Efficient Feeder-Free Episomal Reprogramming with Small Molecules

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.

Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides

“…An important application would be in the driving of reprogramming, self-renewal, and differentiation of stem cells. iPSC technology has been swiftly developed to allow genome nonintegrating DNA-based (19), RNA-based (20), and protein-based (11,12) technologies to supersede the original retroviral protocols (21) (Fig. 3).…”

CPAP promotes timely cilium disassembly to maintain neural progenitor pool

“…At day 10, cells were trypsinized and maintained on irradiated mouse embryonic fibroblasts (MEFs) in human iPS cell medium. Colonies were selected based on morphology (Okita et al , 2011; Yu et al , 2011), further passaged, and expanded. As quality control for iPS clones, we performed alkaline phosphatase (Vector Blue Alkaline Phosphatase Substrate Kit; Vector Laboratories, SK5300) and pluripotent markers staining.…”

Potential for pharmacological manipulation of human embryonic stem cells

“…These include small molecules which modulate important pathways such as the WNT, TGF-b, MEK and FGF pathways, such as GSK3b (CHIR99021, LiCl) (Ying et al, 2008;Li et al, 2009a,b;Yu et al, 2011;Wang et al, 2011b), MEK (PD0325901) (Ying et al, 2008;Lin et al, 2009;Li et al, 2009a;Zhu et al, 2010;Yu et al, 2011), FGFR (SU5402, PD173074) (Ying et al, 2008), TGF-b1 ALKs (SB431542, A83-01) (Li et al, 2009a;Lin et al, 2009;Yu et al, 2011), the lysine specific demethylase LSD1 (parnate/tranylcypromine) (Li et al, 2009b) and HDACs (NaB, VPA, TSA) (Huangfu et al, 2008;Mali et al, 2010;Zhu et al, 2010). Other small molecule compounds promote survival (thiazovivin) (Lin et al, 2009), dampen the senescence response during reprogramming (vitamin C) (Esteban et al, 2010) or activate pyruvate dehydrogenase kinase 1 (PDK1), facilitating a conversion from mitochondrial oxidation to glycolysis (PS48) .…”