Page 1 |
Save page Remove page | Previous | 1 of 221 | Next |
|
small (250x250 max)
medium (500x500 max)
large ( > 500x500)
Full Resolution
All (PDF)
|
This page
All
Subset |
INVESTIGATING THE ROLE OF OCT4 ISOFORMS IN HUMAN EMBRYONIC
STEM CELL SELF-RENEWAL AND DIFFERENTIATION
by
Steven Tsai
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)
August 2011
Copyright 2011 Steven Tsai
Object Description
| Title | Investigating the role of OCT4 isoforms in human embryonic stem cell self-renewal and differentiation |
| Author | Tsai, Steven C. |
| Author email | stevents@usc.edu;stevectsai@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Systems Biology and Disease |
| School | Keck School of Medicine |
| Date defended/completed | 2011-04-18 |
| Date submitted | 2011-06-30 |
| Date approved | 2011-07-01 |
| Restricted until | 2011-07-01 |
| Date published | 2011-07-01 |
| Advisor (committee chair) | Johnson, Deborah |
| Advisor (committee member) |
Lu, Wange Pera, Martin |
| Abstract | Human embryonic stem cells (hESC) derived from the inner cell mass of pre-implantation human blastocysts have two unique properties—indefinite self-renewal in culture and pluripotency, or the ability to differentiate into tissues from all three embryonic germ layers. As a result, hESC are a promising source of cells for regenerative medicine applications and have enormous potential in modeling human embryonic development. To realize this potential, a deeper understanding of the basic biology of hESC, especially of the genes that regulate self-renewal and differentiation, will be necessary. ❧ The focus of our study is on Oct4, a POU domain transcription factor and critical regulator of pluripotency whose levels are precisely controlled in mouse embryonic stem cells (mESC). In contrast to the single murine Oct4 isoform, which is better understood and more widely studied, three alternatively spliced isoforms exist in humans—OCT4A, OCT4B, and OCT4B1. Studies of human OCT4 are further confounded by the presence of six expressed pseudogenes. OCT4A is expressed in the nuclei of hESC and inner cell mass cells of human embryos and functions as a transactivator of self-renewal genes and repressor of differentiation genes. The functions of OCT4B and OCT4B1, however, are currently unknown, although OCT4B has been shown to exhibit cytoplasmic localization in trophectoderm cells of human embryos. These different subcellular localization patterns point to possible differential functions for OCT4A and OCT4B in hESC. In addition, significant differences between the biology of mESC and hESC, such as growth factor requirements and cell surface antigen profiles, suggest that human OCT4 may have different functions in hESC compared to its murine ortholog. Our overall hypothesis is that human OCT4 isoforms have differential biological functions in hESC, and the goal of these studies is to determine the role of OCT4A and OCT4B in hESC self-renewal and differentiation. ❧ In this thesis, we first examine the functional role of the OCT4A isoform in hESC by using an inducible lentiviral overexpression and knockdown strategy to manipulate OCT4A above or below physiologic levels. Additionally, we demonstrate the utility of an effective and efficient method to achieve conditional gene expression in hESC. We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by changes in morphology, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile that returns to baseline within 5 days. In addition, sustained expression of OCT4A enhances the cloning efficiency of hESC, suggesting that higher levels of OCT4A are beneficial for hESC self-renewal. Our results demonstrate that OCT4A dysregulation in hESC does not result in the same differentiation patterns seen in mESC and highlight the importance of conducting isoform-specific studies for human OCT4. ❧ We next examine a mechanism for regulating self-renewal and differentiation of hESC that involves the core transcriptional regulators NANOG, SOX2, and OCT4A. These proteins occupy and transactivate the promoters of genes expressed during self-renewal and repress the promoters of developmental genes required for differentiation. Nuclear localization signals (NLS) have previously been identified in mouse Oct4 and Sox2, and both NLS and nuclear export signal (NES) sequences were recently identified in human NANOG. In this study, we hypothesize that nucleocytoplasmic shuttling of core ESC factors contributes to regulation of self-renewal and differentiation. We determine that amino acids (aa) 190-204 in the POUB domain of human OCT4A and aa 71-84 in the HMG domain of human SOX2 contain functional NES sequences. Furthermore, these NES from OCT4A and SOX2 depend on the CRM1 nuclear export pathway. Using an endoderm differentiation protocol of H9 hESC, we investigate whether localization of core hESC factors changes during differentiation. As predicted, NANOG, OCT4A, and SOX2 are found in the nucleus during the undifferentiated, self-renewal state, but four days after endoderm induction, differentiating cells at colony edges demonstrate pan-cellular distribution of NANOG, OCT4 and SOX2. By day 5, the core hESC factors are undetectable in differentiating cells. In summary, we show that the core hESC transcription factors have functional NES and propose that such signals function to rapidly remove core factors from the nucleus at the time of lineage commitment, thereby halting the self-renewal program and allowing differentiation to proceed. ❧ Finally, in the last section of this thesis, we describe our studies examining the function of OCT4B in hESC. Analysis of OCT4B expression in hESC shows that OCT4B transcript is expressed at low levels in hESC and human teratocarcinoma cell lines but that endogenous OCT4B protein cannot be detected by western blotting. In addition, OCT4B transcript levels do not change as hESC differentiate into embryoid bodies over the course of five weeks. Transient transfection of siRNA oligos specifically targeting the unique N-terminus of OCT4B do not result in efficient OCT4B knockdown, although siRNA targeting the C-terminus is effective. There is no change in hESC phenotype by morphology or cell surface marker expression after OCT4B downregulation. Inducible overexpression of V5-tagged OCT4B in hESC results in low levels of transgene transcript and undetectable levels of V5-tagged protein by western blotting and immunofluorescence staining. In contrast, inducible overexpression of OCT4B-V5 in 293A cells results in abundant OCT4B-V5 protein, suggesting a silencing mechanism specific to hESC. Lastly, heat shock assays fail to confirm a role for OCT4B as a stress response protein in hESC, contrary to previously reported results. ❧ Taken together, our studies helped to clarify the role of OCT4 isoforms in hESC biology and enhance our understanding of the mechanisms underlying self-renewal and differentiation. A deeper understanding of the basic biology of hESC will ultimately support the advancement of hESC therapies into the clinic and reveal insights into the mechanisms of early human embryonic development. |
| Keyword | human embryonic stem cells; self-renewal; pluripotency; OCT4; OCT4 isoforms; OCT4 splice variants |
| 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 | Tsai, Steven C. |
| 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_Volume71/etd-TsaiSteven-37.pdf |
Description
| Title | Page 1 |
| Full text | INVESTIGATING THE ROLE OF OCT4 ISOFORMS IN HUMAN EMBRYONIC STEM CELL SELF-RENEWAL AND DIFFERENTIATION by Steven Tsai 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) August 2011 Copyright 2011 Steven Tsai |
Comments
Post a Comment for Page 1
