Abstract
Mouse somatic cells can be reprogrammed to pluripotency by the ectopic expression of four pluripotency transcription factors, Oct4, Sox2, cmyc, and Klf4. Usually, silencing of the exogenous reprogramming factors is considered to be essential for complete reprogramming and differentiation. In the vast majority of studies, murine pluripotency transcription factor sequences have been used for the reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSC). The effectiveness of xenogeneic transcription factors in miPSC generation has not yet been investigated in detail. Here, we evaluated transposon-based vectors with four porcine pluripotency factors for their ability to reprogram mouse fetal fibroblasts (MEFs) harboring an Oct4-EGFP reporter construct to pluripotency. Additionally, we examined the effects of the non-silenced heterologous transgenes on the expression levels of key endogenous pluripotency markers and the differentiation capacities of the miPSC. Within 8 days of transfection with porcine reprogramming transcription factors the MEFs acquired typical compact miPSC morphology and upregulated expression of endogenous Oct4 and other critical pluripotency genes. Consequently, the transgenes under the control of the TetO promoter became silenced, while the CAG-controlled constructs were expressed throughout the period of culture. Despite the continuous transgene expression, the CAG-miPSC showed normal morphology and were capable of differentiation into the three primary germ layers in vitro and in vivo. However, the expression levels of important endogenous pluripotency markers, Klf4, c-myc, Rex1, and Utf1, were significantly lower in CAG-miPSC compared with TetO-miPSC with silenced reprogramming cassettes. Surprisingly, the endogenous Oct4 and Sox2 expression levels were not affected by the residual transgene expression. Our results suggest that porcine reprogramming transcription factors are suitable for production of miPSC, but silencing of the heterologous transgenes may be necessary for complete reprogramming to pluripotency.
Highlights
The fate of a terminally differentiated somatic cell can be reversed by ectopic expression of pluripotency-related factors, as demonstrated by Takahashi and Yamanaka[1]
Our results suggest that porcine reprogramming transcription factors are suitable for production of miPSC, but silencing of the heterologous transgenes may be necessary for complete reprogramming to pluripotency
The balance between the expression levels of the endogenous pluripotency genes and the transgenes is thought to be critical for the success of the reprogramming of somatic cells to induced pluripotent stem cells (iPSC), with the epigenetic silencing of the retroviral transgenes coinciding with the up-regulation of the endogenes[4]
Summary
The fate of a terminally differentiated somatic cell can be reversed by ectopic expression of pluripotency-related factors, as demonstrated by Takahashi and Yamanaka[1]. The balance between the expression levels of the endogenous pluripotency genes and the transgenes is thought to be critical for the success of the reprogramming of somatic cells to iPSC, with the epigenetic silencing of the retroviral transgenes coinciding with the up-regulation of the endogenes[4]. The net concentration levels of Oct and Sox in the iPSC would need to be maintained within a narrow range characteristic for ESC. In support of this hypothesis, it was demonstrated that following upregulation of the corresponding endogenous pluripotency factors, the transgenes became gradually silenced in successfully reprogrammed iPS cells[7,8]. In a recent report, miPSC produced with transposon-delivered transgenes that were continuously expressed by supplementation with doxycycline (DOX) were reversed to an alternative pluripotency state (F-state), characterized by aberrant colony morphology and dependency on the continuous transgene expression for their maintenance[12]
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