Engineered Human Hematopoietic Stem Cells Generated in Developing Chimeric Animals from a New Class of Blastomere-like Human Totipotent Stem Cells

Although immune-deficient mice engrafted with human CD34 + HSC are widely employed in human immunology studies, these models have limitations. Generation of a fully diverse human lympho-hematopoietic system in research animals would radically change the landscape of biotechnology. Human HSC generated within developing murine AGM, fetal liver, and thymic niches of chimeric animal embryos can give rise to a full repertoire of human lymphocytes, myeloid cells, and HLA-expressing APC. We have reported Tankyrase/PARP1 Inhibitor-Regulated Naïve Stem Cells (TIRN-SC) by chemically reverting conventional hiPSC to an improved stem cell state that contributes differentiating human cells into developing mouse embryos. TIRN-SC are a new class of human blastomere-like totipotent stem cells with high epigenetic plasticity. In directed differentiation protocols, TIRN-SC generated 5-to-50-fold greater numbers of granulocyte-macrophage progenitors per culture volume and input cell than conventional hiPSC. TIRN-SC hematopoietic cultures produced two-fold greater purities of cultures of CD34 + CD45 + progenitors and with >90% CD11b + and >85% CD33 + myeloid progeny. To evaluate in vivo developmental potential, TIRN-SC were injected into murine embryos to develop human-murine chimeras. TIRN-SC i njection into 8C-16C-staged murine embryos resulted in efficient integration of human cells to both extra-embryonic (placenta) and embryonic (neural, hematopoietic) lineages of blastocyst and fetal chimeras , including human CD34 + CD45 + hematopoietic progenitors that developed within murine fetal livers. TIRN-SC chimerism efficiencies could be further optimized to high levels of 20-50% using immunosuppression or ectopic expression of a murine-E-Cadherin (mECad) transgene; the latter strategy promoted enhanced human cell targeting to murine hematopoietic organs ( e.g ., AGM and fetal liver; Fig. 1 ). Transgenic mECad-TIRN-SC achieved human cell chimerism rates of 2%-80% and 2%-18% in whole murine E14.5 feti and fetal livers, respectively. Toward our goal of generating humanized pigs that can produce unlimited amounts of engineered cells in their peripheral blood, we prepared CAR-expressing TIRN-SC lines recognizing the glioblastoma-specific disialoganglioside surface tumor antigen GD2 . Direct and adaptor-based anti-GD2 CAR-expressing TIRN-SC-derived myeloid cells were evaluated for tumor cell-dependent activation, cellular killing, and phagocytosis. TIRN-SC will allow the development of animals with humanized immune systems and cellular immunotherapy animal biofactories. For example, chimeric animals with human lymphohematopoietic systems from engineered TIRN-SC could provide unlimited sources of patient-specific or transgenic CAR-expressing NK,T, and myeloid cells in their peripheral blood.