Human transcription factors responsive to initial reprogramming predominantly undergo legitimate reprogramming during fibroblast conversion to iPSCs

Abstract

The four transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) together can convert human fibroblasts to induced pluripotent stem cells (iPSCs). It is, however, perplexing that they can do so only for a rare population of the starting cells with a long latency. Transcription factors (TFs) define identities of both the starting fibroblasts and the end product, iPSCs, and are also of paramount importance for the reprogramming process. It is critical to upregulate or activate the iPSC-enriched TFs while downregulate or silence the fibroblast-enriched TFs. This report explores the initial TF responses to OSKM as the molecular underpinnings for both the potency aspects and the limitation sides of the OSKM reprogramming. The authors first defined the TF reprogramome, i.e., the full complement of TFs to be reprogrammed. Most TFs were resistant to OSKM reprogramming at the initial stages, an observation consistent with the inefficiency and long latency of iPSC reprogramming. Surprisingly, the current analyses also revealed that most of the TFs (at least 83 genes) that did respond to OSKM induction underwent legitimate reprogramming. The initial legitimate transcriptional responses of TFs to OSKM reprogramming were also observed in the reprogramming fibroblasts from a different individual. Such early biased legitimate reprogramming of the responsive TFs aligns well with the robustness aspect of the otherwise inefficient and stochastic OSKM reprogramming.

Figure 1

Profiling reprogramming of human transcription factors for fibroblast-to-iPSC conversion. (A) Classification of differential expressions for the entire human transcription factor (TF) repertoire. (B) Number of TF genes in different degrees of enrichment in human embryonic stem cells (ESCs) as compared to fibroblasts. (C) A heat map showing enrichment of 310 human TF genes in hESCs. (D) Number of TF genes in different degrees of enrichment in human fibroblasts as compared to hESCs. (E) A heat map showing enrichment of 279 human TF genes in human fibroblasts compared with that in hESCs.

Figure 2

A group of transcription factors in the TF reprogramome is resistant to OSKM reprogramming at the early stages. (A) A heat map showing that 108 fibroblast-enriched TFs were irresponsive to OSKM reprogramming at the initial stages. (B) A heat map showing that 188 PSC-enriched TFs were irresponsive to OSKM reprogramming at the initial stages. OSKM, OCT4, SOX2, KLF4 and MYC. 48 and 72 after OSKM indicate time for RNA harvest post OSKM transduction of fibroblasts. OSKM reprogramming cells are highlighted in red. Human embryonic stem cells (ESCs) are highlighted in green.

Figure 3

The majority of the initial TF downregulation by OSKM is legitimate reprogramming. (A) Logic for evaluation of reprogramming legitimacy. (B) Classification of the 70 TFs downregulated by OSKM into different expression statuses in PSCs and the reprogramming starting fibroblasts. The orange sector indicates that 2 TFs are expressed at least 2-fold higher in ESCs. (C) Further characterization of reprogramming status for the 56 fibroblast-enriched genes downregulated by OSKM. (D) A heat map showing proper downreprogramming of the 21 fibroblast-enriched TFs. (E) A heat map showing insufficient downreprogramming for the significantly downregulated 33 fibroblast-enriched TFs by OSKM.

Figure 4

Most of the initial TF upregulation by OSKM is legitimate reprogramming. (A) Classification of the 49 TFs upregulatd by OSKM into different expression statuses in PSCs and the reprogramming starting fibroblasts. (B) Further reprogramming characterization of the 33 ESC-enriched genes upregulated by OSKM. (C) A heat map showing proper upreprogramming of 18 ESC-enriched TFs. (D) A heat map showing insufficient upreprogramming for the 11 ESC-enriched TFs significantly upregulated by OSKM. (E) A heat map showing wrong upreprogramming by OSKM for the 7 fibroblast-enriched TFs.

Figure 5

The TFs that were responsive to OSKM reprogramming in an independent fibroblast (CRL) are predominantly legitimate. (A) 219 TFs were significantly upregulated by at least 2-fold by OSKM (q < 0.01, fibroblast = 4, OSKM = 4). (B) 129 out of the 219 OSKM-upregulated TFs are enriched in ESCs by at least 2-fold. (C) 118 TFs were significantly downregulated by OSKM in CRL by at least 2-fold (q < 0.01, fibroblast = 4, OSKM = 4). (D) 89 out of the 118 OSKM-downregulated TFs were enriched in fibroblasts by at least 2-fold.