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

To date, all human embryonic stem cells (hESCs) available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Activation of STAT3 by leukemia inhibitory factor is required to maintain "stemness" in mouse embryonic stem cells, but not in hESCs, suggesting the existence of alternate signaling pathways for self-renewal and pluripotency in human cells. Here we show that activin A is secreted by mouse embryonic feeder layers (mEFs) and that culture medium enriched with activin A is capable of maintaining hESCs in the undifferentiated state for >20 passages without the need for feeder layers, conditioned medium from mEFs, or STAT3 activation. hESCs retained both normal karyotype and markers of undifferentiated cells, including Oct-4, nanog, and TRA-1-60 and remained pluripotent, as shown by the in vivo formation of teratomas. Stem Cells 2005;23:489-495