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) iPSCs from skin fibroblasts, marrow stromal cells, and hematopoietic cells, with cre excision of a polycistronic lentiviral reprogramming cassette leaving a residual genetic tag for in vivo tracking. 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 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. We have also now begun to investigate the regenerative potential of autologous iPSC-derived tissues in vivo. We have achieved robust differentiation of rhiPSC to mesenchymal stromal-like cells in vitro, and when these cells are implanted autologously in the presence of a ceramic matrix subcutaneously in vivo, they form bone structures comparable to those formed from primary MSCs, stable and robust now for over one year. The differentiated cells do not form teratomas, in contrast to the parental iPSC. We have achieved in vitro hepatic and hematopoietic differentiation from rhesus iPSC, however greater efficiency and more complete differentiation must be achieved before moving to in vivo studies. Using protocols comparable to those developed for human iPSC hepatic differentiation, we have achieved AFP+ albumin+ rhesus hepatocyte-like cells. With extensive optimization, we have finally achieved more robust hematopoietic differentiation of rhiPSC to CD34+/CD45+ cells, and now plan to optimize neutrophil differentiation of these cells, in order to test the preclinical safety and efficacy of iPSC-derived neutrophil infusions. 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. 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. 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.
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