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Deconstructing the lung: a novel assay to evaluate lung stem/progenitor cell potential in vivo

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Deconstructing the lung: a novel assay to evaluate lung stem/progenitor cell potential in vivo

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Content DECONSTRUCTING THE LUNG: A NOVEL ASSAY TO EVALUATE LUNG STEM/PROGENITOR CELL POTENTIAL IN VIVO by Soula Danopoulos A Thesis Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (EXPERIMENTAL AND MOLECULAR PATHOLOGY) August 2011 Copyright 2011 Soula Danopoulos 08 Fall   ii  ACKNOWLEDGEMENTS   First and foremost, I would like to thank the members of my committee: Dr. Saverio Bellusci, PhD (chair of committee and mentor); Dr. David Warburton, MD; and Dr. Krzysztof Kobielak, MD/PhD. The passion you each have for science is extremely evident and contagious. Each of you was able to teach me something new during the time frame of my thesis; whether it was the introduction of new techniques, or methods by which to improve ones already in practice. Asides from providing me with technical support, you also taught me how to be independent and truly appreciate all the challenges research offers. Next, I would like to thank all the members of my lab. The two post docs, Caterina Tiozzo, MD/PhD and Denise Al Alam, PhD, were each a great source of support and knowledge in helping me achieve and understand my data. You both taught me so much and I am greatly appreciative for all the encouragement and assistance provided by you both. I would also like to thank the lab’s assistants: Sheryl Baptista, Melissa Green, Jonathan Branch, and Maria Pearce for providing me help whenever requested. You each were so willing to help me whenever I needed it, which most definitely alleviated some of the stress I may have been experiencing at the time. I would also like to thank Clarence Wigfall, the lab’s information technology expert, and former lab-mate Sara Parsa, PhD, for giving me a great deal of moral support and encouragement during times of struggle.   iii  I would also like to thank the two surgeons who provided their expertise during the time frame of my research. Without them, none of the in vivo experiments would have been possible. Dr. Jamil Mathews, MD, assisted me in some of the subcutaneous implants as well as the implant of the polymer within the omentum. Dr. Edwin Jesudason, MD, also performed some of the subcutaneous implants, as well as all of the very complicated subcapsular implants. Furthermore, I would like to thank Dr. Tracy Grikscheit, MD, for allowing me the use of her lab’s polymer so that I could conduct the polymer scaffold implanted within the omentum experiment. Finally, I would like to thank my family for all their support and understanding within these past couple of years. When times would get tough they always gave me the strength I needed to push through. They made sure I never gave up on myself. THANK YOU EVERY ONE OF YOU!!!! You each played such an important role these past few years and I appreciate each of you immensely.       iv  TABLE OF CONTENTS Acknowledgements ............................................................................................... ii List of Tables ........................................................................................................ vi List of Figures .......................................................................................................vii Abstract ................................................................................................................. x Chapter 1: Introduction ..........................................................................................1 1. Lung Development..................................................................................4 2. Lung Progenitor Cells ...........................................................................10 2.1 Embryonic Progenitor Cells vs. Adult Stem Cells...........................10 2.2 Lung Epithelial Progenitor Cells .....................................................12 2.3 Lung Mesenchymal Progenitor Cells..............................................15 3. Organotypic Models and Animal Xenografts ........................................15 Chapter 2: Aim of the Study ................................................................................18 Chapter 3: Materials and Methods ......................................................................20 1. Mice ......................................................................................................20 2. Lung Single Cell Suspension................................................................20 3. Counting Cells ......................................................................................22 4. Labeling the Cells .................................................................................22 5. Cell Culture Conditions .........................................................................23 A. Cells Directly Atop of Carbon Filter..................................................23 B. Cells Mixed with Matrigel Atop of Carbon Filter ...............................23 C. Hanging Droplet...............................................................................24 6. Embedding............................................................................................24 A. In Vitro Samples...............................................................................24 B. In Vivo Samples ...............................................................................25 7. Stainings ...............................................................................................26 A. Hematoxylin and Eosin (H&E) .........................................................26 B. PAS..................................................................................................27 C. Alcian Blue Staining.........................................................................27 D. DAB..................................................................................................27 E. Immunofluorescence........................................................................28 8. Kidney Capsule Surgery .......................................................................31   v  Chapter 4: Results...............................................................................................32 1. E12 Lung Implant..................................................................................32 2. E 14.5 Lung Single Cell Suspension Plated .........................................37 Upon a Carbon Filter 3. E14.5 Lung Single Cell Suspension Plated ..........................................41 with Matrigel Upon Carbon Filter 4. E14.5 Lung Single Cell Suspension Plated ..........................................45 with Matrigel and Implanted Subcutaneously 5. E14.5 Lung Single Cell Suspension Placed .........................................50 in Polymer and Implanted in Omentum 6. Fresh E14.5 Lung Single Cell Suspension ...........................................54 Injected into Kidney Capsule 7. E14.5 Lung Single Cell Suspension Plated with...................................56 Matrigel and Implanted Within the Kidney Capsule 8. E14.5 Lung Single Cell Suspension Plated as .....................................61 Hanging Droplet 9. E14.5 Lung Single Cell Suspension Plated as .....................................65 Hanging Droplet and Implanted Underneath the Kidney Capsule Chapter 5: Discussion .........................................................................................72 1. Progenitor Cell Expression ...................................................................72 2. How p63 Indicated Differentiation.........................................................74 3. How T1α Indicates Differentiation.........................................................75 4. Cell Aggregation as Opposed to Proliferation.......................................75 5. Subcutaneous vs. Subcapsular ............................................................77 6. Matrigel vs. No Matrigel ........................................................................80 Chapter 6: Conclusion / Future Directions...........................................................84 References ..........................................................................................................86   vi  LIST OF TABLES Table 1: List of Antibodies...................................................................................29 Table 2: Summary of Results and Conclusion....................................................68  vii  LIST OF FIGURES Figure 1: Different Stages of Lung Development. ................................................6 Figure 2: Biochemical and Biomechanical Regulators of .....................................9 Lung Growth. Figure 3: Classical Stem Cell Hierarchy. ............................................................11 Figure 4: Schematic of the Main Cell Types Along the.......................................13 Proximodistal Axis of the Mouse Lung. Figure 5: Summary of Probable Stem/Progenitor Cell .......................................14 Relationships in Trachea, Intralobar Airways and Alveoli. Figure 6: E12 Lungs Implantation Underneath Kidney.......................................33 Capsule Leads to Formation of Embryonic-Type Proximal/Distal Airways Structures Figure 7: Cartilage Formation Shows Advancement .........................................33 in Lung Development in E12 Lungs Subcapsular Implantation Post 1 Month Figure 8: Change in Expression of Lung Epithelial.............................................35 Progenitor Markers From E12 Lungs Subcapsular Implantation Post 1 Month Figure 9: Epithelial Differentiation Shows Advancement....................................36 in Lung Development in E12 Lungs Subcapsular Implantation Post 1 Month Figure 10: Progressive Lumen Formation Post 72 Hours ..................................38 of Plating E14.5 Lung Single Cell Suspension Upon Carbon Filter Figure 11: Expression of Lung Epithelial Markers in ..........................................40 E14.5 Lung Single Cell Suspension Plated Upon Carbon Filter  viii  Figure 12: Progressive Spheroid Formation During 1 ........................................42 Week Incubation of E14.5 Lung Single Cell Suspension Plated with Matrigel Figure 13: Mixture of Cells Expressing Lung.....................................................43 Progenitor Markers Within Single Matrigel Spheroid Suggests Cell Aggregation as Opposed to Proliferation Figure 14: Mixture of Cells Expressing Lung Epithelial ......................................44 Markers Within Single Matrigel Spheroid Suggests Cell Aggregation as Opposed to Proliferation Figure 15: E14.5 Matrigel Spheroids Subcutaneous ..........................................46 Implantation Leads to Minimal Formation of Airway Structures Post 1 Month Figure 16: Expression of the Three Progenitor Cell ...........................................47 Markers Remain Stagnant Post 1 Month Subcutaneous Implantation, Suggesting Negligible Differentiation Figure 17: Gradual Deterioration of Epithelial Marker ........................................49 Expression Post 1 Month Subcutaneous Implantation Indicates Inadequate System Figure 18: Structural Development From Omentum...........................................50 Implanted Scaffold of E14.5 Single Cell Suspension Post 1 Week Figure 19: Lack of Lung Progenitor Marker Expression .....................................52 Within Omentum Implanted Polymer Implies Minimal Differentiation Figure 20: Lack of Lung Epithelial Cell Markers .................................................53 Expression Within Omentum Implanted Polymer Implies Minimal Differentiation Figure 21: No Structural Formation During Subcapsular....................................55 Single Cell Injection Implied No Cellular Aggregation (Progenitor Markers) Figure 22: No Structural Formation During Subcapsular....................................56 Single Cell Injection Implies No Cellular Aggregation (Epithelial Markers)   ix  Figure 23: Change in Expression of Lung Epithelial Progenitor .........................58 Markers and Structural Development of Subcapsular Matrigel Spheroid Figure 24: Epithelial Marker Expression Indicates Differentiation ......................60 of Matrigel Spheroids Did Not Take Place Post 2 Weeks Subcapsular Implantation: Detrimental Effects of Matrigel Figure 25: Gravity Allows the Cells of the Hanging Drop to ...............................62 Aggregate Constructing Airway-Like Structures Figure 26: Lung Epithelial Marker Expression Indicates that the .......................64 Hanging Drop Cells Gradually Organize into Airway-Like Structures Representative of the Native Lung Figure 27: Progenitor Cell Marker Expression Emphasize.................................66 Development of Various Lung-Like Structures Indicating Hanging Drop Differentiation and Growth Post 1 Month Subcapsular Implantation Figure 28: Epithelial Cell Marker Expression Emphasize...................................67 Development of Various Lung-Like Structures Indicating Hanging Drop Differentiation and Growth Post 1 Month Subcapsular Implantation   x  ABSTRACT Rationale: The lung is an organ essential for survival, however, due to its exposure to the outside environment, it is continuously subjected to multiple injuries. As a result, lung diseases are the leading cause of human death worldwide. The majority of these diseases are chronic and incurable, therefore representing a considerable financial burden. An ideal way in which such diseases could be effectively treated would be via regenerative medicine; however, for such medicine to be available, lung stem/progenitor cells must first be characterized. Objective: To develop an in vitro/in vivo assay, which would prove to be the simplest, yet most informative, in testing the contribution of given cells to different lung cell lineages. We have composed an adequate organoids system made from dissociated epithelial and mesenchymal embryonic 14.5 lung cells. These organoids were then grafted in vivo in different locations. Results: We constructed a number of different organoids systems so as to achieve the ideal conditions. These varying approaches included: single cell suspension plated directly atop carbon filters, single cell suspension mixed with matrigel plated atop carbon filters, and hanging droplets. These different systems were then implanted in different locations within 2 month-old NOD/SCID female mice, thus reducing chances of rejection by the host. These implantation sites included: the omentum, subcutaneous, and subcapsular. Via the use of immunohistochemistry, we would determine whether or not the organoids were   xi  able to develop and differentiate during implantation. Identification of progenitor cells was done using Sox2, Sox9, and Id2 antibodies. Cell markers include CC10, αSMA, SpC, TTF-1, p63, and T1α. Our results indicate that the subcapsular engrafted hanging droplets are the most promising. All of the aforementioned markers stained positively in their appropriate locations, indicating that the E14.5 cells advanced in the organoids in their differentiation status, generating both proximal- and distal-like airway structures, as well as alveoli-like structures. Conclusions: The results of the different organoids assays within different implantation sites indicated that the subcapsular hanging droplets would be the ideal approach. Once the conditions for this in vivo system are optimized, it will prove to be an elegant, yet powerful system in determining lung stem-cellness. With the use of different genetically modified mice and additional methods by which to separate the assorted cells in the lung, a powerful assay will have been constructed that may test how different signaling pathways and cell-types effect the construction of mature lung tissue.                           1  CHAPTER 1: INTRODUCTION The lung is an organ essential for survival. It is the means by which respiration takes place so that oxygen enters the bloodstream to support the body’s cells and tissues. Although the lung’s main and most pertinent role is its ability for a being’s respiration, the lung has numerous other non-respiratory functions. For instance, it may alter the blood’s pH, by facilitating alterations in the partial pressure of carbon dioxide, and it may also influence the concentration of some biologic substances and drugs used in medicine in blood. (http://www.lungusa.org). However, the lung is peculiar in comparison to the other organs of the body because it is directly connected to the outside environment. As a result, anything one breathes in from their surroundings, such as germs, smoke, and pollutants may pose a threat to the lungs, and ultimately affect the organ’s functionality. Because of this constant and immediate interaction with the external environment, the lung conveniently has a natural defense system designed for its sole protection. Yet, this “in-grown” protection system may only shield the lung to a certain extent, and for this reason there are several conditions which are capable of affecting some aspect of the lung, and in due course impact a person’s ability to breathe (http://www.lungusa.org.) These conditions include diagnoses such as lung cancer (which is the second most commonly diagnosed cancer in both men and women), cystic fibrosis, asthma, and bronchiolitis. The   2  majority of these diseases are incurable and progressive, thus leaving the patient in discomfort until they die, often due to the respiratory condition itself. This is the reason why respiratory diseases are the leading cause of death worldwide  (http://www.lunguk.org). Although expensive and laborious, studying the lung is crucial in today’s society. Scientists have already discovered so much in regards to molecular pathways and the construction process of the lung. However, a comprehensive understanding of the lung progenitor cells in the epithelium and mesenchyme: where they are, how to activate them and how they act, has yet to be determined. With this discovery, such novel therapeutic approaches as regenerative medicine may be applied to lungs; ultimately saving the lives of millions of people. There are two types of stem cells that are often referenced: embryonic stem cells (ESCs) and adult stem cells (ASCs). ESCs are located in the inner cell mass of the blastocyst, and tend to disappear after the 7 th day in normal embryonic development (Evans and Kaufman, 1981). These pluripotent cells have the ability to give rise to practically every tissue in the body, given the adequate conditions, and have the remarkable ability to self-renew continuously. However, there are an obnoxious number of debates regarding the ethics of working with such cells. For this reason, studying ASCs and their ability in regards to   3  regenerative medicine appear more plausible at the moment. ASCs are naturally poised to generate a particular tissue, being that they are specific to each organ. This allows the body to replenish its own tissues either due to homeostasis or injury. They are rare and difficult to isolate due to their being hidden inside a niche, and because of this, are still poorly characterized. Yet, ASCs decrease in number as individuals age, meaning they have a naturally limited replacement/repair capacity (Warburton et al, 2008). This could be a result of simple exhaustion of the stem cell pool, or acquired mutations that impede proper stem cell function. However, it has been speculated that the stem cell’s abilities may be reversed and reactivated if the endogenous stem cell pool is infiltrated by exogenous stem cells. For this reason, if ASCs are localized, they may be reactivated inside a diseased environment and may contribute to the regeneration of damaged tissues and organs by stimulating previously irreparable organs to heal themselves (Warburton et al, 2008). A recent study conducted by Kajstura et al (2011) further supports this concept. The group isolated c-kit-positive cells from adult human lung, and injected them into the left lung of cryoinjured adult immunosuppressed mice. After 10 to 14 days the human c-kit-positive lung cells formed human bronchioles, alveoli, and pulmonary vessels within the injured areas of the mouse, indicating self-renewal and multipotentiality in vivo.   4  This concept of regenerative medicine is ideal because it has the potential to solve the current problem of the shortage of organs available for donation, as well as complications associated with organ transplant rejection, since the organ's cells match that of the patient. Although there are a number of organs in which ASCs have been localized, such as in gut and skin, that of the lung have yet to be isolated. This is why studying lung in mouse is so important. 1. Lung Development The pulmonary system is very organized and efficient in allowing the exchange of oxygen and carbon dioxide during cellular respiration. It consists of the tracheal tube, located most proximally, which then branches off into the bronchi and bronchioles. These structures then allow the inhaled gas to reach the more distal arrangements, known as the alveolar structures. The capillaries integrated into these peripheral configurations facilitate the exchange of oxygen with waste gas from the blood. Although the lungs are spatially organized along both the cephalo-caudal axis and dorsal-ventral axis, they are usually asymmetrically lobulated. For instance, in the mouse the right bud gives rise to four lobes (cranial, medial, causal, and accessory) whereas the left bud gives rise to only one (Warburton et al, 2010). The branching pattern in mouse lung is extremely reproducible from one embryo to the next, implying that there is a tight temporal-   5  spatial genetic control. This is also true in Humans, where the left lung is trilobed in comparison to the bilobed right lung. This change in branching is regulated by the interacting growth factors being released by both epithelium and mesenchyme during the five different structural stages of lung formation. These five stages are as follows: Embryonic (2-4 weeks of Human pregnancy; E9-E11.5 days in mouse embryo), Pseudoglandular (5–17 weeks of Human pregnancy; E11.5-16.5 days in mouse embryo), Canalicular (16–25 weeks of Human pregnancy, E16.6–17.4 days in mouse embryo), Saccular (24 weeks to late fetal period in Human, E17.4 to postnatal day 5 (P5) in mouse), and Alveolar (late fetal period to childhood in Human, P5–P30 in mouse) (Figure.1) (Maeda et al, 2007; Warburton et al, 2010).   6  The lateral-esophageal sulcus located between the thyroid and stomach of the foregut endoderm is what gives rise to the lung (Serls et al, 2005). Being that more than one organ arises from this general region; the lung and thyroid originate from the area containing the Nkx2.1 homeodomain-containing transcription factor, also called TTF1. Fibroblast growth factor (FGF) is what differentiates the formation of one organ from the other. Cells that receive less FGF stimulation form the thyroid, whereas those that receive more form the lung.   7  Often times the embryonic and pseudoglandular stages may be combined. During this time, formation of the lung buds and major bronchi take place, as well as division of the tracheal-esophageal tube. Furthermore, proliferation of bronchial branches, acinar tubules and buds takes place alongside vasculogenesis and innervations (Maeda et al, 2007). At this time there is a great deal of autocrine-paracrine signaling amongst and between the mesenchyme and endoderm, such as FGF-10 from the mesenchyme (Min et al, 1998) and its receptor FGFR2 in the endoderm (De Moerlooze et al, 2000). The pulmonary cells still retain considerable plasticity, so as to allow reprogramming of their differentiation. At this point in time the primitive branched tubes resemble an exocrine gland filled with fluid as opposed to gas (Warburton et al, 2010). Next is the canalicular stage, during which transcriptional factors control differentiation within the conducting airway epithelium (Maeda et al, 2007). During the pseudoglandular stage, all of the epithelial cells lining the airways are undifferentiated, with the differences ranging from pseudostratified epithelium lining the trachea and main bronchi, and the more distal airways being lined by simple columnar-cuboidal epithelium. At this time thyroid transcription factor-1 (TTF-1), nuclear factor-1β (NF-1β), GATA-binding protein-6 (GATA-6), retinoblastoma (RB), E-Twenty Six (ETS), SRY (sex determining region-Y)-box (SOX), and forkhead box (FOX) family transcription factors play a role in cell specific differentiation, which varies along the cephalo-caudal axis of the airways   8  (Maeda, 2007). Due to this cross-talk between all the different transcription factors within the mesenchyme and epithelium, the diameter and length of the respiratory tree expands, and the terminal bronchioles get divided into respiratory bronchioles and alveolar ducts (Warburton et al, 2010). At the end of E17.5 days in mouse, or 25 weeks in human pregnancy, the lungs advance into the saccular stage. The lung is now preparing itself for transition from a fluid-filled lung to an air-filled lung. This third/fourth period is characterized by thinning of the mesenchyme and alveolar epithelial cells differentiating into the squamous type I pneumocytes and the secretory type II pneumocytes (Warburton et al, 2010). As the lung saccules dilate, the increasing amounts of pulmonary capillaries are able to closely integrate with the epithelium, thus facilitating the ability of gas exchange. By the end of this stage, the lung is now capable of sustaining gas exchange without collapsing. The transcription factors that regulate the expression of genes that are necessary for respiratory adaptation at birth include thyroid transcription factor-1 (TTF-1), forkhead box A2 (FOXA2), nuclear factor of activated T-cells, cytoplasmic 3 (NFATc3), CCAAT/enhancer binding protein a (C/EBPa), and glucocorticoid receptors (Maeda et al, 2007). Finally, the last morphological step involved in lung formation is the alveolar stage. At this stage alveologenesis takes place, implying that the distal epithelial   9  cell types are differentiating, thus leading to the development of mature alveoli and alveolar ducts. This pertinent step, which assures proper processing of gas exchange, occurs postnatal in mice, but during gestation in humans. Alveolarization is a complicated process that is influenced by myriads of transcription factors. The fact is that the signaling mechanisms behind these transcription factors are not fully understood, but some of the ones involved in the process include GATA-6, TTF-1, SMAD3, FOXA2, and FOXF1 (Maeda et al, 2007). This brief morphological overview clearly explains that molecular cross-talks between the mesenchyme and endoderm are many and pertinent in lung development (Fig.2).      10  2. Lung Progenitor Cells 2.1 Embryonic Progenitor Cells vs. Adult Stem Cells It has been established that organ morphogenesis is controlled by cell-cell signals between the epithelium, mesenchyme, mesothelium and vascular smooth muscle cells. This is a result of both intercellular and intracellular signaling from both the progenitor cells and the differentiating cells. Embryonic progenitors are often multipotent cells located in the developing organ, that give rise to adult cells before turnover begins. They have the ability to affect organ size, shape and cellular composition (Rawlins et al, 2008). Progenitor cells have the ability to divide both symmetrically or asymmetrically. During symmetrical division, the progenitor cells may divide into two new progenitor cells, or two cells that will differentiate (Fig. 3). The course of division taken by the progenitor cells is what determines the shape and size of the organ itself (Rawlins et al, 2008).  11  One of the recently proposed models is that as the lung branches, the daughters of the distal tip progenitors get left in the stalks to differentiate, and the self- renewing progenitors remain within the epithelial budding tips (Rawlins et al, 2008). The progenitor cells have a unique pattern of gene expression, which include the transcription factors etv5, id2, sox9 and high levels of activity from the Wnt, Bmp, Fgf, and Shh signaling pathways (Shu et al, 2005; Liu et al ,2002; Bellusci et al, 1996). Embryonic progenitor cells are different from adult stem cells, in that they self- renew throughout the lifetime of the animal, and give rise to one or more different differentiated cell types within a particular organ. In reference to the other mature cells within the organ, the adult stem cells are considered less differentiated and divide infrequently. Embryonic progenitor cells mediate  12  branching morphogenesis by sending signals to pattern the mesenchyme and allowing rapid epithelial proliferation. On the other hand, adult stem cells only proliferate to maintain homeostasis, or assist in injury, and thus divide slowly (Rawlins et al, 2008). However, in regards to the lung, a question has been posed that leads to some controversy: are the adult lung epithelial stem cells a distinct cell population, or have some of the multipotent embryonic progenitors persisted to adulthood (Rawlins et al, 2008)? Although not confirmed, recent investigations have suggested that the two are distinct cell populations, but lineage related. 2.2 Lung Epithelial Progenitor Cells Lung development originates from two cell populations, one of which gives rise to the larynx and trachea, and the other, which generates the peripheral bronchi and alveolar surfaces (Warburton et al, 2010). The cell types in these two regions vary extensively in that within the most proximal conducting airways columnar epithelium consisting of ciliated cells, secretory cells, basal cells and submucosal glandular epithelium is expressed. Then, in comparison, the distal airways have no basal cells, and no secretory cells nor submucosal glands. Furthermore, the epithelium is not columnar but rather contains peripheral squamous cells with more proximal cuboidal cells with an increasing ratio of secretory (Clara) cells to ciliated epithelial cells (Kotton and Fine, 2008) (Fig. 4 shows a schematic of where in the lung these cells are located).  13  With the assistance of various cell injury models, and the use of either experimental labeling of proliferating cells or cell lineage tracing, it has been discovered that the type of airway that is injured determines which set of progenitor cells will be activated. For instance using naphthalene injury, in transgenic mice expressing inducible cre recombinase under the control of the cytokeratin K14 promoter, it was deduced that the basal cells may give rise to both secretory and ciliated cells in the proximal airways (Hong et al, 2004a; Hong et al, 2004b). Thus, in the proximal lung, it is suggested that these K14-positive basal cells behave like the dedicated stem cells. Moving away from the proximal region, basal cells start to disappear. In turn, cells known as ‘variant’ Clara cells  14  (Clara V cells), which are clustered next to the neuroendocrine bodies, assume the stem cell role in response to injury (Hong et al, 2001). The Clara V cells are thought to give rise to Clara cells and ciliated cells. The Clara cells may then act as committed progenitors, being able to self-renew, and give rise to both ciliated and goblet cells. Another set of putative stem cells of the distal lung are located in the BADJ (bronchio-alveolar duct junction), which is a transition region between the terminal bronchioles and the alveoli. Via in vitro culturing, a rare group termed bronchioalveolar stem cells (BASCs) was discovered in this region (Kim et al, 2005). These cuboidal cells co-express CC10, which is a marker of Clara cells, and SpC, which is expressed by type II alveolar cells. These cells are resistant to naphthalene, and are bipotential, giving rise to both Clara cells and alveolar type II cells. Finally, in the alveoli, injury models have shown that the type II cells may give rise to the type I cells (Evans et al, 1975) (Fig. 5 Summary).  15  2.3 Lung Mesenchymal Progenitor Cells For a long time the lung mesenchyme was thought to solely provide inductive properties on the lung epithelium to initiate branching morphogenesis. It was deduced that FGF9 activation within the mesothelium would activate and control FGF10 within the peripheral mesenchyme via FGFR2c, SHP2, Grb2, Sox, and Ras (Del Moral et al, 2006; Bellusci et al, 1997; Teft et al, 2002). However, now it has been understood that the mesenchyme expressing Fgf10 may operate as a progenitor cell population for peripheral airway smooth muscle. These Fgf10 expressing cells undergo transdifferentiation so as to express α-smooth muscle, under the control of SHH and BMP4, both of which are expressed in the distal epithelium. Furthermore, it has been observed that mesothelial-mesenchymal- epithelial-endothelial cross-talking leads to vascular progenitor function, which is critical in lung regeneration (Ramasamy et al, 2007). 3.Organotypic Models and Animal Xenografts A model design should be capable of recapitulating both the three dimensional organization and the differentiated function of an organ, while still allowing the possibility for experimental intervention. In order for the model to act in accordance to its organ of origin, it should be exposed to adequate biochemical cues from its surrounding environment. The individual conducting the experiment has the power to manipulate both the model system and the environment. Thus, a collection of experimental models with varying conditions may help deduce  16  molecular markers and targets, as well as the physiological relevance of the environment. It is important that the ECM and its molecules reflect the physiology of the tissue of origin (Schmeichel and Bissell, 2003). Furthermore, in order for a total organ environment to be reproduced in culture, adequate autocrine and paracrine interactions are pertinent. For this full physiological effect to take place, a mouse xenograft needs to be conducted with the three dimensional culture. By using a combination of 3D culture and animal xenograft strategies, it has become evident that the stroma is an important regulator for epithelial function (Schmeichel and Bissell, 2003). In regards to understanding lung development, many researchers have grafted lungs at different developmental stages in such environments as the subcutaneous region of mouse back and the renal capsule. Although both sites of implantation have proven adequate models for the development of human fetal lung tissue (Pavlovic et al, 2008), they each have their pros and cons. The subcutaneous implants provided ease in regards to the engraftment itself, and the size of the grafts were large; however, they would also be less uniform in the tissue they constructed (Pavlovic et al, 2008). On the other hand, the subcapsular grafts posed a more difficult procedure, during which a smaller graft size would be used; however, the results were uniform and similar to what is produced in the native lung, following an in-utero timeline (Pavlovic et al, 2008; Vu et al, 2003). From these studies it has been discovered that the blood  17  vessels in the lung graft develop from residential endothelial progenitor cells with no vascularization from the host itself. The embryonic lungs are able to stimulate host angiogenesis and recruit host vessel connections, which are a requirement for late fetal lung development to take place (Vu et al, 2003). In other words the host vessels do not actually vascularize the grafts, but the graft vessels are connected to the host circulation. Keeping with this concept, the same must be true for organoids of the tissue as well.  18  CHAPTER 2: AIM OF THE STUDY Over the past few decades the formation of the various pulmonary epithelial cell lineages have been studied in great detail (Rawlins et al, 2006), whereas the mesenchyme has been slightly neglected. Via the assistance of mouse lung injury models, such as the use of naphthalene and sulfur dioxide, combined with the labeling of proliferative cells and lineage tracing, lung embryonic epithelial progenitor cells and their control mechanisms have been determined (Rawlins et al, 2008), yet not fully understood. Furthermore, stem cell regulation in the adult lung has yet to be clearly appreciated. All in all, a comprehensive understanding of the lung progenitor cells in the epithelium and mesenchyme: where they are, how to activate them, and how they act, must still be ascertained. For instance, it is pertinent that work on the reciprocal interactions between mesenchymal, endothelial, and epithelial cells during development are conducted so that interplay between niche cells and stem cells in the adult tissue may be determined (Rawlins et al, 2006). As a result to such findings, effective cell- based therapies for the treatment of lung conditions may become a practicable possibility. However, in order for all these things to be determined a proper assay needs to be developed. That is what the work in this thesis is attempting to deduce; the simplest yet most informative system in testing lung cell lineages. The aim of the study was that if we were able to compose an adequate organoids system made  19  from dissociated epithelial and mesenchymal embryonic lung cells, then it would give rise to a mature lung tissue when grafted. If such a system was achieved, then with the use of different genetically modified mice and different methods by which to separate the assorted cells in the lung, a powerful assay will have been constructed that may test how these different signaling pathways and cells effect the construction of mature lung tissue. A vast array of approaches will be described in this work in regards to the organoids system itself, ranging from the injection of single cell suspension to the construction of matrigel supported spheroids, to the location of the implantations, ranging from the omentum to the kidney. At the end of explaining these multitude attempts, the reader will be introduced to the most promising, and elegant system, that will ultimately allow researchers to test the potential stem cellness of cells within the lung.  20  CHAPTER 3: MATERIALS AND METHODS 1. Mice All the animals used in this study were purchased from the Jackson Laboratories. In order to get embryos, we purchased time-pregnant C57BL6 mice. The mice mated in the evening and the following morning was considered embryonic day 0.5 or E0.5. The embryos we used were at E12.5 and E14.5. To be able to track our cells, we used actin-GFP mice, which constitutively express green fluorescent protein in all their cells. For graft surgeries, we used NOD/SCID mice to avoid graft rejection. All our animal protocols, including the survival surgeries, were approved by IAACUC at Children’s Hospital, Los Angeles. The mice were euthanized using CO 2 inhalation, in accordance to our approved protocol 31-08. 2. Lung Single Cell Suspension After euthanizing the pregnant females, the embryos were removed from the uterus and immediately placed on ice in Hank’s solution. The lungs of the embryos were then dissected out one by one using a Leica dissecting microscope to be able to visualize the lungs, and placed on ice in Hank’s solution. Once all the lungs were collected, the tracheas were removed and the lungs were washed in 5 mL Hank’s solution in a 15 mL conical tube to remove all the blood. The tube was then centrifuged using a Beckman GS-6R Centrifuge at 2000rpm for 5 minutes at 4˚C. The HBSS solution was then decanted and the lungs were suspended in 500µl 0.5% Collagenase+DNase. The lungs and  21  enzyme solution were then transferred to a 50ml conical tube containing a total of 30ml 0.5% Collagenase/DNase. The lungs were then vigorously cut into small pieces within the tube via repetitive scissoring movements with a sharp/sharp Student Iris Scissor (Fine Science Tools). The dissociated lungs were then placed into a 37°C 5% CO 2 incubating chamber for 50 minutes. Every 15 minutes the cells would be briefly vortexed, providing additional mechanical force to assist in the dissociation of the cells. Once the tissue was totally dissociated, the solution was centrifuged as previously mentioned, and the majority of the enzyme solution was decanted. To dissociate the remaining cell aggregates, the solution was pipetted up and down a few times, first by a p1000, then a p200, followed by 3 different syringe gauges (19g, 21g, 25g). Finally, the cells were passed through a 70µm nylon strainer, followed by a 40µm strainer. The final strained solution was centrifuged under the same settings as previously mentioned, and the remaining 0.5% Collagenase+DNase solution would be decanted. The cells were then washed in 5mL culture media containing 10% FBS /DMEM/F12 + 1% Penicillin/Streptomycin (P/S) three times. After the final wash, the cells were resuspended into a known amount of culture media, and the cells were counted.  22  3. Counting the Cells After pipetting the cell solution up and down multiple times to assure that the cells were dispersed uniformly throughout the solution, 0.5µL cells were added to a Trypan Blue (SIGMA)/PBS solution at a dilution factor of 100x. About 15µL of this Trypan Blue-cell solution were then gently expelled into a hemacytometer (Hausser Bright-Line). After counting the total number of cells located in the four large outer corner squares, the final cell concentration per milliliter was determined by taking the total number of cells counted in the four squares and multiplying that by 2500 and the dilution factor (100). After determining the number of cells within a milliliter, the actual total number of cells could be deduced. Once the total number of cells was known, they would then be suspended into the appropriate volume of culture media based on the specifications of each model. 4. Labeling the Cells Cells from WT, non-GFP mice were dyed using a Cell Tracker CM-Dil (Invitrogen) stain. One vial of the Cell Tracker Cm-Dil was diluted with N’N’- Dimethylformamide (Acros Organics) according to the manufacturer’s protocol. The microcentrifuge in which the suspended, washed cells were located would be centrifuged using the Eppendorf Mini Spin Plus, at 2000rpm for 5 minutes. The culture media would be decanted and the cells would be resuspended into 500µl PBS (GIBCO). 10µl of the CM-Dil dye was then added to the cells (this  23  amount could be added for up to 10 million cells). The cells were then incubated in a 5% CO 2 37˚C incubator for 5 minutes. After that, the cells were placed for 15 minutes inside of a 4˚C refrigerator. Finally, the cells were washed twice in PBS, and resuspended in the adequate volume of culture media based on which model system was being used. 5. Cell Culture Conditions A. Cells Directly Atop of Carbon Filter 1ml of culture media was pipetted into each well of a 4-well plate (Nunc). A 13mm diameter 8µm Nuclepore Track-Etch Membrane (Whatman) was then placed atop the medium. 15µl of the final cell suspension, containing approximately 2*10 6 cells, was slowly released atop a single carbon filter. The 4-well plate was then placed into a 5% CO 2 37˚C moist environment incubator. B. Cells Mixed with Matrigel Atop of Carbon Filter The carbon filter was set up as previously described in a 4-well plate. 5µl Basement Membrane Matrix (Growth Factor Reduced (GFR), Phenol Red- free, 10 ml *LDEV-Free) (BD Biosciences) was pipetted, and spread atop each carbon filter. The plate was then placed into the 37˚C incubator for about half an hour, allowing the matrigel to polymerize. After the matrigel polymerized, we added 10 µL of a mixture matrigel and culture media 3:2  24  containing a total of 2*10 6 cells to each well. The plate was then placed in the incubator for 1-7 days. C. Hanging Droplet 1ml of culture media was placed into each well of the 4-well plate (Nunc). The total cells were brought to a concentration of 500,000 cells per 100µl. 20µl of cells were gently expelled onto the inner portion of the lid, atop of a dot located on the opposite side, to identify the placement of the cells. The lid was then flipped over, closing the 4-well plate, and enclosing the droplets inside of a well. This allowed the droplets to hang without getting in contact with the medium in the well. The culture time varied for each condition depending on the cell growth and the outcome of the culture. It could go from 24hrs up to 7 days. 6. Embedding. A. In Vitro Samples The samples were fixed in 4% PFA for 1 hour at room temperature and then washed 3 times 10 min in PBS (1x). The samples were then gradually dehydrated through increasing ethanol gradient (40%, 70%, 80%, 90% and 100%) then cleared in xylene. The samples were processed in each solution 2x for 15 minutes, except for 70% EtOH and  25  100% EtOH, each for which the samples were washed 2x for 30 minutes. The samples were then incubated in 50:50 paraffin/xylene bath at 64°C for one hour and transferred in 100% paraffin in new glass vials where they were left 3 hours prior to being set into a cast. Sections (5 µm) were cut using a LeicaRM2135 microtome and mounted on slides for histological analysis. B. In Vivo Samples The samples were fixed in 4% PFA for 3 hours at room temperature and then washed 3 times 10 min in PBS (1x). The samples were then gradually dehydrated through increasing ethanol gradient (40%, 70%, 80%, 90% and 100%) then cleared in xylene. The samples were processed in each solution 2x for 30 minutes, except for 70% EtOH and 100% EtOH, each for which the samples were washed 2x for 1 hour. The samples were then incubated in 50:50 paraffin/xylene bath at 64°C for 2x for 30 minutes and transferred in 100% paraffin in new glass vials where they were left overnight prior to being set into a cast. Sections (5 µm) were cut using a LeicaRM2135 microtome and mounted on slides for histological analysis.  26  7. Stainings Sections were cleared with two changes of xylene and hydrated with a successive gradient ethanol series (100%x2, 95%, 80%, 70%, 50%, and 30%) to water. The slides were washed for 10 minutes in each solution. Further steps would change based on the type of stain being conducted. A.Hematoxylin and Eosin (H&E) After bringing the slides to water, Harris Hematoxylin (Electron Microscopy Sciences) was added and left on the slides for ten minutes. The slides were then washed in running tap water for ten minutes, and then washed in 70% EtOH for another ten minutes. The slides were dried off, after which Eosin (Sigma-Aldrich) was then placed atop the sample for two minutes. After the two minutes, the slides were immediately washed in 70% EtOH for ten minutes. The slides then continued to follow the dehydration process, being placed in the following sequential solutions, for ten minutes each solution: 95% EtOH, 100% EtOH, and twice in histochoice. Finally, the slides were mounted using, Mounting Medium Xylene (Protocol). This stain is conducted to show the histology of the sample. In result, the nuclei stain blue/purple from the hematoxylin, whereas eosin, which is an acidic fluorescent red dye, stains the basic structures of the cells, which are located in the cytoplasm.  27  B. Periodic Acid Schiff (PAS) PAS staining was performed according to the manufacturer’s protocol (Sigma 395B). The method is used for staining structures containing a high proportion of carbohydrate macromolecules. In result, the glycogen- rich areas stain a bright magenta color, whereas the cell nuclei stain purple from hematoxylin. C. Alcian Blue Staining After the slides were brought to water, they were covered in Alcian Blue solution for 30 minutes. The slides were then washed in running tap water for 2 minutes, followed by a rinsing in distilled water for another 2 minutes. The slides were then counterstained with nuclear fast red for another 5 minutes, and from there were dehydrated with a series of increasing ethanol concentrations, and mounted. It is a dye that stains acid mucopolysaccharides and glycosaminoglycans a blue color. These structures are often found in goblet cells within the cartilage of the trachea and bronchi. D. DAB After bringing the slides to water, the next step was based on whether the antibody required antigen retrieval or not. Antigen retrieval was performed by boiling the samples for 20 minutes in Na-citrate buffer (10 mM ph6.0).  28  After completely cooling the slides down gradually, the slides were washed in 0.1% TBST 3x, 10 minutes each wash. From this point on the steps were the same for all slides. The tissue was then incubated in 0.3% H 2 O 2 (2.5ml 30% Hydrogen Peroxide by Fisher Science + water until 50 ml) for twenty minutes. The slides were then washed again in 0.1% TBST and covered in 10% normal goat serum blocking solution (Invitrogen) for at least one hour. Finally, the slides were incubated overnight at 4˚C with the primary antibodies at requested concentrations. The following day the signal was visualized with the DakoCytomation EnVision+Dual Link System-HRP (DAB+) kit as recommended by the manufacturer. After developing the color, the slides were then dehydrated with a series of increasing ethanol concentrations, and mounted. The Photomicrographs were taken using Leica DMRA microscope with a Hamamatsu Digital Camera CCD camera and Zeiss Axioplan, Germany. E. Immunofluorescence After bringing the slides to water, the next step was based on whether the antibody required antigen retrieval (Same steps as with DAB were followed). The slides were then covered with blocking solution for at least one hour. Incubation of primary antibodies for immunofluorescence was  29  performed in TBS with 3% Bovine serum albumin and 0.1% triton overnight at 4°C. The following day the slides were incubated for one hour with the appropriate secondary antibody. Secondary antibodies were from Jackson Immunoresearch, and were all used at a 1:200 concentration. After the hour incubation, the slides were washed in 0.1% TBST and mounted using DAPI (Vector Laboratories). In order to maintain the longevity of the slides, they would be kept in a 4°C refrigerator when not in use. Table 1: List of Antibodies Antibody Animal in Which Generated Dilution Antigen Retrieval Company αSMA Mouse 1:100 No Dako Cytomation CC10 Goat 1:100 No Santa Cruz CCSP Rabbit 1:200 No Seven Hills Collagen II Mouse 1:200 Pepsin Millipore E-Cadherin Mouse 1:200 Microwave in Na+ Citrate Buffer BD Biosciences  30  Table 1: Continued Antibody Animal in Which Generated Dilution Antigen Retrieval Company GFP Mouse/Rabbit 1:500/1:500 Microwave in Na+ Citrate Buffer (both) Abcam Id2 Rabbit 1:50 Microwave in Na+ Citrate Buffer Cal Bioreagents p63 Mouse 1:100 Microwave in Na+ Citrate Buffer Santa Cruz Sox2 Rabbit 1:200 Microwave in Na+ Citrate Buffer Seven Hills Sox9 Rabbit 1:100 Microwave in Na+ Citrate Buffer Santa Cruz SpC Rabbit 1:150 No Seven Hills TTF1 Mouse 1:200 Microwave in Na+ Citrate Buffer Seven Hills T1α Syrian Hamster 1:200 Microwave in Na+ Citrate Buffer Developmental Studies Hybridoma Bank  31  8. Kidney Capsule Surgery The animal would be anesthetized with the use of isofluorane and placed on its side. After adequately cleaning and shaving of the area, a 2cm long incision would be made approximately 3cm from the base of the tail. The skin would then be separated from the body wall via blunt dissection. The body wall would then be cut, and squeezing the incised area would pop the kidney out. With either fine forceps or a needle, the capsule would be lifted from the kidney parenchyma, and with a blunted glass rod, a small hole would be produced between the capsule and parenchyma. The sample would then be picked up via forceps and placed directly in the location where the hole was constructed. Once the sample was placed inside the kidney capsule, the kidney would be placed back into the body cavity and both the body wall and outer skin would be sutured.  32  CHAPTER 4: RESULTS 1. E12 Lung Implant It has been previously shown that when E12 embryonic lung rudiments are grafted underneath the renal capsules of either syngeneic or immunodeficient mice for 8 days, the epithelium develops extensively and appears to go through all stages of morphological development (Vu et al, 2003). From these previous results, it was determined that the development of the lung renal capsule graft follows in utero lung development. Being that the experiments to follow were to be based on the results from this work, it was critical to first conduct the same experiment and assure that the results were replicable. Comparing the results we obtained to those of the previous work, it was evident that our lung implant did not advance in the same manner. When lungs are implanted underneath the kidney capsule, a large white expansion is to be observed in the gross histology. Often times, the growth is so vast that the implantation itself may equal the size of the kidney in which it was implanted. However, this extensive expansion was not observed in our implantations (Fig. 6A): the growth of an iridescent white patch beneath the kidney capsule was a fraction of the size of what Vu and colleagues would grow, suggesting our attempt obsolete.  33  Yet, upon sectioning this specific region, and conducting an H&E stain (Fig. 6B & 6C), it was apparent that this was where the lungs had been implanted and that their structural development had advanced. Numerous airway structures (bronchiole-like and bronchi-like structures) started to develop, with cartilage also growing at the perimeter of some of these structures, showed by collagen II staining (Fig. 7).  34  In order to determine where in the kidney the lungs were implanted, we decided to use actin-GFP E12 lungs and implanted them underneath the kidney capsule of a NOD/SCID mouse. As such, we would then be able to determine the location of the lungs via the use of an anti-GFP antibody when conducting immunoflourescence (Fig.9). Upon sacrificing the animals at different time points, and analyzing their development via immunohistochemistry, we were able to further support the idea that the lungs had advanced in development, as suggested by the structural data of the H&E. Compared to E14.5 lung expression, progenitor cell marker expression changed over implantation time (Fig.8): Sox2 expression decreased over time, being practically non-existent in the post 30 day implantation, whereas both Sox9 and Id2 appeared to increase (Fig.8: compare C to G and K). Thus, indicating that the E12 lung developed passed the stage of an E14 lung.  35  Asides from analyzing the progenitor cell markers, we also checked epithelial and mesenchymal markers to observe changes in cell differentiation. Both CC10 and SpC were present throughout the different sacrificial time points. An increase of both p63 (marker for basal cells) and T1α (marker for epithelial alveolar type I cells) expression truly showed that the appropriate differentiation was taking place (Fig. 9). Figure 8: Change in Expression of Lung Epithelial Progenitor Markers From E12 Lungs Subcapsular Implantation Post 1 Month A-C: Change in Sox2 expression in E12 lung post 1 month subcapsular implantation. Starts off as specific, nuclear staining and by post 1 month is no longer specific. E-G: Change in Sox9 expression in E12 lung post 1 month subcapsular implantation. Starts off as non-nuclear and then by post 30 days nuclear staining intensifies. I-K: Change in Id2 expression in E12 lung post 1 month subcapsular implantation. D, H, L: Control staining for Sox2, Sox9, and Id2 (respectively) in E14 lung.  36  Based on these results, it may be determined that a subcapsular E12 implant post 30 days allowed the lung to differentiate and advance at least to the point of a E17.5 lung where basal cells and type I pneumocytes are present.  37  2. E14.5 Lung Single Cell Suspension Plated Upon a Carbon Filter. After seeing that the E12 lung was able to grow past the point of an E17.5 lung post 1 month implantation, we wanted to try to construct a lung organoids system able to respond in a similar fashion. For this we used single cell suspension obtained from E14.5 lungs. Post 24 hours incubation, the cells already started aggregating (Panel 1 of Fig. 10), departing their initial condition of being in single cell suspension. At this time, the aggregations lacked structure, and appeared to rally somewhat haphazardly. However, by 72 hours (Panel 4 of Fig. 10), it becomes apparent that the aggregation of these cells was done resolutely, with the formation of branch- like/tubule-like configurations (reminiscent of the pseudoglandular stage). Furthermore, at some regions it also appears as though saccule-like structures are forming, with the presence of secondary septa (Fig. 11C).  38  Via immunohistochemisry, one may clearly see that many of the cells containing lung specific markers are able to aggregate and construct structures similar to those located in the lung. For instance, the CC10 (+) cells are the Clara cells, which are located in the proximal epithelial tissue, contributing to the formation of proximal airways, such as bronchi (which starts to be expressed at E16). Within the organoids, we observe that the CC10 (+) cells were able to localize in a single location (Figure 11B and C) and construct an airway-like structure, similar to those formed by the Clara cells in the lung. As for SpC, it represents the  39  distal lung, being expressed in the type II cells of the alveoli. These cells were also able to localize. The αSMA positive cells associated themselves around the lumen, which is how they are also localized within the native lung being that they are first to appear in the proximal bronchial tubes during the glandular/canalicular stage of development. Asides from expressing lung specific markers, the organoids were also able to structurally develop into lung-like structures, aggregating into lumen with the structure of secondary septa (Fig. 11C) further supporting that the cells are able to reconstruct themselves similarly to their native lung. All in all, the in vitro aspect of this system proved to be fairly promising, however things went awry when we introduced the in vivo phase. Each time we attempted transferring the carbon filter with the organoids, the branching network would fall apart. Thus, adding a greater mechanical force, as is provided by implantation, would not be a possibility. What this suggested was that some type of scaffold/ support system needed to be introduced to our model to assure the integrity of the organoids.  40   41  3. E14.5 Lung Single Cell Suspension Plated With Matrigel Upon Carbon Filter Based on the lack of structural support in the previous attempt, we decided to incorporate matrigel with the single cell suspension, to act as a type of scaffold and provide mechanical support for the cells. There was significant gross advancement of the cell aggregation over time. Due to the spatial and a mechanical support provided by the matrigel, the cells were able to form 3- dimensional spheroid type of aggregations over the time (Fig. 12). By 168 hours, the cells constructed well-defined spheroid structures that were capable of being transferred from their location of incubation to a potential site of implantation (Fig. 12N).  42   43  When staining for the progenitor cell markers Sox2, Sox9, and Id2, the organoids stained positively for all three (Fig. 13). This was expected, being that E14 lungs stain positive for each of these markers (Fig. 8 panel 4). Interestingly, each cell spheroid was able to stain positively for all three of the markers.  44  When looking at epithelial marker expression within these spheroids, TTF1, CC10 and SpC were present (Fig. 14). The markers that did not appear to be as pronounced were p63 and T1α, as was expected (Fig. 14). The reason for this is because although it was expected that the cells would be able to form aggregates upon incubation, not much was expected in terms of differentiation. In other words the cells did not advance in their embryonic timeline, but rather remained at the point of E14.5-E16.5. This is probably due to the lack of any type of outside inducing signal that would allow the cells to continue in their structural formation. This clearly validated that an in vivo portion was required for what was wished to be accomplished.  45  4. E14.5 Lung Single Cell Suspension Plated With Matrigel and Implanted Subcutaneously Knowing that some type of vascularized environment was needed to allow the organoids to differentiate past the E14.5 stage, we decided to take the aforementioned matrigel spheroids and implant them subcutaneously. Post 3 days incubation, the spheroids were subcutaneously implanted on the dorsal region of a NOD/SCID mouse. The mice were then sacrificed at different time points, so as to observe how growth and differentiation continued over time. The H&E stains (Fig.15) show that post implantation, the organoids continued to aggregate and grow, which is visible when comparing the structures that formed when the cells were solely in matrigel to those of the subcutaneously implanted organoids (Fig. 13 and Fig. 15). When solely incubated in matrigel the resulting structures were very frail and somewhat flimsy. Each spheroid was uniform in appearance and would express the same markers. Once the cells were implanted subcutaneously, the aggregated structures appeared more solid and lung-like. Some regions represented a bronchiole-like formation, whereas others seemed somewhat alveolar-like. Grossly comparing the structure of the in vitro organoids to that of the in vivo structural advancement was evident, but immunohistochemistry proved minimal differentiation.  46  Progenitor cell marker expression, Sox2, Sox9, and Id2, remained consistent between one week implantation through four weeks implantation (Fig.16). We expected the expression of Sox9 and Id2 to remain consistent throughout the implantation time frame, while Sox2 diminished over time. These expectations were based on the in-utero development that an implanted E12 lung follows when subcapsularly implanted. This suggested that the subcutaneous environment was not providing the adequate conditions for the cells to differentiate and advance into further lung development.  47   48  The epithelial marker data (Fig. 17) further supported that the subcutaneous environment did not provide adequate conditions for cell differentiation. The αSMA/CC10 panel (Fig. 17A-C) shows that as time progressed the CC10 looked as if it deteriorated. The same results may be seen with the SpC and p63 (Fig. 17D-F and Fig. 17G-I respectively), whereas with the T1α, there was an increase in signal over time, but the majority of the structures staining positive were red blood cells. Combining the results from both the epithelial and progenitor marker data, it was evident that not only did differentiation and growth of the subcutaneously implanted matrigel organoids not progress, but, there had been deterioration.  49   50  5. E14.5 Lung Single Cell Suspension Placed in Polymer and Implanted in Omentum Another approach we attempted was the use of a polymer scaffold that was implanted within the omentum of a NOD/SCID mouse. The E14.5 lung fresh single cell suspension was seeded into the opening of a polymer scaffold. Lung cells continued to be added until the scaffold was covered (Fig. 18A). The polymer was directly implanted into the omentum of the animal post seeding the cells. The implant was left inside the animal for one week, after which the results were once again analyzed via immunohistochemistry.  51  Structurally, at a microscopic level, the single cell suspension was able to aggregate and form tubule/bronchiole-like structures (Fig. 18B). This was fairly interesting being that no prior structure gave rise to this growth, as with the matrigel and the spheroids that had formed during the time of incubation. This showed that the cells obtain the ability to aggregate and construct lung-like structures, whilst implanted, with no prior organization from which to build upon. However, although there was structural progress cell differentiation did not occur. The expression of the progenitor cell markers in the polymer implanted organoids showed that only Sox2 was clearly present (Fig. 19A). Because there was no positive staining for either Sox9 or Id2 (Fig. 19B and C respectively), the implanted cells were incapable of constructing any distal based lung-like structures.  52  These results were further supported by the expression of the epithelial cell markers (Fig.20). The robust expression of CC10 clearly shows that proximal structures were compiled upon cell aggregation (Fig. 20A). Whereas the abundance of SpC suggests Type II alveolar cells were present and that primary buds developed. The lack of T1α implies that Type II cells did not differentiate to form Type I pneumocytes. Furthermore, the lack of p63 indicates that the bronchi-like structures have also not developed in their entirety. Thus, this system proved to be ideal superficially, but did not provide the molecular profile required by the system we were attempting to achieve.  53   54  6. Fresh E14.5 Lung Single Cell Suspension Injected Into Kidney Capsule Based on the fact that the E12 lungs developed fairly well when placed underneath the kidney capsule, and seeing that the cells were able to aggregate fairly well when implanted into the omentum as a fresh single cell suspension, we combined these two methods by injecting the fresh single cell suspension directly into the kidney capsule. Immunohistochemistry analysis proved this to be a poor supposition. Looking at the counterstain, and the location of the positively stained progenitor cells (Fig. 21), it was clear that the kidney capsule did not provide the adequate confined space we were seeking. Failing to take into consideration that the kidney capsule was continuous and that the cells were suspended in 10% FBS, we disregarded that the cells had the ability to flow throughout the kidney capsule and not remain in the confined space of injection. In result the cells were incapable of aggregating and remained as a single cell suspension.  55  All the epithelial markers (Fig. 22) lacked any true staining, furthering the fact that neither aggregation, nor differentiation took place. However, due to the fact that the cells remained in single cell suspension, this proves that the organoids are a result of aggregation as opposed to colonization.  56  7. E14.5 Lung Single Cell Suspension Plated with Matrigel and Implanted Within the Kidney Capsule We then implanted the matrigel spheroids underneath the kidney capsule. These structures had already showed a fair deal of promise in terms of structure and growth on their own, so the vasculature of the kidney would just allow improvement. In order to assure that we were obtaining the optimal conditions under which to implant these matrigel spheroids, we decided to implant them post 7 days incubation. This was the time during which the most robust growth in the spheroids was observed. In the end, the results were more promising than the subcutaneous implant, but not successful enough to become the ultimate  57  system. Comparing the counterstain of the post 3 day implant to that of the post 14 day, it is noticeable that bronchiole-like structures started to form post 3 days, and became more defined in the post 14 day sections (Fig. 23); showing an improvement in growth. In regards to the progenitor cell markers, Sox2 was present at both implantation time frames (Fig. 23C-D), whereas Sox9 and Id2 were not as defined. The Sox9 staining of the post 3 days implantation was unspecific, localizing in the cytoplasm when it is a nuclear staining (Fig. 23E). Although, this type of unspecific staining was also evident in the post 14 day implantation, there was also fair specific nuclear staining present (Fig. 23F). In continuance, comparing Id2 expression in the post 3 days implantation to that of the post 14 days implantation, the nuclear staining in the post 3 days is very scattered and ill defined whereas in the post 14 days all of the nuclei are solidly stained (Fig. 23G-H). This implied an increase in development and differentiation the longer the organoids remained under the kidney capsule.  58   59  This promising thought was mitigated upon observing the expression of the epithelial cell markers (Fig. 24). There was no αSMA at the circumference of the CC10 (+) cells, indicating the bronchi-type structures were failing to form (Fig. 24C-D). This was further supported by the fact that very little p63 expression was observed in either sample (Fig.24 G-H). Additionally, SpC (+) cells were present in the post 3 day implantation but practically non-existent in the post 14 day (Fig. 24E-F). This implies that Type II alveolar cells were incapable of surviving an extensive implantation period. This was further encouraged in examining the expression of T1α, being that there was slight expression in the post 3 day implantation, but none in the post 14 day (Fig.24 I-J). This implies that without the presence of Type II alveolar cells in the post 14 day implantation, differentiation of these cells could not take place so that Type I alveolar cells may arise, and as result no T1α could be produced. As had been the circumstance with the matrigel’s subcutaneous implant, the differentiation of the cells seemed to revert, or perhaps desist altogether.  60   61  8. E14.5 Lung Single Cell Suspension Plated as Hanging Droplet Convinced that underneath the kidney capsule was the ideal place in which to conduct the in vivo portion of the organoids system, the organoids themselves had to be improved. The cells needed to remain in a primitive yet aggregated state, without the introduction of foreign substances, such as matrigel. Thus, it was evident that hanging drops would be the idyllic setup. The droplets were analyzed at different time points, which unfolded interesting results. By post 24 hours incubation, spherical aggregates had already developed at the bottom of the droplet (Fig. 25A). At post 4 days incubation, there was a more intricate aggregation, constructing airway-like structures, as observed in the lung. Both hanging droplets were able to stain positively for Sox2 and Sox9, indicating that they still had the ability to potentially differentiate and provide both proximal and distal structures (Fig. 25). Although the results provided by the progenitor marker stains were fairly basic, those observed in the epithelial markers were quite intriguing (Fig. 26).  62   63  Comparing the TTF1 results of the two incubation periods, at post 1 day there was very little specific TTF1 staining, whereas at post 4 days the presence of the transcription factor was fairly abundant (Fig. 26A-B). Looking at the staining of αSMA with CC10, it is quite evident that in the post 1 day section, no definite structures have yet formed. The positively stained cells are spread throughout the organoid, having yet aggregated into the desired lung-like structures. Then, at post 4 days, it is quite evident that the staining is more abundant and organized (Fig. 26D). The αSMA is located at the periphery of the airway-like structures that were constructed. This same general staining pattern may also be observed in the SpC stain when comparing the post 1 day to the post 4 day incubations (Fig.26 E-F). All in all, the structure and localization of the epithelial markers at post 4 days incubation closely follow the pattern observed in the native lung, whereas in post 1 day everything appears to be very scattered.  64   65  9. E14.5 Lung Single Cell Suspension Plated as Hanging Droplet and Implanted Underneath the Kidney Capsule Having determined a system that was solely composed of cells, and easily transferrable without compromising the aggregates, we wanted to see how it would perform when transplanted underneath the kidney capsule. After having produced the hanging droplet and incubated it for one day, we took the aggregated cells and implanted them under the kidney capsule for a month. A series of immunohistochemistry was conducted to determine development in terms of differentiation. Before analyzing the specific stainings in terms of what they represented, the varied structures that were constructed were noteworthy to observe. In Figure 27A a bronchi-like structure seemed to be forming. Then, in Figure 27B, the structures appear more distal-like, with some saccule-like structures surrounding. The structure observed in Figure 27C appears more like a bronchiole-like structure, and in Figure 27D branching structures, similar to the airways spreading from the proximal region to the distal, such as when the bronchioles move to the alveoli may be observed. Asides from these structural features, one may observe that, whereas Sox2 expression appears to be fairly nonspecific (Fig. 27B), both the Sox9 and Id2 appear to be fairly specific (Fig. 27C-D). Furthermore, it is interesting that these markers stained positive in the structures representative of where they would stain in the natural lung itself (the distal structures).  66  Epithelial marker expression further supported that differentiation took place. Although the CC10 staining appears to show the cells being clumped together (Fig. 28A), the rest of the markers, such as SpC (Fig. 28B), showed to be localized in their supposed adequate structures. Both p63 and T1α staining was substantial (Fig. 28C & D respectively), emphasizing that the hanging drop aggregates were able to differentiate and give rise to multiple basal cells in bronchi-like regions and type I alveolar cells, in areas that had saccule features.  67  These results ultimately suggest that this model is ideal. It is the most simple and elegant method attempted, eliminating all foreign materials, ultimately allowing the cells to behave more naturally. Furthermore, it allowed the desired differentiation and growth of cells to take place so that lung stem-cellness could now be tested.  68  Table 2: Summary of Results and Conclusions Markers Expressed Progen itor Differentiation Type of Manipulation Structures Formed Sox2 Sox9 Id2 TTF1 SpC αSMA CC10 p63 T1α Conclusions In Vivo: Subcapsular Implant of E12 Lung Post 1 Month; n=4 Trachea (Cartilage); Proximal-like airways (bronchi- like structures); Some bronchiole-like structures; Alveolar- like saccules - + + N/A + + + + + Lung developed at least up to the saccular stage (E17.4-P5); Although not properly organized, all structures resident in native lung were able to develop and differentiate In Vitro: E14.5 Lung Single Cell Suspension Plated Upon Carbon Filter Post 3 Days; n=6 2-D structures; Lumen formed due to aggregating and branching of cells; 2° septa formed N/A N/A N/A N/A + + + N/A N/A Lumen and branching structures; Cells lacking support and thus not transferrable In Vitro: E14.5 Lung Single Cell Suspension Plated with Matrigel Upon Carbon Filter Post 1 Week; n=4 3-D spheroids; Airway-like structures + + + + + + + - - Matrigel contributed as scaffold to spheroid organoids, making them easier to transfer; Structures were fairly homogenous and uniform; Lacking in development and differentiation  69  Table 2: Continued Markers Expressed Progen itor Differentiation Type of Manipulation Structures Formed Sox2 Sox9 Id2 TTF1 SpC αSMA CC10 p63 T1α Conclusions In Vivo: E14.5 Lung Single Cell Suspension Plated with Matrigel and Implanted Subcutaneously Post 1 Month; n=4 Proximal-like airways (bronchi-like airways); Some bronchiole-like airways; No alveoli- like structures + + + + + - - + - Progenitor and epithelial marker expression appears to deteriorate over time; No advancement in structure formation; Limited differentiation and development; Inhibiting effects In Vivo: E14.5 Lung Single Cell Suspension Placed in Polymer and Implanted in Omentum Post 1 Week; n=3 Proximal-like airways (bronchi- like airways); Some bronchiole-like airways; No alveoli- like structures + - - N/A + + + - - Scaffold held cells in place; Some p63 and T1α start to express; Structural development and some differentiation In Vivo: Fresh E14.5 Lung Single Cell Suspension Injected into Kidney Capsule Post 1 Month; n=4 None + + + + + - - - - No structures formed; Cells remained in single cell suspension; No development nor differentiation  70  Table 2: Continued Markers Expressed Progen itor Differentiation Type of Manipulation Structures Formed Sox2 Sox9 Id2 TTF1 SpC αSMA CC10 p63 T1α Conclusions In Vivo: E14.5 Lung Single Cell Suspension Plated with Matrigel and Implanted within the Kidney Capsule Post 2 Weeks; n=8 Proximal-like airways (bronchi- like airways); Some bronchiole-like airways; No alveoli- like structures + + + + - - + - - Structural development increases with implantation time; Epithelial marker expression decreases over time = Deterioration; Some type of inhibiting effect is taking place; Comparing to subcutaneous implanted matrigel spheroids, implies that matrigel may be the inhibitory component In Vitro: E14.5 Lung Single Cell Suspension Plated as Hanging Droplet Post 4 Days; n=3 Proximal-like airways (bronchi- like airways); Some bronchiole-like airways + + N/A + + + + N/A N/A Aggregates start forming by 1 day but more advanced airways form at 4 days; Transferrable and no foreign substance affecting the cells’ interactions with on another  71  Table 2: Continued Markers Expressed Progen itor Differentiation Type of Manipulation Structures Formed Sox2 Sox9 Id2 TTF1 SpC αSMA CC10 p63 T1α Conclusions In Vivo: E14.5 Lung Single Cell Suspension Plated as Hanging Droplet and Implanted Underneath the Kidney Capsule Post 1 Month; n=1 Proximal-like airways (bronchi- like airways); Some bronchiole-like airways; Distal-like airway and alveolar- like saccules + + + N/A + + + + + Both proximal and distal-like airways formed (Structural Development); Markers expressed indicate differentiation; No foreign substance implanted to alter functionality of cells; The most promising results  72  CHAPTER 5: DISCUSSION 1. Progenitor Cell Expression Sox2, Sox9 and Id2 are indicative of the progenitor cells located within the lung. They are each expressed at different developmental time points during lung organogenesis, and are localized in different regions. Sox2 is an Sry related HMG box protein that is expressed in non-branching regions, and absent in branching regions (Gontan et al, 2008). Obtaining this role suggests that down- regulation of Sox2 is required for airway epithelium to respond to branch inducing signals, which usually occurs around E15. Sox9 is another Sry related HMG box protein that is first expressed in the lung at around E12.5 and continues to be present until adulthood (Perl et al, 2005). However, unlike Sox2, as development takes place, Sox9 tends to increase its expression and is localized more within the distal structures, for example around the bronchiole-alveolar duct. Id2 is located at the distal tips of what is believed to be undifferentiated epithelial progenitor cells. The tip cells can self-renew and contribute descendents to all epithelial cell bronchiolar and alveolar compartments (Rawlins et al, 2009). Being that it is expressed in the bronchioles, branching must first take place for its expression to be present. Therefore, in regards to the model systems with which we experimented, it was suggested that differentiation was taking place when Sox2 was diminished and expression of Sox9 and Id2 was elevated. A good example of such results was when we conducted the subcapsular implantation of the E12 lung. Figure 2 clearly shows that as the implantation time  73  increased, the Sox2 expression gradually decreased whereas the Sox9 and Id2 expression increased. By post 30 days implantation, it was evident that Sox2 was practically nonexistent, whereas Sox9 and Id2 expression was more specific and abundant. According to Vu and colleagues, the embryonic lung follows in- utero development when implanted underneath the kidney capsule, and therefore by post 30 days implantation should be significantly passed the E12 stage, with branching taking place. Using the E12 lung as a control, we were then able to compare its results to those of our model systems. Based on this understanding of Sox2, Sox9, and Id2 expression, and having a control (E12 lung subcapsular implantation) with which to compare the results from each of the models, it was apparent that the majority of the systems did not allow adequate differentiation of the cells to take place. Both the matrigel spheroids and the subcutaneous implant of the matrigel spheroids, stained for all three progenitor markers with the same intensity (Fig.13 and Fig. 16 respectively). This indicated that the structures did not advance past the point of an E14.5 lung. The subcapsular implanted matrigel spheroids showed potential promise, in that there was an increase in Sox2 expression and a decrease in Id2 expression, but Sox9 showed a decrease as opposed to an increase, indicating that there was some flaw in the differentiation. Finally, implantation of the hanging drop showed the most promise, and was most in sync with the results provided by the control E12 lung subcapsular implantation. In Figure 22 it is  74  apparent that Sox2 is losing its specificity within the nucleus, providing a more nonspecific stain, whereas both Sox9 and Id2 appear to be staining structures within the system that appear to be similar to distal structures within the lung. Basically, it appears as though the markers are actually staining the structures that are representative of what they would be staining in the native lung. This is idyllic because the system is not only able to provide structures represented in the native lung, but is also able to adequate markers in these newly developed structures. 2. How p63 Indicates Differentiation p63 is a marker located in the basal cells, which are fairly undifferentiated cells that make up about 30% of the pseudostratified mucociliary epithelium in the lung (Rock et al, 2009) . Within Humans, these cells are located throughout the airways, reaching such distal structures as the small bronchioles. However in mouse, p63 positive cells are solely located in the most proximal regions, such as the trachea and some of the bronchi (Rock et al, 2009). The presence of p63 indicates commitment of the pulmonary epithelium to tracheal and bronchial airway cell lineages. This usually occurs during the transition from pseudoglandular (E11.5-16.5 days) to canalicular (E16.6–17.4 days) phases of lung development (Hong et al, 2004a). For this reason very little p63 expression is seen post 6 days implantation of the E12 lung, but a significant amount more is observed post 30 days implantation (Fig. 8). Understanding when and where  75  p63 is expressed in the lung helps reveal when differentiation and growth of the lung is taking place. For this reason we would often look at the expression of p63 in the models to observe whether differentiation took place, or not. 3. How T1α Indicates Differentiation T1α is a protein that is expressed by alveolar type I cells. For these type I pneumocytes to exist, they must have differentiated from the alveolar type II cells. This generally takes place in the saccular stage of lung development, which is from E17.5 to P5. By post 30 days implantation of the E12 lung, it is clear that these type I alveolar cells are present (Fig. 8L) and that they are forming potential alveolar like structures. Just as was the case with p63, by knowing when and where T1α is expressed in the lung, it helps reveal when differentiation and growth of the lung is taking place. Thus, in conjunction with p63, the expression of T1α our models provides the ability to deduce whether differentiation has taken place, and if the cells are capable in forming mature lung tissue. 4. Cell Aggregation as Opposed to Proliferation Although the matrigel spheroids were not able to provide any useful information regarding progenitor markers and differentiation, they were able to give an explanation as to how the single cells were interacting with one another. What we noticed was that these cells were all able to localize in a single region, meaning  76  that one spheroid had all three positive cell types present (Fig.13). Being familiar with where these positively expressed cells are located in the lung (Sox2 is proximal whereas Sox9 and Id2 are distal), we believe that this supports the idea that these cells are aggregates, and not a product of cell proliferation. If the spheroids were constructed as a result of proliferation, there would be very little chance that the cells would be able to proliferate and differentiate whilst incubating, with no external signals. Therefore, being that all three progenitor cell transcription factors are found in an E14.5 lung, for the proximal cell types to construct a spheroid with distal cell types, it is likely that these cells aggregated. This was also evident in the hanging drop setup (Fig. 25), being that Sox2 and Sox9 positive cells appeared to be localized in the same areas. Observing the epithelial marker expression in any of the in vitro systems could further support this. Whether in seeding directly atop the carbon filter (Fig. 11), seeding the cells with matrigel (Fig. 14), or forming a hanging drop (Fig. 26), the cells were always able to show expression of CC10 and SpC. However, not all the cells in a positively stained structure experienced expression. If the structures, such as the spheroids for example, were all uniformly expressing the same markers, such as CC10, then it would suggest that the structures were a result of proliferation. Thus, because this is not the case, then it is more suggestive that the cells are capable of re-aggregating.  77  5. Subcutaneous vs. Subcapsular When we had originally started working with the matrigel and the cellular spheroids, we believed that this would be the most promising system we would achieve. This was because cell aggregation was clearly taking place, and, furthermore, it provided a transferrable system, allowing implantation of the organoids. Keeping with the idea that simplicity is key, we decided to first implant the organoids system subcutaneously, underneath the skin located on the dorsal region of a two month NOD/SCID female mouse. This provided an easily accessible, well vascularized, site fore implantation. Because it had not been a very invasive procedure, the surgery often took no longer than half an hour. Also, because the site of implantation was so large, it allowed the organoids system to be implanted without disruption. This is because the carbon filter itself would be transferred in its entirety, thus avoiding compromising the spheroids. However, the system was not as promising as hypothetically suggested. The subcutaneous region proved to be a more strenuous environment than expected. First off, it provided very little protection and support to the organoids. The fact is that skin has the flexibility to move when touched. For example, if the back of a mouse were rubbed, the skin would glide atop that general area. Therefore, keeping in mind that mice are not very calm animals, there is the high probability that the skin on the mouse’s back would move, resulting in shearing and tearing of the organoids (a lack of protection). This is most likely the reason why the only growth that could be observed in these  78  subcutaneous implantations were isolated lumen structures (Fig. 15). The other downfall of this sight of implantation is that there was a lot of infiltration of the host animal upon the organoids themselves. Looking at the figures representing the subcutaneous implants, it is evident that a great deal of fat surrounds the “bronchiole-like” structures. This may have also been another reason why growth was limited in this system. Thus, although an easily accessible, well- vascularized environment, the subcutaneous region was clearly a poor site of implantation. Being aware of the downfalls associated with the subcutaneous implantation, we decided that a subcapsular implantation would be better. The kidney is one of the most highly vascularized organs in the body. In order to conduct a subcapsular graft, the membrane surrounding the kidney needs to be separated from the parenchyma, and the desired substance of implantation needs to be placed within the newly formed pocket. Therefore, unlike in the subcapsular implantation, the implanted specimen solely interacted with a single thin membrane. There was no fat or other foreign material from the host with which the organoids had to interact. Because the organoids were only interacting with a thin membrane, and not the excessive forces that are provided by the “sticky” properties of fat, shearing of the implanted organoids was likely very minimal. The only downside to this approach was that the volume of material being implanted had to be significantly smaller than what had been implanted during  79  the subcutaneous implantation. Being that the hanging drops were already small enough to be implanted as they were, some creativity had to come into effect so as to implant the matrigel spheroids. Instead of implanting the spheroids still attached to the carbon filter, we came up with a means to slowly detach the matrigel from the carbon filter using the tips of a pipette, and constructing it into a miniature ball. This was done very gently so as to assure that the spheroids within the matrigel were minimally disturbed. In result to the recipient volume in the kidney being significantly smaller than the subcutaneous, it assured to keep the implanted material in a confined space so that locating it and excising the growth was easier to achieve. Although the kidney capsule is a confined space, it was not compact enough to support the direct injection of a single cell suspension. We had been under the impression that because the allotted space was so limited, it would be able to provide an adequate support system so that the cells could aggregate as in the previous systems, however that was clearly not the case. In the end the cells remained as a single cell suspension. We had disregarded that that the membrane surrounding the kidney is continuous, thus providing the cells the liberty to spread throughout the capsule, and not just at the site of injection. This proved that the cells needed some type of aggregation prior to implantation. Having made these comparisons, and noting the quality of results obtained from  80  each practice, it was evident that the subcapsular technique of pre-aggregated cells was the most successful and logical approach for future attempts. 6. Matrigel vs. No Matrigel The matrigel provided an adequate scaffold, assuring that the cells remained localized within the general vicinity of their implantation, whether subcutaneous or subcapsular. It allowed the cells to aggregate in a three dimensional manner and showed promise in regards to its potential. However, once the matrigel spheroids were actually implanted, unexpected results were observed. These spheroids were first implanted subcutaneously, where they were left up to one month. Based on the loss of epithelial marker expression over time, and the stabilized state of the progenitor cell markers, it was evident that the cells were not differentiating and advancing on into mature lung tissue. Being that this was the first attempt we had made in regards to implanting the matrigel, there were multiple factors that needed to be taken into consideration regarding the inadequacy of development. For instance: the ill-vascularized, fat abundant environment may have been what caused the deficiency, or perhaps it was the vast space in which the implant was able to move throughout, thus causing shearing of the structure that led to reversion of growth, or it could have been the matrigel in which the cells had been seeded that cause the hindrance. Trying to focus on each of these potential downfalls separately, we next implanted the  81  matrigel spheroids into the kidney capsule. This was technically observing two of the questionable failures regarding the subcutaneous attempt: the fat abundant environment and the large space. The kidney capsule is a highly vascularized region, with solely a thin membrane encapsulating the contents of the organoids, implying that no fat could cause shearing or damage to the structure of the system. Furthermore, it is a fairly confined space, so the organoids had less maneuverability, which would also decrease their chances in somehow damaging their structure. However, as with the subcutaneous implantation, the immunohistochemistry suggested that perhaps reversion took place. Comparing the results of the post 3 days implantation to those of the post 14 days, the post 3 days had more “promising” results (Figs. 23&24). Having changed the location and spacing of the surrounding environment, with no improvement in results, suggested the matrigel had some type of hindering role upon the survival of the cells. Two possibilities by which it may be accomplishing this is that it is either preventing the adequate signals from the surrounding environment to enter the vicinity of the cells so that they may continue growing and differentiating, or the matrigel itself is providing its own inhibiting factors that are influencing the cells in a more direct manner. However, something contradictory that may stir attention is that ultimately the general structure of the post 14 day implantation appears to be more advanced  82  than that of the post 3 day. A suggestion to this is that during the first few days of implantation, the inhibitory effects of the matrigel are being overpowered by the stimulatory ones of the surrounding environment; or, perhaps, it itself is providing the adequate stimulation for the cells. This belief is based on the positive development that was observed in the post 3 day implantation. However, there is some point, after 3 days implantation but before 14 days, where the inhibitory effects start taking control, thus causing the cells to perhaps die or plainly lose their abilities. As such, they are able to maintain the advanced structural growth, but not differentiate past that stage, perhaps losing molecular expression altogether. Being sure that the kidney capsule provided the proper vascular support and cell signaling to enhance growth and differentiation, we needed to develop a means by which to sturdily aggregate the cells without the use of matrigel. In result, we came up with the hanging drop technique. The cells were now adhering via the assistance of gravity, and were transferrable. Observing the products of implantation, it was evident that this was the most promising system as of yet. From this implantation, the cells were able to construct both proximal and distal- like structures, each expressing the appropriate markers in accordance to the native lung (Figs. 27 & 28). This successful system finalized that the matrigel was causing some type of inhibitory effect on the lung cells, and that the idyllic means by which get these organoids to reach a state of mature lung tissue would  83  be via the use of cells aggregated in their most natural state, thus allowing them to function as they would in their native environment.  84  CHAPTER 6: CONCLUSION / FUTURE DIRECTIONS Our overall attempt was to construct an organoids model from dissociated epithelial and mesenchymal embryonic lung cells so that when grafted, would give rise to a mature lung tissue. Although it took multiple approaches, we believe that we have finally reached a system that works fairly well: subcapsular implantation of hanging droplets. Though the system itself is not perfected, from the preliminary work provided in this paper, it is evident that it is a fairly promising technique. Due to time restraints, we were unable to test which incubation periods would provide the best results upon implantation. This is based on the fact that we had only implanted cells that had been incubating for 24 hours, yet Figure 26 clearly displays that the structures formed in the hanging droplets post four days had a more organized, airway-like appearance. Once we optimize this system, it will provide an elegant assay by which to observe the functionality and potential stem cellness of the various cells that compose the lung. Upon reaching this level of achievement, there are a multitude of experiments that we would like to observe conducted by this system. First off, with the use of an Fgf10LacZ mouse, we would be able to see how and where the mesenchyme interacts in the construction of organoids. We could also observe how Wnt signaling effects the formation of the organoids by using either Topgal or Batgal mice. However, asides from using different genetically modified mice, and observing how each specific genetic alteration/signaling pathway effects the  85  formation of the mature lung tissue, we can also see how the different layer of the lung contribute to its formation. With the assistance of FACS, we could take the single cell suspension and separate the mesenchymal cells from those of the epithelium. We could then take these separated epithelial cells and form a hanging droplet. Based on previous knowledge, the expected result would be that no mature lung tissue forms. Then we would take the separated mesenchymal cells, and slowly reintroduce them to the epithelial cells within the hanging drop. This would hopefully show how important the mesenchyme is in construction of the lung. Just from the few future experiments mentioned that could be conducted with this assay, it is apparent how strong of a tool this assay could prove. 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Abstract (if available)

Abstract Rationale: The lung is an organ essential for survival, however, due to its exposure to the outside environment, it is continuously subjected to multiple injuries. As a result, lung diseases are the leading cause of human death worldwide. The majority of these diseases are chronic and incurable, therefore representing a considerable financial burden. An ideal way in which such diseases could be effectively treated would be via regenerative medicine

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Generation of patterned and functional kidney organoids that recapitulate the adult kidney collecting duct system

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Development of immunotherapy for small cell lung cancer using novel modified antigens

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DNA methylation markers for blood-based detection of small cell lung cancer in mouse models

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Immunomodulatory and regenerative potential of amniotic fluid stem cells as a treatment strategy for pulmonary fibrosis

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The calcium-sensing receptor in the specification of normal and malignant hematopoietic cell localization in the bone marrow microenvironment

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The function of BS69 in mouse embryogenesis and embryonic stem cell differentiation

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Effects of a GSK-3 inhibitor on retroviral-mediated gene transfer to human CD34+ hematopoietic progenitor cells

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The role of fibroblast growth factor signaling on postnatal hepatic progenitor cell expansion

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Optimizing an immortalized human alveolar epithelial cell line model system to recapitulate lung adenocarcinoma development in vitro

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A novel construct to study the pulsatility of insulin secretion in single cells, islets and whole pancreas

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LINC00261 induces a G2/M cell cycle arrest and activation of the DNA damage response in lung adenocarcinoma

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Role of the bone marrow niche components in B cell malignancies

Asset Metadata

Creator Danopoulos, Soula (author)

Core Title Deconstructing the lung: a novel assay to evaluate lung stem/progenitor cell potential in vivo

School Keck School of Medicine

Degree Master of Science

Degree Program Experimental and Molecular Pathology

Publication Date 07/22/2013

Defense Date 06/09/2011

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

Tag kidney capsule transplant,lungs,mouse survival surgeries,OAI-PMH Harvest,organoids,progenitor cells,stem cells

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Bellusci, Saverio (committee chair), Kobielak, Krzysztof (committee member), Warburton, David (committee member)

Creator Email danopoul@usc.edu,sdanopoulos@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c127-641761

Unique identifier UC1345803

Identifier usctheses-c127-641761 (legacy record id)

Legacy Identifier etd-Danopoulos-155-0.pdf

Dmrecord 641761

Document Type Thesis

Rights Danopoulos, Soula

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu