Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1

Abstract

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming, but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs, which resembled ground-state mouse ES cells in growth properties, gene expression, and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency. The human iPSCs described here should facilitate functional analysis of the human genome.

Fig. 1.

Critical roles of RA signaling in reprogramming. (A) Schematic of the reprogramming strategy. M10 and M15 are media for MEFs and ES cells, respectively. (B and C) Expressing Rara or Rarg, together with 4F, drastically promoted reprogramming, whereas expressing Rara-DN blocked reprogramming. Reprogrammed colonies were visualized by AP staining (**P < 0.005 and ***P < 0.0005). Rara-DN, dominant-negative form of Rara. (D) Temporal requirement of RA signaling in reprogramming. After transfection of 4F, Rarg agonist CD437 (0.1 μM) or Rara agonist AM580 (0.01 μM) was immediately added to the medium for several time lengths. Control, DMSO, the solvent for CD437 and AM580. (E) Six factors increased reprogramming efficiency. AP⁺ colonies were scored on day 10 after transfection. (F) Rapid reprogramming by 6F. Activation of endogenous Oct4 was detected by GFP expression at several time points after transfection. (Scale bars: 10.0 μm.) (G) Six factors improved iPSC quality based on GFP expression from the Oct4 locus. Individual colonies were picked on day 10 after transfection and expanded in 96-well plates for flow cytometry analysis (***P < 0.0005).

Fig. 2.

Rarg and Lrh-1 synergistically promote reprogramming. (A) Schematic of the Tet-On reprogramming strategy. (B and C) Rapid activation of endogenous Oct4 (B) or Rex1 (C) by 6F. Expression of 6F for 4 d was sufficient to fully activate endogenous Oct4 (B) or Rex1 (C) to obtain Dox-independent iPSCs. (D) Morphology and GFP expression of Dox-independent colonies from Rex1-GFP reporter MEFs. (Scale bars: 10.0 μm.) (E) Kinetics of the Oct4 locus activation in Oct4-GFP reporter MEFs. The blue or red dots represent percentages of GFP⁺ cells in 6F or 4F transfections, respectively.

Fig. 3.

Characterization of Dox-independent mouse iPSCs produced by 6F. (A) Nearly complete demethylation in the promoters of Oct4 and Nanog in the iPSCs. (B) Pluripotency of iPSCs in N2B27/2i medium as demonstrated by Nanog and SSEA-1 immunostaining. (Scale bars: 10.0 μm.) (C) Both X chromosomes were active in undifferentiated female iPSCs produced with 6F. The differentiated female iPSCs lost Oct4 expression and had one inactivated X chromosome, which was decorated by H3K27me3 staining. (Scale bars: 40.0 μm.) (D) Contribution of iPSCs to the germline in chimeras. The chimeras were crossed to WT C57BL6 females (albino). Germline transmission pups were the agouti ones.

Fig. 4.

Production and characterization of human iPSCs that are free of exogenous factor expression. (A) Reprogramming HDFn cells using the Tet-On 6F. (B) Typical human iPSC colony morphology on the STO or HDFn feeders in the KSR/2i/LIF medium (passage 10, top two panels). (Scale bars: 4.0 μm.) The bottom two panels are representative human iPSC colonies growing in KSR/2i/LIF medium (compact and 3D) or human ES medium KSR/FGF (monolayer and flattened). (Scale bars: 10.0 μm.) (C) qRT-PCR analysis of key genes in parental HDFn, human iPSCs, and H1 hESC cells. HiPS15-5 and -10 were two independent iPSC lines derived from a male neonatal dermal fibroblast line (passage 20). Expression was relative to Gapdh and normalized against gene expression in H1 hESCs growing in KSR/FGF medium. (D) Immunostaining of human iPSCs for hESC pluripotency markers: SSEA1, SSEA3, SSEA4, TRA-1–60, and TRA-1–81 (cell surface markers) and OCT4 and NANOG. (Scale bars: 20.0 μm.).