Simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells

ObjectiveHuman embryonic stem cells (hESCs) have been shown to generate CD34+ primitive hematopoietic cells after several days of coculturing with the OP9 murine stromal cell line. CD73+ multipotent mesenchymal cells have also been isolated from hESC/OP9 cocultures after several weeks. We hypothesized that generation of CD34+ hematopoietic cells and CD73+ mesenchymal stem cells (MSCs) may follow similar kinetics, so we investigated the generation of CD73+ cells in the first 2 weeks of hESC/OP9 cocultures, at a time when CD34+ cells are generated.Materials and MethodsWe cocultured hESCs with OP9 cells and examined the time course of appearance of human CD34+ and CD73+ cells using flow cytometry. We tested the hematopoietic progenitor potentials of CD34+ cells generated using hematopoietic colony-forming assays, and the multipotent mesenchymal properties of CD73+ cells generated using in vitro differentiation assays.ResultsWe observed that in the first 2 weeks of the hESC/OP9 coculture system CD34+ hematopoietic and CD73+ MSC generation follows a similar pattern. We sorted the CD34+ cells and showed that they can generate hematopoietic progenitor colonies. Starting with cocultured cells on day 8, and through an enrichment procedure, we also could generate a pure population of MSCs. These hESC-derived MSCs had typical morphological and cell surface marker characteristics of adult bone marrow-derived MSCs, and could be differentiated toward osteogenic, adipogenic, and chondrogenic cells in vitro, a hallmark property of MSCs.ConclusionsOP9 cells when cocultured with hESCs support simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ MSCs from hESCs.