Early studies demonstrated that primitive cells

Early studies demonstrated that primitive Senexin A Supplier are readily converted into iPSCs (Kim et al., 2008; Kleger et al., 2012; Tsai et al., 2011). Thus, we expected that Lgr5+ cells would be susceptible to reprogramming. Unexpectedly, Lgr5+ cells from HFs and MEFs showed more resistance to early reprogramming rather than sensitivity, as demonstrated by the result of AP staining (Figs. 1C and 4C). However, by focusing on Nanog expression, their progeny showed more Nanog+ cells than nonprogeny. These findings suggest that the Lgr5+ stage is a barrier to protect unsuccessful reprogramming. Indeed, recent studies reported that transiently induced factors by reprogramming and not stably induced factors could function as an indicator of the successful cell reprogramming process (Koga et al., 2014; Wang et al., 2013). Because of the leakage of FDG, we could not determine the number of Nanog+ cells that express LacZ activity (M-Lgr5+ cell progeny). Instead, SSEA1 expression, which is a surface marker, was examined (Fig. 4B). Contrary to the result of the formation of Nanog+ colonies, merely 8.4% of SSEA1+ cells showed LacZ activity. This result appears to occur because SSEA1+ cells do not always convert into iPSCs (Brambrink et al., 2008).
Conclusion In conclusion, we found that focusing on Lgr5 positive cells from HFs and MEFs provided hints for facilitating the simple generation of Nanog+ cells without selection, such as using a drug. Lgr5+ HFs will be a promising material for the practical application to humans. Indeed, we found that human Lgr5+ cells can also more effectively convert into successful reprogrammed cells than Lgr5− cells. In addition, many efforts have been made to increase the reprogramming efficiency (Feng et al., 2009; Tang et al., 2012; Zhu et al., 2014), with most focusing on increasing the number of successfully reprogrammed cells and not on decreasing the number of unsuccessfully reprogrammed cells. Our findings provide a novel strategy for iPSC derivation by inhibiting unsuccessful reprogramming and contribute to minimizing sorting efforts for obtaining superior iPSCs. The following are the supplementary data related to this article.
Acknowledgments We are grateful to Drs. T Motohashi and T Kunisada for their help in using a flow cytometer. This work was supported by Grant-in-Aid for Research Activity start-up (25893084) and for Young Scientists (B) (26860365) from the Ministry for Education, Culture, Sports, Science and Technology of Japan and by the Sasakawa Scientific Research Grant from The Japan Science Society (25-415). The authors would like to thank Enago (www.enago.jp) for the English language review.
Introduction Generation of induced pluripotent stem cells (iPSCs) from differentiated adult cells like fibroblasts offered a promising means to investigate disease phenotypes in patient-derived cell model (Trounson et al., 2012). Moreover, iPSC technology opened a new era of for future cures of genetic disorders (Singh et al., 2015). Regular homologous recombination, by TALEN and CRISP/Cas9 techniques, offered the possibility to completely correct genetic defects (Hockemeyer et al., 2011; Kazuki et al., 2010; Li et al., 2015). To date, numerous disease-specific iPSCs were generated and iPSC-derived target cells already showed similar phenotype at cellular level, which indicate the potentials of iPSCs to study mechanism of genetic diseases. Glanzmann thrombasthenia (GT) is an autosomal recessive genetic hemorrhagic disorder. The platelets in GT patients have deficiency or functional defect of platelet integrin αIIbβ3 (glycoprotein (GP) IIb/IIIa; CD41/CD61), which is essential to blood coagulation (Solh et al., 2015). Most GT is caused by mutations within the encoding gene ITGA2B and ITGB3, which impairs expression of αIIbβ3 or lead to expression of dysfunctional integrin (Nurden et al., 2015). To date, a major therapy of GT is platelet transfusion. However, platelet transfusion refractoriness may occur due to anti-platelet alloimmunization (Ishaqi et al., 2009). Thus, it is pivotal to find a better way of treating the disease. One possible and promising way is to generate genetically corrected pluripotent stem cells from GT patients. The iPS cell technology can produce patient-specific stem cells for therapeutic use and thus offer advantages over embryonic stem cell (ES cell) gene therapy in terms of immunorejection and ethical concerns (Maherali and Hochedlinger, 2008). After genetic corrections, patient-specific iPS cells can then be induced to produce autologous, normal platelets, which can be used to treat bleeding disorders in GT patients.