In the subsequent sections we

In the subsequent sections we attempt to consolidate and discuss recent findings that have emerged from the study of the reprogramming process. Primarily, this is composed of three phases: initiation, maturation and stabilization and are discussed in greater detail below (Fig. 2).
Second milestone: unveiling the reprogramming pathway
Discussion While the roadmap to reprogramming is now taking shape, further refinement of this process is still needed. Indeed, recent advances have shed light on different road blocks, such as an improved OKSM stoichiometry (Carey et al., 2011) and understanding that TAK-875 can become refractory to reprogramming even by shutting down transgenic expression systems (Polo et al., 2012). There is a molecular connectivity or cascade of expression changes in specific genes during the reprogramming steps which have allowed the use of different surface and genetic markers during the study of the mechanism of reprogramming. As discussed, the initiation stage is marked by Cdh1, alkaline phosphatase, and SSEA1 followed by the maturation phase markers (e.g. Oct4, Nanog) and the stabilization phase markers (e.g. Pecam1, Sox2) which results in the production of bona fide iPSCs (Buganim et al., 2012; Golipour et al., 2012; O\'Malley et al., 2013; Polo et al., 2012). As the transcriptional signatures are now quite defined, it seems that for a given starting cell type, the sequence of events is highly conserved in nature and order. The last question, the time latency issue, points out epigenetic barriers such as, what makes a gene easier to reactivate than others, especially among the pluripotency associated genes? Bypassing the epigenetic barrier(s) is therefore a major challenge and solving this issue will require an in depth analysis of epigenetic regulatory events during reprogramming, including 3D structure of the genome studies, as performed recently with the Nanog promoter (Apostolou et al., 2013). This should yield factor-based or chemically-based reprogramming approaches matching the speed and faithfulness of somatic-cell nuclear transfer (SCNT) reprogramming. This is particularly relevant as human ESCs derived by SCNT, thereby mimicking embryo-derived ESCs, have just been successfully obtained (Tachibana et al., 2013). Importantly, alternative cocktails of reprogramming factors, for example by including Esrrb or knocking-down Mbd3, have demonstrated that overcoming this barrier is possible (Buganim et al., 2012; Luo et al., 2013; Rais et al., 2013). It remains to be shown whether those alternative or accelerated reprogramming strategies are changing the sequence of events, and if they change the faithfulness of reprogramming.
Acknowledgments Laurent David is supported by funds from region Pays-de-la-Loire (2011-12957). Jose Polo is supported by the NHMRC (APP1036587), ARC and the Silvia and Charles Viertel Senior Medical Research Fellowship. We apologize to researchers whose work could not be cited owing to space constraints. The authors thank Vincent Pasque and Sue Mei Lim for their comments and suggestions on the manuscript.
We () and others () previously demonstrated that in fresh renal tissue, adult human kidneys contain a population of renal stem and more committed progenitor cells characterized by co-expression of surface markers CD133 and CD24. These cells can be expanded in culture while maintaining their phenotype, and they exhibit self-renewal potential as well as the capacity to differentiate into podocytes and tubular cells both in vitro and in vivo (). Consistently, CD133+ renal cells represent a subset of CD24+ cells during human kidney development, when they constitute the metanephric mesenchyme-derived primordial nephron (). In their manuscript published in the August issue of , Bombelli et al. describe the isolation of putative novel renal stem cells by culturing nephrosphere suspensions from adult human kidneys (). Based on our previous studies, Bombelli et al. analyze nephrosphere-derived cells for expression of CD133 and CD24. The authors report the existence within nephrospheres of a CD133+ and CD24+ podocyte progenitor and also propose the existence of a CD133+ renal stem cell population that does not express CD24 (). However, in their study the authors limit their analysis to the population that they derived after nephrosphere cultures and do not check for the actual existence of CD133+/CD24− cells in adult human kidney tissues. Indeed, as already reported (), after depletion of hematopoietic cells using the pan-haematopoietic marker CD45, all the CD133+ cells observed in adult human kidney tissue are CD24+ (). Thus, CD133+/CD24− cells do not exist in vivo (). The authors\' results may have two possible explanations: 1) The staining for CD24 performed by direct fluorescent labeling and using a different anti-CD24 antibody in FACS analysis may have reduced sensitivity and what the authors see as CD133+ CD24− are cells that express low levels of CD24, and 2) Prolonged culture in suspension before analysis (at least 10days, as reported by the authors) may have modified the cell phenotype. However, the latter hypothesis is unlikely, since culture of nephrospheres was previously reported to enrich a cell with a CD133+/CD24+ phenotype by Buzhor et al. (). In summary, we would like to underline that although diverse methods may be used to identify putative renal stem or progenitor cells in adult human kidneys and study their properties, it is ultimately important to establish the existence of the putative stem cell population in fresh kidney tissue, to avoid misinterpretations related to technical differences and/or culture manipulation.