Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

Abstract

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:1101-1108 SIGNIFICANCEThe efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells.

Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming

“…Additionally, consistently with previous report of vimentin‐deficient fibroblasts exhibiting a slower rate of proliferation, fibroblasts isolated from the arm skin, along with fibroblasts from neck and breast regions, all mostly vimentin‐positive, proliferated at significantly higher rate than dermal fibroblasts from abdomen or thigh that showed a reduced expression of vimentin (Figure ). Notably, accumulating evidence suggests that low proliferation rates of somatic cells enhance their reprogramming, hence difference in proliferation rate of fibroblasts isolated from different anatomic sites might indirectly suggest a diverse response of fibroblast populations to reprogramming. Despite the common expression of several transcripts of genes typically expressed in mesenchymal cells, there was a heterogeneous expression of CD105 and CD146 when comparing dermal fibroblasts obtained from distinct anatomic sites, with fibroblasts isolated from abdomen skin that had a higher expression of all three markers.…”

Genome-wide gene expression analyses reveal unique cellular characteristics related to the amenability of HPC/HSCs into high-quality induced pluripotent stem cells

“…The Ink4a / Arf locus, which encodes three tumor suppressor genes (p16 Ink4a and p19 Arf from Cdkn2a and p15 Ink4b from Cdkn2b ), was found to be a barrier to reprogramming [ 20 , 21 ], further emphasizing the relationship between immortalization and reprogramming. Furthermore, a negative correlation has been revealed between excessive cell proliferation and reprogramming efficiency in human somatic cells [ 22 ]. Therefore, understanding the molecular events of reprogramming in different cell types to elucidate the core circuit of nuclear reprogramming may allow more effective interventions for the reprogramming of cells.…”

Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes

“…For example, there are several reports suggesting that proliferation is essential for reprogramming and that a higher proliferation in the starting cell population correlates with higher reprogramming efficiency [ 27 ]. Nevertheless, excessive cellular proliferation associated with an upregulation of the growth factor signaling pathway has been reported to have an adverse effect on reprogramming [ 15 ].…”