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


METHODS
We 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.


RESULTS
We 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.


CONCLUSIONS
OP9 cells when cocultured with hESCs support simultaneous generation of CD34(+) primitive hematopoietic cells and CD73(+) MSCs from hESCs.
Trivedi
Parul
P
Department of Medicine, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, USA.
Hematti
Peiman
P
D015720:5'-Nucleotidase; D000818:Animals; D018952:Antigens, CD34; D002454:Cell Differentiation; D002460:Cell Line; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D004622:Embryo, Mammalian / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007700:Kinetics; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D039902:Multipotent Stem Cells / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D017154:Stromal Cells / Q000166:cytology*
D018952:Antigens, CD34; D015720:5'-Nucleotidase