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Title: Functional study of DNMT3B in human pluripotent and nullipotent stem cells
Author: Wongtrakoongate, Patompon
ISNI:       0000 0004 2720 9170
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2011
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Human embryonic stem (ES) cells and embryonal carcinoma (EC) cells are the stem cells derived from human blastocysts and teratocarcinoma germ cell tumors, respectively. The majority of human EC cell lines have been shown to be nullipotent stem cells, which are not competent to be induced to differentiation both in vitro and in vivo compared with their pluripotent counterparts. However, by knocking down OCT4 it has been shown to induce differentiation of nullipotent EC cells. In this thesis, gene expression analysis has shown that these two stem cell types are different in term of inner cell mass- and early germline-specific gene expression profiles. STELLA was found to be up-regulated in EC compared with ES cells. However, an ectopic over-expression of STELLA in ES cells did not promote self-renewal. Rather, it facilitated germline and endodermal differentiation of the stem cells. DNMT3B was observed to be highly expressed in human nullipotent EC cells, and to be down-regulated during differentiation of ES cells. Silencing of DNMT3B by shRNAs did not alter the stem cell state of either ES or EC cells, but led to a reduction of differentiation of ES cells. In addition, reprogramming of differentiated cells in which DNMT3B had been knocked down, to produce induced pluripotent stem cells, was more efficient than reprogramming differentiated cells expressing DNMT3B. These results suggest that DNMT3B promote the differentiation capacity of pluripotent stem cells. Inhibition of DNA methylation by Aza-deoxycytidine (Aza-dC) resulted in differentiation and apoptosis of pluripotent ES and EC cells. However, knockdown of DNMT3B antagonized differentiation and apoptosis induced by Aza-dC, suggesting that the ability of the stem cells to undergo these two fates is DNMT3B-dependent. Moreover, the differentiated EC cells, induced to differentiate by OCT4-knockdown, had a reduced apoptotic response to Aza-dC, implying a stem cell-specific induction of apoptosis by the inhibition of DNA methylation. Since the inhibition of DNA methylation does not lead to differentiation of nullipotent EC cells, DNA methylation might not be important for maintenance of the nullipotent state. To test whether other epigenetic mechanisms are nevertheless involved in the maintenance of nullipotency, N21 02Ep differentiated nUllipotent EC cells generated by OCT4 silencing were reprogrammed to achieve a pluripotent state by transfection with a vector encoding the Yamanaka factors, OCT4, SOX2, KLF4 and c-MYC. Stem cells derived from this reprogramming show a potential to down-regulate stem cell markers such as SSEA-3, and up-regulate differentiation markers for example A2B5, PAX6 and TUJ 1 during retinoic acid-induced differentiation, whereas their nullipotent parental cells did not. These results suggest that epigenetic reprogramming leads to an acquisition of a more relaxed stem cell state of the reprogrammed N2102Ep cells, and that epigenetics is a factor implicated in the maintenance of nUllipotency of human EC cells. Altogether, the finding in this thesis implicates epigenetic factors, in particular DNMT3B, in regulation of stem cell fates and states of human ES and EC cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available