Background: It has been demonstrated that human ES cells can differentiate into hemangioblast and hematopoietic progenitors and enucleated into red cells. The process is very inefficient and needs improvement. We have developed a method to induce erythroid differentiation in vitro using hypoxic supportive cells and specific culture conditions. This methodology is being presently translated into bioreactor for massive production of red blood cells. A. Cell cultures in vitro: Cell Expansion of human ES cells is performed similar to embryonic stem cells mass cultures. By taking advantage of their epigenetic memory, the induction to differentiate to erythrocytes is facilitated through specific culture method and support of hypoxic cells of human origin. The process of differentiation and enucleation is enhanced by these processes. These hematopoietic lineage cells would include cells from hemocytoblasts, common myeloid progenitors, proerythroblasts (pronormoblasts), basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts or normoblasts which differentiate directly into polychromatic erythrocytes (reticulocytes). The erythrocyte end product cell type (RBC) that is derived from reticulocytes has no nucleus and thus cannot be genetically manipulated or form tumors in vivo. The development of long term culture conditions and expansion of these cells simplify the overall induction to the reticulocyte and in vitro formation of RBC. B. Bioreactor Development. It has been demonstrated that the human ES can be cultured as resident adherent cells on matrices that are fed through hollow fiber technology. The stem and progenitor populations are retained within an entrapment niche due to their adherent properties and thus promote the continuous production of RBC. RBC isolation within this system is accomplished by creating conditions that would allow erythrocyte escape from the progenitor entrapment niche as they enucleate and become non-adherent. Furthermore, this technology could make use of the inherent distortional capability of the RBC to enter small capillary-like channels under hypoxic conditions. This allows harvesting of pure populations of RBC in high numbers from the non-adherent compartment while maintaining continuous self renewing long term cultures of human ES cells- derived hematopoietic stem and progenitor populations for the production of RBC. Summary: Thus the differentiation pathway to RBC could be simplified to only a few intermediate cell types and accomplished by controlled culture conditions. Development of bioreactor conditions that mimic the bone marrow niche would provide a scalable system for large scale production of RBC. References: 1. US20070218552 Method for Producing Red Blood Cells. 2. Lu S-J, Feng Q, Park JS, Vida L, Lee B-S, Strausbauch M, Wettstein PJ, Honig GR, Lanza R. Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood. (2008) 112: 4475-4484. 3. Basak GW, Yasukawa S, Alfaro A, Halligan S, Srivastava AS, Min WP, Minev B, Carrier E. Human embryonic stem cells hemangioblast express HLA-antigens.J Transl Med. 2009 Apr 22;7:27.