We have demonstrated that donor specific DNAm patterns are

We have demonstrated that donor-specific DNAm patterns are maintained upon reprogramming into iPSCs (Shao et al., 2013), and these also remain upon redifferentiation into iPS-MSCs. Thus, there is some epigenetic memory after chromatin remodeling—whether these donor-specific DNAm patterns are functionally relevant remains to be elucidated. On the other hand, tissue-specific epigenetic differences were erased during reprogramming and not reestablished in iPS-MSCs. This may explain some of the epigenetic discrepancy of MSCs and iPS-MSCs. DNAm changes that accumulate during in vitro culture of MSCs (Koch et al., 2013) are also induced at a similar level during culture expansion of iPS-MSCs—apparently starting with loss of the pluripotent state. In contrast, age-related DNAm, which accumulates during aging of the organism (Weidner et al., 2014; Horvath, 2013) remains overall reset in iPS-MSCs. Notably, epigenetic rejuvenation does not counteract mutations, which may accumulate during in vitro culture. So far, the functional relevance of age-related DNAm changes and the underlying mechanism are not known, but the finding that they remain reset in iPS-MSCs is interesting and encourages further comparison with MSCs from different aged donors in vitro and in vivo. If age-related modifications contribute to loss of regenerative potential this may suggest higher regenerative potential of iPS-MSCs, which may also be reflected by the higher proliferation rates of iPS-MSCs as compared to primary MSCs.
Experimental Procedures A detailed description of all materials and methods is presented in the Supplemental Experimental Procedures.
Acknowledgments
Introduction The discovery of the technique that induces pluripotency in somatic cells, thereby leading to so-called induced pluripotent stem orexin receptor (iPSCs) (Takahashi and Yamanaka, 2006), raised great hopes within regenerative medicine. The potential application of patient-specific cells for autologous cell replacement therapies is exciting. The methods of iPSC generation have improved greatly since the breakthrough discoveries by Takahashi and Yamanaka; however, certain hurdles must be overcome before iPSC-derived cells can become clinically useful. These major issues include directed differentiation into certain cell types (Yamanaka, 2009) and the high tumorigenic potential of iPSCs (Ring et al., 2012; Yamanaka, 2009). The direct lineage conversion of differentiated cells into neurons (i.e., induced neurons) or expandable multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]) without passing through the pluripotent stage has been achieved (Han et al., 2012; Pang et al., 2011; Ring et al., 2012; Vierbuchen et al., 2010). This technique offers an attractive alternative to current iPSC technology because the tumorigenic potential of these cells might be significantly lower compared with iPSCs. Neural stem cells are self-renewable and generate differentiated cells, including neurons and astrocytes (Gage, 2000). Therefore, neural stem cells have enormous potential for regenerative therapies directed toward neurodegenerative diseases. Recent studies, including those conducted in our labs, have reported the direct conversion of mouse and human somatic cells into functional, expandable iNSCs that show all the major properties of primary NSCs (Corti et al., 2012; Han et al., 2012; Kim et al., 2011; Lujan et al., 2012; Ring et al., 2012; Sheng et al., 2012; Thier et al., 2012). However, in vivo long-term survival rates, multilineage differentiation, and the functional integration of iNSCs have not been analyzed in detail among these cells. The characteristics of transplanted cells after prolonged in vivo periods are crucial for potential cell-replacement approaches. Therefore, we investigated the characteristics of mouse-fibroblast-derived iNSCs 6 months after transplantation into adult mouse brains. We show that iNSCs differentiate into neurons, astrocytes, and oligodendrocytes in vivo. These cells survived for long periods in the mouse brain and functionally integrated into the existing neuronal circuitry. These results provide strong evidence that this iNSC procedure might be a valuable tool for cell-replacement therapies.