The identification of a single, early marker for full developmental potential of induced pluripotent stem (iPS) cells has proven elusive. Recently, however, activation of the imprinted gene cluster, Dlk1-Dio3 has emer...The identification of a single, early marker for full developmental potential of induced pluripotent stem (iPS) cells has proven elusive. Recently, however, activation of the imprinted gene cluster, Dlk1-Dio3 has emerged as a viable candidate in the mouse. To explore the relationship between Dlk1-Dio3 expression and developmental potential more fully, we used murine ear mesenchymal stem cells (mEMSC) for iPS cell induction. Mouse EMSC are easily obtained and share functional characteristics with embryonic stem (ES) cells and therefore, may be a reliable non-embryonic source for iPS cell production. We report that mEMSC express high levels of Gtl2, a maternally expressed gene within the Dlk1-Dio3 imprinted cluster. Moreover, mEMSC produce Gtl2 expressing (Gtl2on) iPSC clones that share functional characteristics with ES cell clones. The production of Gtl2on iPS cell clones from mEMSC provides a new model with which to investigate the regulation of Dlk1-Dio3 cluster activity during direct cell reprogramming.展开更多
Human somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct4, Sox2, and either Klf4 and cMyc or Nanog and Lin28, using virus-based sys...Human somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct4, Sox2, and either Klf4 and cMyc or Nanog and Lin28, using virus-based systems. However, low reprogramming efficiency and the potential for deleterious virus-induced genomic modification limit the clinical potential of this technology. Recent reports indicate, however, that the generation of iPS cells can be enhanced by the addition of synthetic small molecules, including epigenetic modulators. In this report, we demonstrate that the epigenetic modifiers Valproic Acid (VPA) and 5-azacytidine activate the reciprocal transcriptional regulation of endogenous pluripotency transcription factor genes in human dermal fibroblasts and that VPA alone can directly activate endogenous Oct4 in the absence of transgenes. Moreover, using human adipose cells, we demonstrate that histone deacetylase inhibition, prior to reprogramming factor transfection, increases embryonic stem (ES) cell-like colony formation ~2 - 3 fold. In addition, DNA methyltransferase (DNMT) inhibition during human ES cell culture promotes maturation of reprogrammed somatic cells, increasing the yield ~4 fold. These data provide proof of principle that reprogramming efficiency can be improved by inhibiting specific repressive epigenetic regulatory components at the levels of ES cell-like colony formation and maturation. In addition, these studies raise the interesting possibility that a more efficient small molecule-based reprogramming system may provide a superior alternative to current virus-based approaches.展开更多
文摘The identification of a single, early marker for full developmental potential of induced pluripotent stem (iPS) cells has proven elusive. Recently, however, activation of the imprinted gene cluster, Dlk1-Dio3 has emerged as a viable candidate in the mouse. To explore the relationship between Dlk1-Dio3 expression and developmental potential more fully, we used murine ear mesenchymal stem cells (mEMSC) for iPS cell induction. Mouse EMSC are easily obtained and share functional characteristics with embryonic stem (ES) cells and therefore, may be a reliable non-embryonic source for iPS cell production. We report that mEMSC express high levels of Gtl2, a maternally expressed gene within the Dlk1-Dio3 imprinted cluster. Moreover, mEMSC produce Gtl2 expressing (Gtl2on) iPSC clones that share functional characteristics with ES cell clones. The production of Gtl2on iPS cell clones from mEMSC provides a new model with which to investigate the regulation of Dlk1-Dio3 cluster activity during direct cell reprogramming.
文摘Human somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct4, Sox2, and either Klf4 and cMyc or Nanog and Lin28, using virus-based systems. However, low reprogramming efficiency and the potential for deleterious virus-induced genomic modification limit the clinical potential of this technology. Recent reports indicate, however, that the generation of iPS cells can be enhanced by the addition of synthetic small molecules, including epigenetic modulators. In this report, we demonstrate that the epigenetic modifiers Valproic Acid (VPA) and 5-azacytidine activate the reciprocal transcriptional regulation of endogenous pluripotency transcription factor genes in human dermal fibroblasts and that VPA alone can directly activate endogenous Oct4 in the absence of transgenes. Moreover, using human adipose cells, we demonstrate that histone deacetylase inhibition, prior to reprogramming factor transfection, increases embryonic stem (ES) cell-like colony formation ~2 - 3 fold. In addition, DNA methyltransferase (DNMT) inhibition during human ES cell culture promotes maturation of reprogrammed somatic cells, increasing the yield ~4 fold. These data provide proof of principle that reprogramming efficiency can be improved by inhibiting specific repressive epigenetic regulatory components at the levels of ES cell-like colony formation and maturation. In addition, these studies raise the interesting possibility that a more efficient small molecule-based reprogramming system may provide a superior alternative to current virus-based approaches.
