Rat Embryonic Fibroblasts Improve Reprogramming of Human Keratinocytes into Induced Pluripotent Stem Cells

Abstract

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells.

Developmental and Functional Nature of Human iPSC Derived Motoneurons

“…HESCs are usually maintained in an undifferentiated state by culturing on feeder cells layers, mostly mouse embryonic fibroblasts (MEFs) [42,168,214] or rat embryonic fibroblasts (REFs) [108]. Alternatively, hESCs can be cultured permanently on feeder cells from human tissues like human foreskin fibroblasts [127,147], human bone marrow stromal cells or human amniotic mesenchymal cells (hAMCs) [227].…”

“…(gremlin 1) have been shown to be secreted from MEFs and REFs and facilitate hESCs self-renewal [108].…”

“…Recently, somatic cells of murine and human origin such as fibroblasts [192], keratinocytes [1,108], NSCs [92] or CD34+ mobilized human peripheral blood cells [117] have been reprogrammed by addition of selective transcription factors including OCT4, SOX2 in combination with both c-MYC and KlF4, KIF4 alone [140], LIN28 and NANOG [223] or ESRB [55] (Fig. 2).…”

“…Aasen and coworkers could show that the reprogramming efficiency of human keratinocytes is at least 100-fold more efficient and two-fold faster compared with the reprogramming of human fibroblasts [1]. Moreover, reprogramming efficiency has been improved by chromatin modulation [75], application of chemical compounds [126], the use of synthetic modified mRNA [207] and using rat embryonic fibroblasts (REFs) instead of mouse embryonic fibroblasts (MEFs) [108]. HiPSCs generated from fibroblasts or keratinocytes are indistinguishable from hESCs in colony morphology, growth properties, expression of pluripotency associated surface and transcription markers, global gene expression profiles and …”

“…HiPSCs express genes and surface markers characteristic of hESCs including NANOG, OCT4, SOX2, as well as SSEA4, TRA-1-60 and TRA-1-80 [1, 108,117,212]. They express high levels of telomerase activity and are positive for alkaline phosphatase.…”