Human Umbilical Cord Blood Derived IPS Cells as a Source of Hematopoietic Progenitors Cells

Abstract 4790

The ability to generate hematopoietic stem cells (HSCs) from an unlimited source of cells, such as from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable HSCs for therapeutic purposes. Umbilical cord blood is an ideal source of fetal/neonatal cellular material for iPS reprogramming due to the proliferative capacity of the cells as well as the reduced exposure of these cells to environmental factors compare to commonly used skin fibroblasts. In addition, it has been proposed that the cellular starting material imparts an epigenetic memory to the iPS cell line that influences lineage predisposition upon its differentiation. This project seeks to evaluate human umbilical cord blood as a starting cell source for generating iPS cells, with the ultimate goal to generate transplantable HSCs. We have isolated, cultured, and characterized an adherent cell fraction from the hemato-endothelial lineage. These cells were found to have an endothelial progenitor phenotype with CD45^neg, CD133^neg, VE-Cadherin^hi, VEGFR2^med, CD31^hi, CD34^hi cell surface markers as determined by FACS and formed tubules in the matrigel tubular assay. The iPS cell lines were generated using a 5-factor cocktail of inducible lentiviral vectors with an efficiency of 0.02%. The iPS cell lines were then evaluated for blood cell lineage differentiation capacity using our state-of-the-art ES/iPS-to-blood differentiation protocol. From the 5 iPS lines we tested, we saw 30 +/− 20% hematopoietic (CD45+) cells in our differentiation cultures. Moreover, the percentage of hematopoietic progenitors (CD45+ CD34+) of the hematopoietic cell fraction was 19.8+/− 0.6%, and also showed the presence of the more primitive CD45+ CD34+ CD38- progenitor fraction. Clonogenic progenitor cell counts determined by methylcellulose colony assay showed 44 +/− 3 colony forming units per 10,000 plated CD45+ cells. This is in the range of colonies obtained from umbilical cord blood mononuclear cell isolates (mean= 35+/− 2). The efficiency of hematopoietic generation for the lines ranged from 8 to 60% CD45+ suggesting that there are significant differences between the lines in terms of the completeness of reprogramming towards the pluripotent state. Further investigation into the epigenetic status of these lines is being performed. These data demonstrate the utility of human umbilical cord blood derived iPS cells for generating and expanding hematopoietic progenitors. This project advances iPS technologies towards treating life threatening hematological malignancies and diseases.