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  • However the use of an NHP model is hampered

    2018-10-20

    However, the use of an NHP model is hampered by the limited availability of clinically relevant NHP-iPSC lines. While the majority of NHP-iPSCs described in the literature were generated using retroviral vectors, human iPSCs intended for eventual therapeutic use need to be generated using transgene-free technologies. In addition, the efficiency of hematopoietic differentiation from NHP PSCs remains relatively low, and generation of lymphoid SGC707 Supplier from them represents a significant challenge (Gori et al., 2012, 2015; Hiroyama et al., 2006; Shinoda et al., 2007; Umeda et al., 2004, 2006). Here, we describe generation of clinically relevant transgene-free iPSCs from different NHP species, including rhesus, Chinese cynomolgus, and Mauritian cynomolgus monkeys, and demonstrate that GSK3β inhibition is essential to induce rapid and efficient differentiation of the NHP-iPSCs into multipotential hematopoietic progenitors. NHP-iPSC-derived hematopoietic progenitors were capable of differentiating further into mature cell types of myeloid and lymphoid lineages, including natural killer (NK) and T cells. The kinetics and hierarchy of hematopoietic differentiation from NHP-iPSCs was similar to those of human PSCs. Overall, these studies lay the foundation for advancing an NHP model for preclinical testing of iPSC-based therapies for blood diseases.
    Results
    Discussion Several protocols have been described for the induction of hematopoietic differentiation from NHP-iPSCs. These protocols employed co-culture of monkey cells with OP9 or other feeders and an embryoid body method (Abed et al., 2015; Gori et al., 2012, 2015; Hiroyama et al., 2006; Shinoda et al., 2007; Umeda et al., 2004, 2006). However, these studies reported the generation up to 5% CD34+CD45+ hematopoietic progenitors and up to 75 CFU per 105 cells in total cultures. In our studies, we demonstrated that the addition of GSK3β inhibitor potentiates mesoderm induction from NHP-iPSCs and increases the efficacy of clonogenic blood progenitor generation from NHP-iPSCs at least 10-fold, compared with previously published studies. In addition, hematopoietic commitment and differentiation in our system proceeded more rapidly, approximately within 6–10 days, compared with 15 or more days in other studies. Thus, our system simplifies the manufacture of blood cells for preclinical studies. Importantly, our differentiation system induces progenitors with T and NK cell lymphoid potentials. Overall, we were able to generate greater than 2 × 106 CD34+CD45+ multipotential hematopoietic progenitors containing greater than 4 × 103 CFU from 1 × 106 NHP-iPSCs in our differentiation system (Table 1). Recent studies have demonstrated hematopoietic engraftment in NOD/SCID/IL-2 receptor γ-chain-null mice following intrafemoral injection of differentiated NHP-iPSCs (Abed et al., 2015; Gori et al., 2015). Since our differentiation system makes it feasible to produce large numbers of CD34+CD45+ cells that are highly enriched in myeloid and lymphoid progenitors, it opens opportunities for preclinical testing of iPSC-derived blood products in a highly relevant NHP model. Moving artificial blood products into the clinic requires proof-of-concept animal studies and preclinical safety and toxicity assessment of stem cell therapies in animal models before entering into clinical trials (FDA, 2008, 2013; Fink, 2009). Tumorigenicity, biodistribution, and immunogenicity are identified as areas of concern that need to be addressed through in vivo studies (Goldring et al., 2011; Sharpe et al., 2012). The Food and Drug Administration (FDA) considers two types of animal models acceptable for preclinical studies: evaluation of human cells in an immune compromised animal host and evaluation of analogous cells in a species-specific model (FDA, 2013). Both approaches have limitations for the assessment of tumorigenicity and biodistribution because human cells may behave differently in an animal host environment, and intrinsic differences could exist in cell properties between human and animal cells. However, analogous animal models would be a better predictor of immunogenicity and immunotoxicity, because they require immunocompetent hosts.