Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors

Abstract

Human embryonic stem (ES) cells possess the potential to differentiate into all the cell types of the human body and provide potential applications in regenerative medicine [1]. However, the concerns of immune rejection hamper transplantation therapies using human ES cells. To avoid the complications of immune rejection, diverse methods, such as somatic nuclear transfer (also called therapeutic cloning) and fusion of somatic cells with human ES cells [2], have been attempted to produce patient-specific pluripotent stem cells. Most of these approaches have resulted in little success. The generation of human iPS cells (induced pluripotent stem cells) from somatic cells with defined transcription factors makes it possible to produce patientspecific ES-like stem cells for therapeutic purposes [3,4]. Two sets of four-factors, OCT4, SOX2, C-MYC, KLF4 reported by Yamanaka's laboratory and OCT4, SOX2, NANOG, LIN28 reported by Thomson's laboratory, have been shown to reprogram human somatic cells to pluripotency with similar efficiency (10-20 iPS cell colonies from 0.1 million initial fibroblasts) [3,4]. We speculated that C-MYC and KLF4 might synergize with Thomson's 4 factors (OCT4, SOX2, NANOG, LIN28) to reprogram the human somatic cells. Our present study shows that a combination of 6 transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, significantly increases the efficiency of generating iPS cells from human somatic cells.The human genes encoding the transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, were cloned into a lentiviral vector to produce lentivirus.Half million human newborn foreskin fibroblasts were transduced with the lentivirus carrying GFP (serving as negative control), or a cocktail of lentivirus carrying 4 factors (OCT4, NANOG, SOX2 and LIN28) or 6 factors (OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4). Twenty-four hours later, the cells were dissociated with trypsin, transferred to five flasks (0.1 million initial cells per flask) coated with murine embryonic fibroblast (MEF) feeder, and cultured in human embryonic stem cell media. Colonies with a human ES cell-like morphology (iPS cell colonies) first became visible 12 days after transduction with 4 factors, whereas iPS cell colonies became visible 7 days after transduction with 6 factors. In order to quantify the efficiency of the reprogramming, the cells in one flask for each combination of factors were fixed on day 17 to analyze the alkaline phosphatase expression. A total of 16±3 colonies from 0.1 million initial fibroblast cells transduced with 4 factors were alkaline phosphatase-positive, 166±6 colonies from 0.1 million initial fibroblast cells transduced with 6 factors were alkaline phosphatase-positive, and no colonies were observed in GFP-lentivirus transduced controls ( Figure 1A). The efficiency of 6 factors is 10.4-fold more than that of 4 factors. The iPS colonies generated by transduction of 4 factors were picked on day 26. As reported by Yu et al. [4], these cells expressed alkaline phosphatase and undifferentia...

Stem Cell Therapy for Neuromuscular Diseases

“…Simultaneous transduction of c-myc, Oct4, Sox, Klf4, with Nanog and Lin28 increases efficiency dramatically (Liao et al, 2008). Other studies have shown that expression of p53 siRNA and UTF1 dramatically increases iPS colonies .…”

iPS Cells: Born-Again Stem Cells for Biomedical Applications

“…Although all three are required for pluripotency, Nanog overexpression is not required for induced pluripotency. Adding Nanog to the mix, however, increases reprogramming, as does combining the Yamanaka and Thomson reprogramming repertoires (Liao et al, 2008). Fig.…”

Advancements in reprogramming strategies for the generation of induced pluripotent stem cells

“…Partially different set of factors (OCT4, SOX2, NANOG and LIN28) has been reported to be sufficient to reprogram human fibroblasts [18]. For human foreskin fibroblasts, a combination of six transcription factors (OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4) has been shown to significantly increase the reprogramming efficiency [46]. Interestingly, Scholer and his colleagues [22] have demonstrated that OCT4 alone was able to convert human neural stem cells into iPS cells, termed as "one factor (1F) iPS".…”