Several somatic cell types have been

Several somatic cell types have been commonly used for iPSC reprogramming such as fibroblasts or keratinocytes from skin biopsies, lymphocytes and CD34+ hematopoietic stem tizanidine hcl harvested from blood (Ye et al., 2009; Mack et al., 2011; Ye et al., 2013). Recently, cells derived from urine were reported to be able to be reprogrammed into iPSCs (Zhou et al., 2012; Wang et al., 2013a; Guan et al., 2014; Afzal and Strande, 2015; Rossbach et al., 2016). Urine can be easily obtained from patients via non-invasive procedures and only 100mL of urine is sufficient for isolation, culture, and subsequent reprogramming. Therefore, urine cells are a viable somatic cell source for iPSC reprogramming in clinical settings. Protocols for directed neural induction of human iPSCs have been widely adapted and optimized (Chambers et al., 2009; Yuan et al., 2011; Macarthur et al., 2012b; Reinhardt et al., 2013). However, due to the heterogeneity of NSC/NPC differentiation, it is difficult to obtain pure NSC/NPC populations. Residual undifferentiated iPSCs pose perceived risks of tumorigenicity after transplantation. The contamination of cells skewing from the originally desired lineage during differentiation might interfere with neural differentiation or confound data interpretation. To overcome this obstacle, one strategy is to generate lineage reporters of fluorescent proteins compatible with fluorescence-activated cell sorting (FACS) purification. Although several neural lineage reporters have been developed (Ruby and Zheng, 2009; Xue et al., 2009; Li et al., 2015; Pei et al., 2015; Zhang et al., 2016), these reporters cannot be applied directly to clinical settings. A more straightforward methodology is to identify lineage specific cell surface markers to sort for NPCs from differentiated iPSCs, such as a quadruple sorting strategy of CD184+/CD271−/CD44−/CD24+ (Yuan et al., 2011). While the above approach is effective, a simpler sorting method is desirable to facilitate the clinical application of iPSC-derived neural cells. A2B5 is a commonly used antibody which recognizes a glycoganglioside that is specifically expressed on the cell surface of neural lineage cells. Previously, A2B5 has been used to identify glial progenitors in mouse and rat embryos (Rao et al., 1998). A2B5 also labels glial progenitors in human fetal brain derived neural cells (Eisenbarth et al., 1979; Mayer-Proschel et al., 1997; Mujtaba et al., 1999; Liu et al., 2002; Liu et al., 2004; Campanelli et al., 2008; Wu et al., 2008). In the present study, we reported a simple and effective protocol to generate patient specific neural populations for clinical application. We first isolated cells from patients\' urine, and reprogrammed the urine cells into iPSC cells (named as U-iPSCs) with the non-integrating Sendai viral vectors. After neural differentiation and purification with A2B5, a neural progenitor cell population was obtained. The purified A2B5+ progenitor cells were able to proliferate for at least 10 passages and differentiate into both neurons and glial cells in vitro. Importantly, these NPCs survived and integrated well after transplantation into the injured spinal cord. These results suggest that NPCs derived from patients\' U-iPSCs are a valuable source of cell based therapy for SCI.
Materials and methods
Results
Discussion As a promising source for cell-based regenerative medicine, iPSCs have attracted intensive investigations for their potentials in treating SCI and other neurological conditions. Through meticulously designed protocols, iPSCs can be coaxed into a variety of specific cell types, including NPCs, motor neurons, interneurons, oligodendrocytes and astrocytes (Chambers et al., 2009; Hu and Zhang, 2010; Krencik et al., 2011; Liu et al., 2011; Douvaras et al., 2014). These induced cells have been used to reconstruct in vitro disease models, serving as drug screening and testing platforms, and have been grafted to animal models to test their potential to participate in tissue regeneration or repair (Ruff and Fehlings, 2010; Nakamura et al., 2012; Nutt et al., 2013; Rao, 2013; Lu et al., 2014; Sareen et al., 2014; Baghbaderani et al., 2015; Beers et al., 2015; Liu and Deng, 2015). Importantly, patient specific iPSCs provide autologous cell source, which theoretically omit the need for immune suppression.