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Bone morphogenetic protein 4 induces efficient hematopoietic differentiation of rhesus monkey embryonic stem cells in vitro.

A cell culture system consisting of mouse S17 stromal cells supplemented with cytokines was developed for hematopoietic differentiation of rhesus monkey embryonic stem (ES) cells. The differentiated colonies that formed contained clusters of hematopoietic-like cells, as well as structures similar in appearance to embryonic blood islands. When this culture system was supplemented with bone morphogenetic protein 4 (BMP-4), the numbers of primary hematopoietic clusters increased by an average of 15 fold. The primary hematopoietic clusters containing clonogenic precursors (expandable hematopoietic clusters) increased by 18 fold. Immunofluorescence analysis showed that a substantial percentage of the hematopoietic-like cells were CD34(+), with morphologic features of undifferentiated blast cells. Enrichment of the CD34(+) cells was associated with enhanced stromal-dependent, cytokine-driven formation of cobblestone colonies on secondary plating. The hematopoietic identity of the precursors was further indicated by their expression of genes associated with hematopoietic differentiation, as well as morphologic assessments that showed erythroid and myeloid lineages among the progeny cells. In addition, reverse transcriptase-polymerase chain reaction analysis of BMP-4-treated rhesus monkey ES cells demonstrated an up-regulation of early-expressed genes responsible for embryonic hematopoiesis and angiogenesis during the first 7 days of culture. These observations suggest that embryonic mesoderm regulatory protein may mimic physiologic signals that are required for the onset of embryonic hematopoiesis and stem cell formation in rhesus monkey ES cells.