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. We have 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 fully characterized an autologous macaque teratoma model. We have focused on three tissue differentiation targets for testing in the autologous rhesus macaque model. 1) bone-we have shown robust in vivo bone organic differentiation from autologous rhesus iPSC in vivo, after implantation of iPSC differentiated towards the osteoblast lineage mixed with a ceramic matrix. The bone structure appears normal, and no teratoma or other abnormal tissue growth was detected at one year following implantation. 2) cardiac-in collaboration with NHLBI investigators, we have achieved robust cardiac differentiation from rhesus iPSC. We are now optimizing a rhesus macaque surgical infarct induction model, and will begin in vivo experiments this year. 3) Liver-we are working to achieve efficient hepatocyte differentiation from rhesus iPSC, and will deliver these following hepatic lobe embolization. We have also used CRISPR/Cas9 to knockin marker genes into the rhesus iPSC AAVS1 locus, and demonstrated high level consistent expression at all stages of tissue differentiation. 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. We have generated a panel of TERT isogenic wild type and mutant lines to utilize for differentiation studies, given the clonal heterogeneity of individual patient iPSC clones. In collaboration with Dr. Stephen Holland's group, we have generated iPSC from patients with 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. We have generated isogenic pairs of GATA2 mutant and wild type lines in order to investigate the relatively subtle differences observed to date during hematopoietic differentiation from GATA2 patient versus control iPSC.
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