Hematopoietic Development From Human Induced Pluripotent Stem Cells.

Abstract 2530

Poster Board II-507

A decade of research on human embryonic stem cells (ESC) has paved the way for the discovery of alternative approaches to generate pluripotent stem cells. Combinatorial overexpression of a limited number of proteins linked to pluripotency in ESC was recently found to reprogram differentiated somatic cells back to a pluripotent state, enabling the derivation of isogenic (patient-specific) human pluripotent stem cell lines (Park et al, 2008). Current research is focusing on improving reprogramming protocols (e.g. circumventing the use of retroviral technology and oncoproteins) and methods for differentiation into transplantable tissues of interest. In mouse ESC, we have previously shown that the embryonic morphogens BMP4 and Wnt3a direct blood formation via activation of Cdx and Hox genes. Ectopic expression of Cdx4 and HoxB4 enables the generation of mouse ESC-derived hematopoietic stem cells (HSC) capable of multilineage reconstitution of lethally irradiated adult mice. We have asked whether these signaling pathways patterning blood fate are conserved during hematopoietic development from human induced pluripotent stem (iPS) cells generated in our laboratory. Our data showed robust differentiation of iPS cells to mesoderm and to blood lineages, comparable to reports on differentiation of human ESC in this system. We detected robust formation of CD34⁺ (28.9±12), CD45⁺ (26.8±13.4) and CD34⁺CD45⁺ (16.1±13.7) cells, and a high incidence of CFU-initiating cells in functional colony assays, predominantly displaying myeloid but also some mixed CFU-GEMM activity. Similar to our findings in mouse ESC, mesodermal and hematopoietic genes were expressed in waves, and expression was augmented by supplementation of cultures with BMP4. Mesodermal markers (e.g. BRACHYURY ) were induced at day 2, and declined after day 9, when hematopoietic markers (SCL) appeared, indicating conversion of mesoderm to progenitors of the blood lineage. Expression of all three human CDX genes (CDX1, CDX2 and CDX4) peaked at day 6, suggesting that the function of CDX genes to pattern preformed mesoderm to blood fate may be conserved in human embryogenesis. Ongoing experiments in our laboratory focus on genetic modification of human iPS cells to study effects of specific genes during human emrbyonic hematopoiesis. Furthermore we have succeeded in transducing iPS cells with lentiviruses that allow GFP expression and puromycin selection, thus indicating feasibility for genetic modification. Taken together, our results show robust hematopoietic differentiation of human iPS cells and suggest that genetically modified in vitro differentiating iPS cells can be used to study human developmental hematopoiesis. Characterizing genetic pathways governing human embryonic blood formation will direct differentiation of induced pluripotent stem cells into repopulating hematopoietic stem cells, enabling generation of isogenic cell replacement therapies. Moreover, this experimental approach enables modeling of hematologic diseases, opening up a novel platform for gradual studies of genetic mechanisms during disease pathogenesis.