Macaque and human models for preclinical development of iPSCs

通过干细胞指定转录因子的异位表达将出生后体细胞重编程为诱导多能干细胞（iPSCs），在改善人类健康方面具有许多令人兴奋的潜在应用。iPS细胞最初是在小鼠模型中开发的，仅仅几年后，人类iPS细胞就被创造出来了。然而，将iPSC推进临床再生医学应用还有许多障碍。首先也是最重要的是安全问题，如果分化不完全或效率低下，施用原始多能细胞可能有形成肿瘤的潜力，最严重的后果是。其次，多能干细胞有效分化为具有功能的成体组织存在重大挑战。诱导多能干细胞分化为具有良好特征的造血干细胞的方案效率低下，不一致，并导致异常或胚胎造血。设计直接递送或促进诱导多能干细胞或其后代归巢到体内适当位置的方法也将是一项重大挑战。

The re-programming of post-natal somatic cells to induced pluripotent stem cells (iPSCs) via ectopic expression of stem cell specifying transcription factors has many exciting potential applications for improving human health. iPSCs were initially developed in the murine model, and just a few years later, human iPS cells were created. However, there are numerous hurdles to moving iPSC forward into clinical regenerative medicine applications. First and most important are safety concerns, most seriously the consequences of administering primitive pluripotent cells that may have the potential to form tumors, if differentiation is incomplete or inefficient. Second, there are significant challenges to the efficient differentiation of iPSCs into functional adult tissues. Protocols for differentiation of iPSCs towards even well-characterized hematopoietic stem cells are inefficient, inconsistent and result in aberrant or embryonic hematopoiesis. Design of methods for direct delivery or facilitation of homing of iPSCs or their progeny to appropriate locations in the body will also be a major challenge. While murine models are invaluable tools, it is critical to develop more relevant large animal and in vitro models for clinical development of iPSCs. Human iPSCs can be implanted in immunodeficient mouse strains and form teratomas, but the next steps in development, requiring functional differentiation and appropriate delivery or homing, and analysis of immune or inflammatory responses to iPSC and their differentiated progeny are impossible to model accurately in xenografts. The rhesus macaque non-human primate (NHP) model is a valuable resource to clear hurdles preventing clinical development. Teratoma formation and other safety issues can be directly assessed utilizing autologous rhesus iPSCs. Differentiation, homing and other parameters critical for efficacy can be modeled. Tissue damage models such as pancreatic beta cell or hematopoietic stem cell ablation are well established in macaques. Development of rhesus iPSCs at the NIH takes advantage of our unique expertise in NHP transplantation and in the development of novel cell and gene therapies in this valuable model. During the past year we have further optimized a robust protocol for derivation of rhesus macaque (rh)and human iPSCs from skin fibroblasts, marrow stromal cells, and CD34+ hematopoietic cells, with cre excision of a polycistronic lentiviral reprogramming cassette leaving a residual genetic tag for in vivo tracking or use of a non integrating Sendai vector system, all in collaboration with the NHLBI Stem Cell Core. These clones are pluripotent as assayed in a murine teratoma assay, express all pluripotency markers, and can be differentiated to endodermal, mesodermal and ectodermal cell types, and now episomal non-integrating reprogramming methodologies. We have successfully developed and now fully characterized an autologous macaque teratoma model. This required adaption of rhiPSCs to xenofree culture conditions, and development of an autologous clot implantation matrix to replace murine Matrigel for in vivo 3D support. Compared to implantation in immunodeficient mice, autologous teratomas grow more slowly, require a higher cell number injected, and stimulate an inflammatory response that is not seen with injection of mature differentiated autologous progeny cells. In order to better standardize human iPSC generation, we have focused on utilizing barcoding to track the clonal behavior of starting cells as well as iPSC in culture. We have also begun to utilize CRISPR/Cas genome engineering to generate series of isogenic lines with and without disease mutations, in order to over the extraordinary heterogeneity of iPSC clonal behavior and differentiation. Having completed our in vivo studies of osteogenic generation in the macaque model, during the past year we have focused on hepatocyte, hematopoietic and cardiomyocyte differentiation from macaque iPSC. We have achieved robust in vitro hepatic and cardiomyocyte differentiation from rhesus iPSC, however greater efficiency and more complete differentiation must be achieved before moving to in vivo studies. We have also begun to focus on developing delivery and injury approaches to liver and heart in macaques in order to test these cell populations in vivo during the next year. We have developed approaches for genetic modification of rhesus and murine iPSCs and have introduced the inducible caspase 9 suicide gene into these cells and demonstrated effective killing of iPSCs in vitro. Treated cells do not form teratomas when injected in vivo, however, in vivo treatment of already established teratomas is not effective, with growth slowed but not prevented. Differentiated cells are no longer fully susceptible to the AP1903 dimerizer. Lack of sensitivity correlated with down-regulation of suicide gene expression and methylation of the promoter in differentiated cells. More effective suicide genes allowing killing with lower level expression, alternative expression cassettes and demethylating agents are being explored as alternative strategies. We have developed a robust approach to knock-in (or out) specific genes in both rhesus and human iPSCs, and have created stable CD19 and GFP-expressing rhesus and human iPSC. Continuing a collaborative project with Dr. Neal Young's group, our iPSC group has derived a large panel of iPSC clones from patients with telomerase complex abnormalities, including TERT, TERC and DKC-mutant iPSCs. Their telomere dynamics are very abnormal compared to control cells, with accelerated shortening. Hematopoietic differentiation from these mutant iPSCs is very abnormal and diminished, and the degree of abnormality seems to correlate well with the clinical severity of the disease in individual patient from whom the iPSC were derived. These cells are now being used to test possible therapies to improve hematopoiesis in these patients, in collaboration with the NCATS drug screening program. Preliminary work regarding hepatic differentiation also shows a defect, of interest since these patients also develop liver failure in addition to bone marrow and pulmonary failure. In collaboration with Dr. Stephen Holland's group, we have begun to model a second bone marrow failure syndrome, GATA2 deficiency. These patients have abnormal hematopoiesis with loss of monocytes and a specific NK cell subpopulation, a high risk of both marrow failure and leukemia, and lymphatic abnormalities. The relationship between genotype and phenotype are very unclear, and by the time of diagnosis the bone marrows are depleted of HSCs, preventing pathophysiologic studies. We have derived iPSC from multiple GATA2 patients and family members and are now characterizing all steps in hematopoietic differentiation. We have also demonstrated very striking specific defects in NK cell subpopulations in vivo as well as in vitro NK cell differentiation and expansion cultures. For both telomeropathies and GATA2 deficiencies, we have begun to create both knockout and knock-in isogenic pairs of iPSC to directly investigate the role of telomere length, expression of telomere maintenance genes, and GATA2 on hematopoietic and other tissue differentiation.