Profiling of the reprogramome and quantification of fibroblast reprogramming to pluripotency

Abstract

Pluripotent state can be established via reprogramming of somatic nuclei by factors within an oocyte or by ectopic expression of a few transgenes. Considered as being extensive and intensive, the full complement of genes to be reprogrammed, however, has never been defined, nor has the degree of reprogramming been determined quantitatively. Here, we propose a new concept of reprogramome, which is defined as the full complement of genes that need to be reprogrammed to the expression levels found in pluripotent stem cells (PSCs). This concept in combination with RNAseq enables us to precisely profile reprogramome and sub-reprogramomes, and study the reprogramming process with the help of other available tools such as GO analyses. With reprogramming of human fibroblasts into PSCs as an example, we have defined the full complement of the human fibroblast-to-PSC reprogramome. Furthermore, our analyses of the reprogramome revealed that WNT pathways and genes with roles in cellular morphogenesis have to be extensively and intensely reprogrammed for the establishment of pluripotency. We further developed the first mathematical model to quantitate the overall reprogramming, as well as reprogramming in a specific cellular feature such as WNT signaling pathways and genes regulating cellular morphogenesis. We anticipate that our concept and mathematical model may be applied to study and quantitate other reprogramming (pluripotency reprogramming from other somatic cells, and lineage reprogramming), as well as transcriptional and epigenetic differences between any two types of cells including cancer cells and their normal counterparts.

A PIANO (Proper, Insufficient, Aberrant, and NO Reprogramming) Response to the Yamanaka Factors in the Initial Stages of Human iPSC Reprogramming

“…We previously reported that genes involved in cellular morphogenesis have to be extensively and intensely reprogrammed for a successful conversion of human fibroblasts to iPSCs [13]. The current study reveals that these genes fall into three different categories.…”

“…To answer these questions, this study took advantage of the novel concept of reprogramome we proposed recently [13]. This new concept allows one to define two sets of genes that need to be reprogrammed.…”

“…The definitions for reprogramome and subreprogramome have been reported before [13]. In this manuscript, the genes with low and inconsistent expression were removed from the original reprogramome.…”

“…This criterion is stricter than our previous practice, in which only the average normalized DESeq2 read counts were greater than 50 and the individual read counts were greater than 10 only. The rationales to use the cutoffs of 50 and 10 were described previously [13]. Because of this more stringent criterion, the modified reprogramome is slightly smaller (3808 genes in the preprogrammed, and 3366 genes in the downreprogramome)m and the genes with very low expression are more consistent and reliable than those previously defined.…”

“…Recently, we proposed a novel concept of reprogramome, which is the full complement of genes that have to be reprogrammed for a successful conversion of cell fates from one type to another [13]. Using reprogramming of human fibroblasts into iPSCs as a model, we have previously defined the full complement of genes that need to be reprogrammed to the levels of expression found in the embryonic stem cells (ESC), i.e., human fibroblast-to-iPSC reprogramome.…”