Macaque models for preclinical development of iPSCs

National Heart, Lung, And Blood Institute

Investigators

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Abstract

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. Third, many applications require gene editing of iPSC in order to correct genetic defects, add therapeutic goes, or introduce marker genes allow tracking of iPSC-derived cells in vivo. 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 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. We were the first group to derive rhesus macaque iPSC. iPSC clones generated 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 previously successfully developed and fully characterized an autologous macaque teratoma model (Hong et al, Cell Reports) We have optimized CRISPR/Cas9-mediated gene editing of rhesus and human iPSC, demonstrating efficient insertion of marker genes such as CD19 or GFP into the AAVS1 safe harbor locus, placement that we have found ensures high level, constitutive and stable expression of introduced genes during prolonged iPSC passage or following differentiation to cell types from all three germ layers. We have pioneered the use of the sodium iodide symporter (NIS) into rhesus iPSC, and we are excited about the promise of using this gene as a non-invasive marker for tracking of engrafted iPSC-derived cells in vivo. NIS expression is non-toxic, does not change the phenotype of iPSC or differentiated cells such as cardiomyocytes, and allows highly sensitive and specific detection of iPSC or differentiated iPSC-cardiomyocytes in vivo in both mice (Ostrominski Stem Cell Reports) and macaques via PET-CT following tracer administration. We have demonstrated that rhesus cardiomyocytes expressing NIS can be easily detected in vivo in immunodeficient mice via PET/CT and we have shown that NIS expression and exposure to physiologic iodide or to tracer does not impact on cardiomyocyte function, most importantly electrophysiologic properties. We have achieved very robust differentiation of RhiPSC-cardiomyocytes containing NIS or CD19 marker genes and generated sufficient cardiomyocytes for in vivo administration to macaques. We have optimized a rhesus macaque myocardial infarction model, and have carried out two in vivo experiments delivery autologous iPSC-CM to the infarct zone of macaques. We have easily been able to track persistence of the iPSC-CM by PET-CT for up to 9 months to date, the longest time period iPSC-derived cells have been followed in a large animal model. NIS signal in the heart infarct zone increases over time, suggesting maturation and enlargement of the delivered iSPC-CM. We continue to assess safety, primarily regarding potential disturbances of cardiac rhythms, as well as stability of engraftment. The initial animal had to be euthanized at 9 months due to medical issues not related to the cardiac procedures. Histology revealed mature iPSC-CMs intercalated into the myocardium. The second animal is now 7 months post-delivery of iPSC-CMs. We are collaborating with Chuck Murray at the University of Washington to compare autologous and allogeneic delivery.