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The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (>200 fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, non-viral methods for reprogramming human somatic cells.

Conventional mouse and human embryonic stem cells (ESCs) can be typically derived by in vitro culture of blastocysts (Martin, 1981;Thomson et al., 1998), and induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells using defined genetic transduction methods (Takahashi et al., 2007;Takahashi and Yamanaka, 2006;Yu et al., 2007). In both cases, different signaling pathways appear to regulate pluripotency with different characteristics in the two species. However, while rat ESClike cells have been established based on certain traits (Demers et al., 2007;Ruhnke et al., 2003;Schulze et al., 2006;Ueda et al., 2008), to date, these lines fall short of exhibiting true pluripotency (e.g., fail to form teratoma or no/little contribution to chimerism) and thus cannot be considered authentic rat ESCs. Here, we reveal combined genetic reprogramming and chemical conditions that generate and maintain rat iPSCs (riPSCs) that can give rise to teratomas and contribute extensively to chimeric rats. The same strategy is also sufficient to generate atypical human iPSCs (hiPSCs) that exhibit similar colony morphology and self-renewal requirements/signaling responses as those of mESCs.Pluripotent stem cells have also been derived from the postimplantation egg cylinder stage epiblasts of mouse and rat (Brons et al., 2007;Tesar et al., 2007). These populations have been termed epiblast stem cells (EpiSCs). EpiSCs seem to correspond very closely to conventional hESCs with respect to colony morphology and the culture/ signaling requirements that maintain pluripotency but exhibit a range of significant phenotypic and signaling response differences from conventional mESCs. For

Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine. Typically, hESC-based applications would require their in vitro differentiation into a desirable homogenous cell population. A major challenge of the current hESC differentiation paradigm is the inability to effectively capture and, in the long-term, stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and, more importantly, developmental stage-specific differentiation propensity. Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming growth factor β (TGF-β), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within 1 wk under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor, GSK3 inhibitor (CHIR99021), and TGF-β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. Our work uniformly captures and maintains primitive neural stem cells from hESCs.