Differentiating Human ES and Patient Derived IPS Cells to Hematopoietic Stem/Progenitor Cells.

Abstract 2628

The ability to generate hematopoietic stem cells (HSC's) from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to find the molecular determinants of human ES and iPS cell differentiation to transplantable HSC's. We have developed an advanced ES/iPS-to-blood differentiation protocol that mimics embryonic development towards the hematopoietic lineage. Current progress has resulted in up to 84% of human CD45+ hematopoietic cells (mean 41 ± 16%, n = 7) and with significant numbers of hematopoietic progenitor cells (CD45+CD34+, 29 ± 4%, n = 6) as reported by FACS analysis. Moreover, the numbers of hematopoietic progenitor cells generated (mean 20 ± 1 for a keratinocyte derived iPS (kiPS)), as measured by colony forming unit assays were comparable to numbers obtained from fresh whole blood umbilical cord mononuclear cell isolates (mean 35 ± 2) on a per 10.000 CD45+ input cells. We now plan to overexpress key factors predicted to support the generation and maturation of bona fide HSC's with repopulating ability. For this we have generated a tet-regulatable lentiviral based gene expression system to, out of a pool of over 30 genes, express up to 5 cDNA's of interest simultaneously. By clonal selection of lenti-transduced ES and iPS cells we have generated novel pluripotent lines that carry the tet-transactivator gene (m2rtTA), capable of efficient differentiation into blood. FACS analysis of these lines show blood generation of 30.7% and 11.8% of cells positive for CD45 for the human ES line Hues3 and the kiPS line respectively, with progenitor levels (CD45+CD34+) at 8.8% and 20.2%. These values are similar in efficiency to that of untransduced sister lines from which they were derived. These lines strongly express genes of interest following lenti-viral transduction and the addition of Doxycyclin (as reported by GFP). It is our goal to show successful reconstitution with in vitro generated HSC's, and ultimately pave the way for therapeutically relevant HSC's for treating hematological malignancies.