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 will be critical to develop more relevant 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, may be impossible to model in xenografts. Scale-up of laboratory procedures developed in mice to human therapies would also be very difficult to develop solely using murine-murine or human-murine xenograft models. The rhesus macaque non-human primate (NHP) model will be 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, or 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. We have now successfully developed 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. Iterative studies have now resulted in a reproducible rhesus autologous teratoma model. Compared to implantation in immunodeficient mice, autologous teratomas grow more slowly, require a higher cell number injected, and stimulate an inflammatory response that we are in the process of further investigating. 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 MSC-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. The differentiated cells did not form teratomas. We have also begun to work on hepatic and hematopoietic differentiation of rhiPSC, in anticipation of in vivo regenerative studies during the next year. 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. We are investigating methylation patterns and other factors impacting on silencing and loss of sensitivity. 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.

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Hong, So Gun; Lin, Yongshun; Dunbar, Cynthia E et al. (2016) The Role of Nonhuman Primate Animal Models in the Clinical Development of Pluripotent Stem Cell Therapies. Mol Ther 24:1165-9
Jung, Moonjung; Dunbar, Cynthia E; Winkler, Thomas (2015) Modeling Human Bone Marrow Failure Syndromes Using Pluripotent Stem Cells and Genome Engineering. Mol Ther 23:1832-42
Balakumaran, Arun; Mishra, Prasun J; Pawelczyk, Edyta et al. (2015) Bone marrow skeletal stem/progenitor cell defects in dyskeratosis congenita and telomere biology disorders. Blood 125:793-802
Merling, Randall K; Sweeney, Colin L; Chu, Jessica et al. (2015) An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease. Mol Ther 23:147-57
Wu, Chuanfeng; Li, Brian; Lu, Rong et al. (2014) Clonal tracking of rhesus macaque hematopoiesis highlights a distinct lineage origin for natural killer cells. Cell Stem Cell 14:486-99
Wu, Chuanfeng; Hong, So Gun; Winkler, Thomas et al. (2014) Development of an inducible caspase-9 safety switch for pluripotent stem cell-based therapies. Mol Ther Methods Clin Dev 1:14053
Winkler, Thomas; Hong, So Gun; Decker, Jake E et al. (2013) Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs. J Clin Invest 123:1952-63
Wu, Chuanfeng; Jares, Alexander; Winkler, Thomas et al. (2013) High efficiency restriction enzyme-free linear amplification-mediated polymerase chain reaction approach for tracking lentiviral integration sites does not abrogate retrieval bias. Hum Gene Ther 24:38-47
Hong, So Gun; Dunbar, Cynthia E; Winkler, Thomas (2013) Assessing the risks of genotoxicity in the therapeutic development of induced pluripotent stem cells. Mol Ther 21:272-81
Riviere, Isabelle; Dunbar, Cynthia E; Sadelain, Michel (2012) Hematopoietic stem cell engineering at a crossroads. Blood 119:1107-16

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