Abstract
Patient-specific induced pluripotent stem cells (iPSCs) have the potential to be useful in the treatment of human diseases. While prior studies have reported multiple methods to generate iPSCs, DNA methylation continues to limit the totipotency and reprogramming efficiency of iPSCs. Here, we first show the competency of embryonic germ cells (EGCs) as a reprogramming catalyst capable of effectively promoting reprogramming induced by four defined factors, including Oct4, Sox2, Klf4 and c-Myc. Combining EGC extracts with these four factors resulted in formation of more embryonic stem cell-like colonies than did factors alone. Notably, expression of imprinted genes was higher with combined induction than with factors alone. Moreover, iPSCs derived from the combined inductors tended to have more global hypomethylation. Our research not only provides evidence that EGC extracts could activate DNA demethylation and reprogram imprinted genes, but also establishes a new way to enhance reprogramming of iPSCs, which remains a critical safety concern for potential use of iPSCs in regenerative medicine.
Highlights
Induced pluripotent stem cells represent a monumental scientific breakthrough in stem cell biology and regenerative medicine[1,2], capable of breaking down various ethical and logistical obstacles associated with human embryonic stem cell (ESC) research3,4. induced pluripotent stem cells (iPSCs) are generated by inducing the four “Yamanaka” transcription factors Oct[4], Sox[2], Klf[4] and c-Myc (OSKM) into somatic cells[5,6]; and essentially, reprogramming is an epigenetic process for changing the fate of cells[7,8,9]
Previous studies have shown that differentiation-induced de novo DNA methylation can repress a large set of pluripotency genes including Oct[4] and Nanog; whereas, active DNA demethylation is required for reactivation of pluripotency gene[15,16,17]
Results were normalized to GAPDH expression. *P < 0.01, versus other cells. (C-a) Embryoid body formation by suspension culture. (C-b) Teratomas. (C-c) RT-PCR analysis of germ layer markers
Summary
Induced pluripotent stem cells (iPSCs) represent a monumental scientific breakthrough in stem cell biology and regenerative medicine[1,2], capable of breaking down various ethical and logistical obstacles associated with human embryonic stem cell (ESC) research3,4. iPSCs are generated by inducing the four “Yamanaka” transcription factors Oct[4], Sox[2], Klf[4] and c-Myc (OSKM) into somatic cells[5,6]; and essentially, reprogramming is an epigenetic process for changing the fate of cells[7,8,9]. IPSCs are generated by inducing the four “Yamanaka” transcription factors Oct[4], Sox[2], Klf[4] and c-Myc (OSKM) into somatic cells[5,6]; and essentially, reprogramming is an epigenetic process for changing the fate of cells[7,8,9] It involves a number of different mechanisms to overcome the epigenetic barriers that are imposed during differentiation[10,11,12]. In the mouse embryos, migrating primordial germ cells (PGCs) reach the gonads at around 10.5 dpc They undergo an extensive active genome-wide DNA demethylation, including erasure of genomic imprints.
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