![]() ![]() For studies of induction of hematopoietic differentiation, trypsin-treated R366.4 ES cells (4000/well) were seeded on S17 layers in IMDM supplemented with 8% horse serum (Gibco), 8% FCS, 5 × 10 −6 M hydrocortisone, 20 ng/mL BMP-4 (R&D Systems, Minneapolis, MN), and various combinations of recombinant human stem cell factor (SCF 20 ng/mL), interleukin 3 (IL-3 20 ng/mL), interleukin 6 (IL-6 10 ng/mL), VEGF (20 ng/mL), granulocyte colony-stimulating factor (G-CSF 20 ng/mL), Flt3 ligand (10 ng/mL), and erythropoietin (Epo 2 U/mL) (R&D Systems). Layers of murine S17 bone marrow stromal cells (kindly provided by Dr Kenneth Dorshkind, University of California Los Angeles Medical Center) were preformed in gelatin-coated, 6-well culture plates in Iscoves modified Dulbecco medium (IMDM Gibco) supplemented with 15% FCS. ![]() 15-18 In culture systems that included bone marrow stromal cells with or without hematopoietic growth factors, mouse ES cells differentiated to form blood cell precursors in the absence of EB formation. ![]() Under appropriate culture conditions, EBs can further differentiate to form primitive blood islands and can ultimately be stimulated to form each of the definitive hematopoietic lineages. 11, 13, 14 When culture conditions that maintain mouse ES cells in the undifferentiated state are discontinued, the cells typically undergo spontaneous differentiation, with the formation of embryoid bodies (EBs). These studies established the ability of mouse ES cells to form hematopoietic progenitor cells with progeny characteristic of erythroid, myeloid, and lymphoid lineages. 8-12 The potential of ES cells to differentiate in vitro to form hematopoietic precursors has been investigated extensively but almost exclusively in mice. 1-7 These cells have the potential to differentiate to form progeny cells representative of the various tissues and organs of the body. 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.Ĭultures of pluripotent embryonic stem (ES) cells have been established from several mammalian species, including nonhuman primates and humans. 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. 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. Enrichment of the CD34 + cells was associated with enhanced stromal-dependent, cytokine-driven formation of cobblestone colonies on secondary plating. Immunofluorescence analysis showed that a substantial percentage of the hematopoietic-like cells were CD34 +, with morphologic features of undifferentiated blast cells. The primary hematopoietic clusters containing clonogenic precursors (expandable hematopoietic clusters) increased by 18 fold. 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 differentiated colonies that formed contained clusters of hematopoietic-like cells, as well as structures similar in appearance to embryonic blood islands. 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. ![]()
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