Abstract
Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3β inhibitor CHIR99021, TGF-β/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs.
Highlights
Human induced pluripotent stem cells, similar to human embryonic stem cells (ESCs), are capable of unlimited proliferation and have the potential to differentiate into all cell types of the body [1,2]
The reprogramming efficiency with this method, was prohibitively low (,3 induced pluripotent stem cells (iPSCs) colonies from,16106 input fibroblasts). This low reprogramming efficiency was likely due to the loss of oriP/EBNA-1 vectors (.25% per cell generation) and DNA methylation-mediated transgene silencing during the first two weeks post-transfection, a time window required for successful iPSC generation [23]
Small molecules that could either accelerate reprogramming process, or reduce episomal vector loss and transgene silencing during the first two weeks post-transfection, were expected to improve episomal reprogramming. To identify such small molecules, we tested chemical compounds that were previously implicated in reprogramming and epigenetic modifications, using a two-vector episomal combination containing expression cassettes for all seven transgenes (OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4 and SV40LT) (7F-1, Fig. S1A), human foreskin fibroblasts as donor cells and traditional reprogramming conditions (MEF feeder cells and human ESC medium)
Summary
Human induced pluripotent stem cells (iPSCs), similar to human embryonic stem cells (ESCs), are capable of unlimited proliferation and have the potential to differentiate into all cell types of the body [1,2]. Initial methods for iPSC generation employed genome-integrating retroviral or lentiviral vectors [2,3] These approaches could produce tumorigenic insertional mutations, and residual or reactivation of transgene expression during iPSC differentiation could affect lineage choice and the functionality of iPSC derivatives [2,9]. To overcome these problems, various methods were developed to derive iPSCs free of exogenous DNA (footprint-free), including repeated treatments with reprogramming factors (plasmids, minicircle DNA, nonintegrating adenoviral vectors and proteins), transposons and RNA viral vectors [10,11,12,13,14,15,16]. There is a need to develop a simple and efficient feeder-free method to enable the routine derivation of footprint-free iPSCs from many human donor samples, and eventually the derivation of clinical-grade human iPSCs
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