All-Trans-Retinoic Acid Modulates the Proliferation and Differentiation of Yolk Sac Hematopoietic Stem Cells † 758

Abstract

All-trans-retinoic acid (ATRA) affects the proliferation and differentiation of normal and neoplastic hematopoietic cells. We have demonstrated the presence of hematopoietic stem and progenitor cells (HSC) in the murine yolk sac (YS) at day 9 postcoitus (pc) (PNAS 94:6776-6780). In vitro cultures of YS hematopoietic cells have traditionally yielded primitive macrophages and erythroid precursors with little or no granulocyte formation. We sought to explore the effect of ATRA on the proliferation and differentiation of day 9 pc YS cells in an in vitro progenitor assay. We isolated c-Kit+ lineage- CD38+ adult murine bone marrow (BM) and c-Kit+ TER119- CD38+ day 9 pc cells. Cells were cultured in a methylcellulose based system with varying concentrations of ATRA. Two growth factor combinations were utilized: A; GM-CSF, G-CSF, rSCF, mIL-3, hmCSF or B; Epo, IL-3, rSCF, IL-1, hmCSF. Plates were scored for colony number on day 10. Individual colonies were plucked and cell morphology was evaluated. In the presence of ATRA (10-5 - 10-7), a significant inhibition of colony number (control 131± 15; 10-5 RA 64 ± 5 colonies per 1000 cells plated) and an increase in granulocyte formation (control 26% ± 22; 10-5 ATRA 47%± 36 mean% granulocytes) was observed in cultures of c-Kit+ Lin-CD38+ BM cells. Significant inhibition of colony number was observed in the presence of ATRA in cultures of c-Kit+ TER119- CD38+ YS cells in both growth factor combinations whereas an increase in granulocyte formation was observed only in the presence of G-CSF and GM-CSF (control 10% ± 13; 10-6 ATRA 57%± 37). We conclude that the normally restricted in vitro repertoire of primitive yolk sac differentiation can be modified by ATRA in the presence of G-CSF and GM-CSF. The potential relationship between G-CSF, GM-CSF and factors affecting retinoic acid activity (ie retinoic acid receptors and binding proteins) is under investigation.