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DERIVATION AND CHARACTERIZATION OF HUMAN EMBRYONIC
STEM (HES) CELLS AND HUMAN INDUCED PLURIPOTENT STEM
(HIPS) CELLS IN CLINICAL GRADE CONDITIONS
by
Jordan Elliott Pomeroy
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(SYSTEMS BIOLOGY AND DISEASE)
May 2012
Copyright 2012 Jordan Elliott Pomeroy
Object Description
| Title | Derivation and characterization of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells in clinical grade conditions |
| Author | Pomeroy, Jordan Elliott |
| Author email | jpomeroy@usc.edu;jep1780@yahoo.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Systems Biology and Disease |
| School | Keck School of Medicine |
| Date defended/completed | 2012-03-05 |
| Date submitted | 2012-04-30 |
| Date approved | 2012-05-01 |
| Restricted until | 2012-05-01 |
| Date published | 2012-05-01 |
| Advisor (committee chair) | Pera, Martin F. |
| Advisor (committee member) |
Ying, Qilong Lu, Wange Chuong, Cheng-Ming |
| Abstract | Future use of pluripotent cells for regenerative medicine will require the derivation and characterization of new cell lines in clinical grade conditions. Removing potential sources of contamination, such as cell culture components sourced from animals, will aid the regulatory approval for future stem cell based therapeutics. In this dissertation, I describe the development of a xenobiotic-free cell culture system for the derivation and maintenance of human pluripotent cell lines. I have derived >40 hiPSC lines and one hESC line in the xeno-free cell culture conditions with maintenance of pluripotency charcacterized by morphology and immunocytochemistry marker expression for >50 passages and >30 passages respectively. The hESC line, USC-01 and several hiPSC lines derived for this study demonstrate highly similar gene expression patterns although slight differences are apparent. USC-01 and several hiPSC lines demonstrate the ability to differentiate into cells displaying characteristics of all three germ layers in an embryoid body differentiation assay. Further examination of the process of reprogramming somatic cells to a pluripotent state at both the RNA and protein expression levels indicates several genes/markers selective for hiPSCs achieving a pluripotent state most similar to the “gold-standard” of pluripotency possessed by hESCs. This study confirms the usefulness of the markers TRA-1-60, E-Cadherin and EpCAM for live-cell selection of the best hiPSC colonies and also demonstrates the usefulness of the marker GCTM-2. Expression analysis of colonies undergoing reprogramming also indicates that the genes FOXD3, CDH3, LCK, EDNRB, EPHA1, SOX2, and HAS3 are active in only a small subset of colonies 30 days after transfection of the piggyBac transposon reprogramming cassette. Since these genes are active in all hESC and most hiPSC positive control lines tested, confirmation of their activation could be used to select reprogramming hiPSC colonies most likely to achieve a pluripotent state similar to hESCs. Increased selection and derivation efficiencies of hiPSC lines demonstrating high fidelity to the hESC pluripotent state will streamline the generation of hiPSC lines for future testing as a replacement for hESCs in regenerative medicine. |
| Keyword | embryonic; stem; induced; pluripotent; cells; clinical; human |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Rights | Pomeroy, Jordan Elliott |
| 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 author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume4/etd-PomeroyJor-692.pdf |
Description
| Title | Page 1 |
| Full text | DERIVATION AND CHARACTERIZATION OF HUMAN EMBRYONIC STEM (HES) CELLS AND HUMAN INDUCED PLURIPOTENT STEM (HIPS) CELLS IN CLINICAL GRADE CONDITIONS by Jordan Elliott Pomeroy A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (SYSTEMS BIOLOGY AND DISEASE) May 2012 Copyright 2012 Jordan Elliott Pomeroy |
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