Generation of Human Hematopoietic Stem/Progenitor Cells From Pluripotent Stem Cells Through Modulating Retinoic Acid Signaling

Abstract 1203

Hematopoietic stem and progenitor cells generated from patient derived induced pluripotent stem (iPS) cells could provide an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to identify novel pathways involved in hematopoietic stem and progenitor cell generation and expansion from human ES and iPS cells. By using small molecules to modulate retinoid signaling, we report enhanced in vitro generation of hematopoietic progenitors cells (CD45/43+CD34+) and cells with an adult hematopoietic stem cell phenotype (CD45/43+CD34+CD38-CD90+CD45RA-). By inhibiting the cellular synthesis of retinoic acid (RA) and subsequent retinoid signaling we significantly increased the output of cells generated possessing an adult HSC surface phenotype by 2.7-fold (p-value: 0,024, n=7) compared to DMSO carrier controls, and increased clonogenic progenitors (CFUs) by 2.5-fold (p-value: 0,065, n=7). This improvement is consistent when using both a human ES line (Hues3) and a human iPS-line (RB9-CB1 generated from cord-blood derived endothelial cells). We also increased total blood cell output by increasing the frequency of embryoid bodies (EB) that successfully give rise to blood generating colonies. Conversely, and in support of our findings, directly adding RA was found to severely decrease the blood generation efficiency of our protocol. When comparing the colony forming potential of sorted cells gated on umbilical cord blood (CB), our system enabled the generation of CFU's at efficiencies on par with that of CB (preliminary data). Following plating equal numbers of sorted CD45/43+CD34+ blood progenitors, we saw 79 CFU/500 cells for the CB control, 65 CFU/500 cells from iPS + retinoic acid signaling inhibition, and 23 CFU/500 cells from iPS + DMSO control. The hematopoietic cells generated with our ES/iPS-2-Blood system also possess lymphoid lineage potential following plating of CD45/43+CD34+ cells into an in vitro based lymphoid assay, generating cells expressing markers of T-cells, B-cells, and NK-cells. We are currently performing transplantation experiments to assess the repopulating potential of these cells. We are also investigating additional factors that also have demonstrated significant increases in hematopoietic stem/progenitor cell output, with one additional modulator of RA signaling showing additive improvement together with the first molecule. We are further investigating the molecular mechanisms of these different factors for hematopoietic stem and progenitor cell generation with the aim to advance iPS cell technology towards treatments for hematological diseases and malignancies.