Pluripotent Stem Cells Induced From Mouse Somatic Cells by Compounds Reprogramming the Cardiac Field Commentaries on Cutting Edge Science

Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field ever since their introduction by Takahashi and Yamanaka in 2006, as reviewed in Robinton and Daley. iPSCs can be generated from a variety of mouse and human somatic cells through lentiviralmediated overexpression of pluripotency-related genes such as Oct4, Sox2, Klf4, and cMyc (OSKM). Although phenotypically comparable with embryonic stem cells in that they can give rise to most cell types in the body, the fact that iPSCs are generated from adult patient tissues means that their differentiated derivatives can be used to model congenital disorders or even as autologous cells for transplantation. Reprogramming somatic cells to a pluripotent state has involved gene transfer to transiently express key pluripotency genes such as OSKM. The potential safety risks of genome modification have engendered concern and, consequently, have driven the development of nonintegrating methods for expressing OSKM and similar reprogramming cocktails. More recently, several groups have succeeded in replacing ≥1 of the reprogramming factors with small molecules, but to date these approaches still required transfer of ≥1 gene or mRNA. In a recent article in Science, Hou et al described the screening of small molecules to replace OSKM and, in the end, attained full reprogramming of mouse somatic cells to pluripotency. Achieving a small molecule–only protocol marks an important milestone and, probably, also a peak in efforts to develop integration-free reprogramming methods because most have been easy to replicate and adequately serves the majority of applications. Nevertheless, a tremendous amount remains to be learned about the mechanisms whereby a somatic cell can be made pluripotent, reflected by the rather low incidence regardless of technique (0.001% to 1% of somatic cells). The second recent article, by Abad et al, showed that it is possible to reprogram most organs and tissues to pluripotency in vivo through transgenic expression of OSKM in engineered mice. Two conclusions stand out. First, in vivo reprogramming drives formation of pluripotent stem cells in most organs and tissues in situ and, predictably, gives rise to teratomas. Second, and perhaps unexpectedly, the native milieu efficiently reprograms cells to a primitive stem cell state such that the iPSCs can form trophectoderm. Normally, embryonic stem cells and in vitro–generated iPSCs do not efficiently yield extraembryonic lineages, indicating that they are not truly totipotent. Based on transcript profiles, embryonic stem cells and in vitro–generated iPSCs closely resemble pluripotent epiblast cells of the blastocyst; however as shown by Abad et al, in vivo iPSCs more closely resemble developmentally earlier morula-stage embryos. These results suggest that, after the initial reprogramming event, the cellular milieu might guide progression toward totipotency versus pluripotency. Evidence is emerging that reprogramming occurs when OSKM initiates a stochastic phase followed by a meta-stable phase during which incorrectly or partially reprogrammed cells can arise. Finally, stable endogenous expression of Sox2 initiates a deterministic phase that culminates in the induction of Nanog and acquisition of pluripotency. Hou et al identified small molecules by systematic exclusion of individual reprogramming genes, screening for molecules that replaced the omitted gene. By meticulously doing this for each of the 4 OSKM reprogramming genes, they found a set of 7 small molecules that allowed full reprogramming, with an efficiency of ≈0.2%, which is comparable with that with transgenic methods, reflecting an advance during previous attempts that always needed ≥1 factor. Although the chemically induced iPSCs overtly resemble transgene-induced iPSCs in terms of differentiation potential, there remains significant concern that the compounds might induce genes that adversely affect the physiological function of the differentiated derivatives of the iPSCs. On the contrary, a major advantage of the chemical biology approach is that it can be used to identify relevant targets of the small molecules, potentially revealing new insights into the reprogramming process such as commonalities and differences related to the somatic cell species and tissue of origin. This was not performed by Hou et al; however, 2 of the Reprogramming the Cardiac Field