therefore, selection by intensity of staining could aid isolation of the most “fully” reprogrammed hiPSCs. Additionally, staining for a combination of markers illustrates relationships between the two markers. In each combination study, the percentage of foci staining positive for one marker was higher than the percentage staining for both markers. The one noticeable exception is the marker combination EpCAM/E‐Cadherin which demonstrates a high level of concordance between single positive and double positive foci. This concordance supports the idea that EpCAM and E‐Cadherin are under the same temporal control in the reprogramming process such that a similar pluripotency regulatory network modulates the expression of both surface receptors. Consequently, marker combinations demonstrating a lack of concordance are most likely under control 128 Figure 41: Embryoid Body Analysis of Live‐Cell Selected hiPSCs These images depict the pluripotency of the Xeno‐Free hiPSC line G4F through its differentiation potential. Embryoid body formation is a standard characterization assay utilized to identify progenitor and fully differentiated cells. Positivity for Nestin (red) indicates cells with a neural phenotype arising from the ectoderm (A). Alphafetoprotein (green) is indicative of liver progenitors arising from the endoderm (B). Smooth Muscle Actin is a contractile protein found in cells arising from the mesoderm (C). of different regulatory networks. Interestingly, when one categorizes the markers for intense staining the level of concordance is very high. For example, if one marker stains intense, it is highly likely that the second marker in the combination will also stain intense. This data supports the hypothesis that intense staining is representative of foci who have strong reactivation of all the pluripotency networks necessary to reach a “fully” reprogrammed state. A strong positive reaction in the ICC assay requires activation not only of the relevant transcriptional network but also appropriate post‐ transcriptional control and post‐translational modification. Further support for the link between intense staining and reactivation of pluripotency regulatory networks is evident in the expression data arising from the Fluidigm Biomark HD experiments. Live‐cell staining by nature is a less sensitive imaging technique than the highly sensitive staining images coming from the fixed samples on 129 glass slides presented in the timeline experiments. The decrease in sensitivity results from the challenge of imaging through a plastic cell culture dish and the overlying culture medium. Only the most intense staining foci will be adequately visualized. The plastic dish, consequently, acts as a high pass filter. The expression data reflects this relationship as double positive foci selected at Day 30 have an expression profile that cluster with the pluripotent positive controls while less intense staining foci and negative staining foci cluster with the fibroblast negative controls. It is possible that some of the negative staining foci selected for expression analysis would have been scored in the positive (not intense) group if they were imaged on high resolution glass slides. This data supports the hypothesis that intense staining for a combination of late‐ markers select for hiPSCs exhibiting a more “fully” reprogrammed expression profile. The quantitative expression data from this study provides a wealth of information beyond the selectivity of live‐cell staining with a combination of pluripotency markers. As described in the results section, three major clusters of genes exist among the samples. The Downregulated Cluster illustrates genes expressed highly on the fibroblast negative controls that must be downregulated to achieve a “fully” reprogrammed state. The Early Upregulated Cluster contains genes (OCT4, Nanog, NODAL, CDH1, EpCAM, LEFTY1, CRIPTO1, and DNMT3b), all of which have well known roles in the maintenance of pluripotency (Nichols, Zevnik et al. 1998; Chambers, Colby et al. 2003; James, Levine et al. 2005; Vallier, Alexander et al. 2005; Li, Pu et al. 2007; Lu, Lu et al. 2010; Redmer, Diecke et al. 2011), that must be upregulated to achieve “full” 130 reprogramming. Researchers commonly look at the activation of these genes to describe “full” reprogramming, but our data suggests that expression of these genes alone does not signify complete reactivation of the pluripotency network. A more intriguing cluster of genes, consequently, is the Late Upregulated Cluster. All of the positive controls have high expression of these genes, but only some of the selected reprogramming foci exhibit expression. I hypothesize that the activation of this group of genes which includes FOXD3, CDH3, LCK, EPHA1, EDNRB, HAS3 and SOX2 indicate a more complete reprogramming process and could thus be used to isolate reprogramming colonies that have the highest probability of becoming “fully” reprogrammed hiPSCs. There are data to implicate FOXD3 (Hanna, Foreman et al. 2002), SOX2 (Avilion, Nicolis et al. 2003), CDH3 (Kolle, Ho et al. 2009), LCK (Meyn, Schreiner et al. 2005), and HAS3(Tang, Barbacioru et al. 2010) in the maintenance or description of the pluripotent state. Expression analysis of these genes indicates a highly statistically significant difference between Day 30 double positive reprogramming foci and the positive control “fully” reprogrammed hiPSCs and hESCs. Over half of the double positive foci were negative for expression of these genes suggesting that they have not concluded the reprogramming process at this point. It is possible that the foci lacking expression of these genes would later exhibit expression, but the absence of expression after 4‐5 weeks of reprogramming indicates a prolonged process that could be abortive. Selection of reprogramming foci that have rapidly re‐activated expression of these late upregulated genes could improve the derivation of “fully” reprogrammed 131 hiPSCs. Future studies could look at a timeline of appearance for these genes as markers to see if they identify a smaller percentage of colonies during late reprogramming with a more “fully” reprogrammed expression profile. Moreover, one could create a reporter construct with GFP driven by the promoters of these genes such as FOXD3 or CDH3. This reporter could be included on the reprogramming cassette if it is insulated from the constitutive promoter. Alternatively, an extra transposon plasmid containing the reporter construct could be added to the transfection protocol. Additionally, such a reporter line could help the production of XF‐hiPSCs since live‐cell staining with animal sourced antibodies would not be necessary. Among the upregulated genes not within the previously mentioned Downregulated, Early Upregulated and Late Upregulated clusters are the early lineage genes CER1, GSC, BRACHYURY, GATA4, and MIXL1. I hypothesize that the elevated expression of these genes indicates the progression of reprogramming foci through lineage intermediates during the reprogramming process. This could either occur as they are still in transition, or there may be a significant subset of cells in a reprogramming focus that are pluripotent and undergoing early stages of lineage commitment. This lineage expression data underlies the unstable, dynamic process of reprogramming that is most likely incomplete even at Day 30. It usually takes several passages after foci isolation for a stable “fully” reprogrammed hiPSC line to appear. This points to a heterogenous population of cells existing within a single reprogramming focus. Even though the entire reprogramming focus originated from a single cell with 132 integrated transposon(s), each cell arising from a mitotic event can experience an individual reprogramming environment such that some progeny cells will reach a “fully” reprogrammed state while others may divert along another “partially” reprogrammed intermediate state. Staining data supports this idea since intense staining may be limited to small areas of a reprogramming focus. With time, the more “fully” reprogrammed cells within the focus may compete with the intermediates such that the majority of the focus exhibits intense staining. Supporting this is the observation that Day 20 foci often have limited areas of intense staining while Day 30 foci have a much more focus‐wide, intense staining. Additionally, I have been able to split two hiPSC cell lines into clonogenic populations of “fully” and “partially” reprogrammed hiPSCs. All of these observations support the idea of a dynamic reprogramming process. It would be very interesting to perform single‐cell expression analysis on an entire reprogramming focus to illustrate the different states of reprogramming present among each of its cells. After confirming the link between intense staining for the marker combinations GCTM‐2/EpCAM and TRA‐1‐60/E‐Cadherin with a more pluripotent expression profile, I wanted to determine the usefulness of these markers for increasing the derivation efficiency of hiPSCs. At Day 30, I live‐cell stained several dishes and manually dissected marker positive/negative foci. The foci were transferred to xeno‐free cell culture dishes for observation. Of 53 double negative foci, 30 attached and proliferated. All of these double negative foci gave rise to “partially” reprogrammed hiPSCs with most failing to propagate after a few passages. Conversely, 19 of the 73 double positive foci exhibited 133 attachment/proliferation. 14 of these selected foci resulted in “fully” reprogrammed hiPSCs while 5 resulted in “partially” reprogrammed hiPSCs. The live‐cell selected cells exhibiting a “fully” reprogrammed state stain strongly positive for my standard ICC panel and illustrate differentiation into cells with characteristics of all three germ layers by embryoid body analysis. When taken as a percentage of total colonies selected, the “fully” reprogrammed derivation efficiency of double positive foci is 14/73 (19.2%). Compared to derivation efficiency of selection by morphology alone which averages 1/50 ‐1/100 (1‐2%), live‐cell selection represents a nearly 20‐fold selection advantage. This dramatic improvement has the potential to save enormous amounts of time dedicated to initial derivation and subsequent cell culture of hiPSCs. Live‐cell selection utilizing a combination of GCTM‐2, TRA‐1‐60, EpCAM, and E‐Cadherin provides a significant advantage for the derivation of “fully” reprogrammed hiPSCs and could easily be incorporated into automated selection protocols for high throughput methodology. 134 Conclusion The data presented in this dissertation represents several contiguous and interconnected research projects. I have successfully established a xeno‐free pluripotent cell culture system that can be translated to a GMP facility for future clinical derivation of pluripotent cell lines for drug discovery or regenerative medicine. The xeno‐free cell culture system successfully supported the derivation of several XF‐hiPSC lines as well as the derivation of a XF‐hESC line, USC01. I will soon begin the process of submitting the paperwork for inclusion of USC01 on the NIH Stem Cell Registry to enable federal funding support and distribution for future research. While the original goal of deriving several XF‐hiPSC and XF‐hESC lines for characterization of equivalency was not achieved, comparison between USC01 and the XF‐hiPSC lines indicates strong similarity among all assays, but it is apparent that there are also slight differences between hiPSCs and hESCs in morphology, proliferative capacity, pluripotency marker expression, gene expression profile and differentiation capacity. The cell culture platform I have developed provides a basis for comparison of the two cell types generated under conditions fully compatible with clinical use. 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Cell Stem Cell 4(5): 381‐4. 144 Appendix A: Sperm/Egg Donor Consent Form 145 146 147 148 149 150 151 Appendix B: Embryo Donor Consent Form 152 153 154 155 156

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Creator Pomeroy, Jordan Elliott (author)

Core Title Derivation and characterization of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells in clinical grade conditions

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Systems Biology and Disease

Publication Date 05/01/2012

Defense Date 03/05/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Cells,clinical,embryonic,human,induced,OAI-PMH Harvest,pluripotent,STEM

Language English

Advisor Pera, Martin F. (committee chair), Chuong, Cheng-Ming (committee member), Lu, Wange (committee member), Ying, Qi-Long (committee member)

Creator Email jep1780@yahoo.com,jpomeroy@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-20555

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Document Type Dissertation

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Abstract (if available)

Abstract Future use of pluripotent cells for regenerative medicine will require the derivation and characterization of new cell lines in clinical grade conditions. Removing potential sources of contamination, such as cell culture components sourced from animals, will aid the regulatory approval for future stem cell based therapeutics. In this dissertation, I describe the development of a xenobiotic-free cell culture system for the derivation and maintenance of human pluripotent cell lines. I have derived >40 hiPSC lines and one hESC line in the xeno-free cell culture conditions with maintenance of pluripotency charcacterized by morphology and immunocytochemistry marker expression for >50 passages and >30 passages respectively. The hESC line, USC-01 and several hiPSC lines derived for this study demonstrate highly similar gene expression patterns although slight differences are apparent. USC-01 and several hiPSC lines demonstrate the ability to differentiate into cells displaying characteristics of all three germ layers in an embryoid body differentiation assay. Further examination of the process of reprogramming somatic cells to a pluripotent state at both the RNA and protein expression levels indicates several genes/markers selective for hiPSCs achieving a pluripotent state most similar to the “gold-standard” of pluripotency possessed by hESCs. This study confirms the usefulness of the markers TRA-1-60, E-Cadherin and EpCAM for live-cell selection of the best hiPSC colonies and also demonstrates the usefulness of the marker GCTM-2. Expression analysis of colonies undergoing reprogramming also indicates that the genes FOXD3, CDH3, LCK, EDNRB, EPHA1, SOX2, and HAS3 are active in only a small subset of colonies 30 days after transfection of the piggyBac transposon reprogramming cassette. Since these genes are active in all hESC and most hiPSC positive control lines tested, confirmation of their activation could be used to select reprogramming hiPSC colonies most likely to achieve a pluripotent state similar to hESCs. Increased selection and derivation efficiencies of hiPSC lines demonstrating high fidelity to the hESC pluripotent state will streamline the generation of hiPSC lines for future testing as a replacement for hESCs in regenerative medicine.