Embryonic stem cells can be maintained in culture indefinitely as undifferentiated cells when in the presence of leukemia inhibitory factor (LIF). When LIF is removed, ES cells spontaneously proliferate and differentiate to form colonies known as embryoid bodies (EBs). The EBs consist of differentiated cells from various lineages, including the cardiomyocyte and hematopoietic lineages (myeloid, erythroid, megakaryocyte).

(76 & 65)

(66) Phase contrast photgraphy of an undifferentiated rhesus monkey ES cell colony (magnification 100)

The frequency of EBs containing hematopoietic cell lineages can be altered by the added presence of different regulatory molecules.

(85)

Coculture of human ES cells with stromal cells derived from mouse hematopoietic tissue (yolk sac and bone marrow) has been proven to cause differentiation into hematopoietic cells. (65)

Another experiment showed after 10 days of culture in fetal calf serum, more than 40% of all EBs contained visible erythropoietic cells. In the presence of erythropoietin (Epo) the number of EBs with erythropoietic activity increases to over 60%, as well as a prolonging of erythropoietic activity. The addition of interleukin-3 (IL-3) does not increase frequency of EBs containing erythroid cells, but does increase the number of erythropoietic cells associated with them, as well as other lineage-derived cells like macrophages and mast cells. (76)

An in vitro experiment with rhesus monkey ES cells especially demonstrated the crucial role bone morphogenetic protein 4 (BMP-4) plays in hematopoietic development. BMP-4, known to play a pivotal role in patterning of embryonic ventral mesoderm, was show to also trigger hematopoietic induction. A culture system made up of mouse stromal cells and cytokines was developed for rhesus monkey ES cell differentiation. The EBs that formed contained clusters of hematopoietic-like cells and structures that appeared similar to embryonic blood islands. Researchers found that when the culture system was supplemented by BMP-4, the number of primary hematopoietic clusters increased by an average of 15 times. The hematopoietic-like clusters also emerged earlier (day 13 versus day 14) and the structures similar to blood island appeared too be larger as well. Thus BMP-4 is critical to hematopoietic induction. (66) BMP-4 is also important in neural development.

Another in vitro experiment compared supplementation with just BMP-4, and then BMP-4 in addition to mesoderm inducers activin and fibroblast growth factor (FGF). Significantly greater numbers of differentiated erythroid cells were induced when the ectoderm was dealt with BMP-4 in conjunction with activin or FGF. These results suggest blood formation may require at least three functions: mesoderm induction (activing or FGF), mesoderm patterning (BMPs), and cell specification. (67)