Methods for making induced pluripotent stem cells: reprogramming à la carte

Key Points Direct reprogramming enables the generation of pluripotent stem-cell lines from almost any somatic tissue and mammalian species, thereby avoiding the ethical issues associated with human embryonic stem cells. Although direct reprogramming is conceptually and technically simple, it is an extremely slow and inefficient process. It is influenced by several variables that affect its efficiency and reproducibility, and the quality of the resulting induced pluripotent stem cells (iPSCs). Depending on the donor cell type, reprogramming is achieved with different efficiencies and kinetics. These differences are attributed to variations in the endogenous levels of certain reprogramming factors, differentiation status and/or intrinsic epigenetic states that are more amenable to reprogramming. Different factors are able to promote reprogramming, including genes that are normally expressed in early development, factors that directly or indirectly affect cell proliferation, chromatin remodellers or microRNAs. At present in the iPSC field, it is still difficult to unambiguously designate a reprogramming strategy that is fitting for all purposes. In each case, one will have to evaluate the most appropriate starting cell types, factors, culture conditions and delivery method. Reprogramming methods can be divided into two classes, those involving the integration of exogenous genetic material and those involving no genetic modification of the donor cells. Among these methodologies, retroviral delivery of OCT4 , SOX2 , KLF4 and MYC (the OSKM set) into fibroblasts is still the most widely used. Integrative reprogramming approaches generate heterogeneous iPSC lines, which could obscure comparative analysis between lines. The use of Cre-deletable vectors has partially solved this problem. Among non-integrative reprogramming systems, the recently published RNA-based approach seems promising on the basis of the high efficiency it achieves. Although appealing, the high gene dosages of the reprogramming factors resulting from direct messenger RNA delivery may represent an oncogeneic risk owing to higher expression levels of MYC . A crucial challenge in the iPSC field is to evaluate how these various methodologies affect the quality of iPSCs in terms of transcriptional signatures, epigenetic status, genomic integrity, stability, differentiation and tumorigenic potential. Whole-genome sequencing platforms will probably have an important role in the future in assessing the