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 wi th cytokines was developed for hematopoietic differentiation of rhesus monk ey embryonic stem (ES) cells. The differentiated colonies that formed conta ined clusters of hematopoietic-like dells, as well as structures similar in appearance to embryonic blood islands. When this culture system was supple mented with bone morphogenetic protein 4 (BMP-4), the numbers of primary he matopoietic clusters increased by an average of 15 fold. The primary hemato poietic 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 m orphologic features of undifferentiated blast cells. Enrichment of the CD34 (+) cells was associated with enhanced stromal-dependent, cytokine-driven f ormation of cobblestone colonies on secondary plating. The hematopoietic id entity of the precursors was further indicated by their expression of genes associated with hematopoietic differentiation, as well as morphologic asse ssments that showed erythroid and myeloid lineages among the progeny cells. In addition, reverse transcriptase-polymerase chain reaction analysis of B MP-4-treated rhesus monkey ES cells demonstrated an up-regulation of early- expressed genes responsible for embryonic hematopoiesis and angiogenesis du ring tile first 7 days of culture. These observations suggest that embryoni c mesoderm regulatory protein may mimic physiologic signals that are requir ed for the onset of embryonic hematopoiesis and stem cell formation in rhes us monkey ES cells. (C) 2001 by The American Society of Hematology.