Generation of a Common Progenitor Population from Human Embryonic Stem Cells That Gives Rise to Both Embryonic Erythropoiesis and Definitive Hematopoiesis.

Human embryonic stem cells (hESC) provide a valuable new tool for dissecting the earliest developmental events of human hematopoietic-stem progenitor cell (HSPC) genesis. We have recently reported the efficient step-wise differentiation of hESC to embryonic (primitive) erythroid cells followed by definitive erythro-myeloid hematopoietic cells from human embryoid bodies (hEB). Hematopoiesis proceeds spontaneously from hEB-derived cells and appears to model the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development.

We now extend our studies to define conditions which may favor differentiation into definitive hematopoietic cells from hEB. We previously demonstrated that hEB-derived primitive hematopoiesis requires fetal calf serum, but proceeds in the absence of supplemented recombinant growth factors to developing hEB’s. We now show that supplementing hEB differentiation cultures with a broad array of hematopoietic growth factors dramatically enhances the number of primitive erythroblasts, definitive myeloid, BFU-E, CFU-E, and multi-potent mixed colonies in methylcellulose CFC assays while not affecting the kinetics of hematopoietic differentiation. The inclusion of VEGF-165 during hEB differentiation was found to have an exceptionally potent effect in increasing the multilineage generation of both primitive and definitive hEB-derived hematopoietic cells. To further define the hEB-derived population which gives rise to primitive and definitive hematopoiesis we FACS-purified a population of CD45-CD31+CD34+ hEB cells which we and others have shown are capable of both endothelial and hematogenous differentiation. Co-culture of this purified population in serum-containing OP9 stromal layers gave rise primarily to definitive-type erythro-myeloid cells including mature beta-globin-expressing erythroid cells, neutrophils, monocytes/macrophages, CD41+ megakaryocyte precursors, and CD56+ NK-like cells. Alternative culture of this purified CD45-CD31+CD34+ hEB population in serum-free, stromal-free cultures supplemented with erythropoietin and VEGF-165 produced an abundant population of embryonic (nucleated gamma, epsilon, zeta chain-hemoglobin-expressing) erythroblasts.

To isolate a clonogenic, expandable human hemangioblastic progenitor of primitive and definitive hematopoieisis as well as endothelial cells in our system, we have prepared transgenic hESC lines expressing a GFP reporter cDNA under the hemato-endothelial regulatory sequences of the human genomic SCL/TAL1 locus. Our hESC model reveals, for the first time, the putative existence of a population of human hEB progenitors capable of both embryonic-type and definitive blood cells depending on the differentiation environment.