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 (#3). 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 (#1 for continued development of this system). 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, and we are currently developing analysis of levels of specific serum miRNAs as teratoma biomarkers, permitting non-invasive, whole body testing for engrafted residual pluripotent cells. We have also utilized genetic barcoding (see Project HL006063-08) to analyze clonal dynamics of iPSC clones maintained and expanded in vitro, and we have discovered markedly different behavior of individual clones, which we can now track molecularly. We have also shown that differentiation of each clone is highly variable utilizing the barcode system. Single cell studies on barcoded clones should allow identification of gene expression patterns that predict effective differentiation. We have also collaboratively shown that mutations found in iPSC are present in starting fibroblast populations, and are not substantively increased in iPSC (#5). 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 (see #2 and manuscript accepted). We recently introduced 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 via PET-CT following tracer administration (manuscript in preparation). This year we have focused on utilizing the rhesus macaque model to to test iPSC-derived cardiomyocyte cell therapy following myocardial infarction, in collaboration with a number of NHLBI investigators. We have achieved very robust differentiation of RhiPSC-cardiomyocytes containing NIS or CD19 marker genes (manuscript submitted). We have optimized a rhesus macaque myocardial infarction model, and have begun in vivo experiments. The use of autologous Rh-iPSC cardiomyocytes carrying the non-immunogenic rhesus NIS gene will allow very long-term follow-up of engrafted cells via cardiac MRI and via PET/CT. We will assess safety, primarily regarding potential disturbances of cardiac rhythms, as well as stability of engraftment. maturation and integration of engrafted cardiomyocytes, and impact on infarcted tissue function. In collaboration with Dr. Stephen Holland's group, we have generated iPSC from patients with the 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. Murine models do not recapitulate the human phenotype. We have derived iPSC from multiple GATA2 patients and family members and have characterized each step in hematopoietic differentiation. We have also 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. We find a moderate defect in output of CD34+/CD45+ HSPC from GATA2 heterozygous iPSC, and a profound loss of HSPC output from homozygous deficient IPSC. However, we do not find specific defects in differentiation of monocytic or natural killer cell progeny from GATA2-deficient iPSC. This suggests extrinsic factors or secondary mutations may be required for eventual development of the abnormal clinical phenotype, or that the embryonic-type hematopoiesis modeled by iPSCs may not reflect adult hematopoiesis regarding the role of GATA2. We have further investigated the NK cell compartment in GATA2 deficiency via in vitro expansion and differentiation of NK cells from circulating blood precursors. GAT2 patients as compared to controls have profoundly decreased CD56bright NK cells and relatively preserved CD56dim NK cells. With our collaborator Yenan Bryceson, we have shown that adaptive NK cells defined by expression of a set of signaling molecules and NKG2C are able to proliferate/renew independently of HSPC, and account for residual CD56dim NK cells in GATA2 deficiency. However, production of new CD56bright NK cells from precursors is profoundly impaired in these patients (#4).
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