Human erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. Here we show that it is feasible to differentiate and mature human embryonic stem cells (hESCs) into functional oxygen-carrying erythrocytes on a large scale (10 10 -10 11 cells/6-well plate hESCs). We also show for the first time that the oxygen equilibrium curves of the hESCderived cells are comparable with normal red blood cells and respond to changes in pH and 2,3-diphosphoglyerate. Although these cells mainly expressed fetal and embryonic globins, they also possessed the capacity to express the adult -globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of -globin (from 0% to > 16%) after in vitro culture. Importantly, the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to more than 60% of the cells generating red blood cells with a diameter of approximately 6 to 8 m. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale. (Blood. 2008;112:4475-4484) IntroductionHuman embryonic stem cells (hESCs) can be propagated and expanded in vitro indefinitely, providing a potentially inexhaustible and donorless source of cells for human therapy. Hematopoietic differentiation of hESCs has been extensively investigated in vitro, and hematopoietic precursors as well as differentiated progeny representing erythroid, myeloid, macrophage, megakaryocytic, and lymphoid lineages have been identified in differentiating hESC cultures. [1][2][3][4][5][6][7][8] Previous studies also generated primitive erythroid cells from hESCs by embryoid body formation and coculturing with stromal cells. [8][9][10] However, the efficient and controlled differentiation of hESCs into homogeneous red blood cell (RBC) populations with oxygen-carrying capacity has not been previously achieved.Mammalian erythropoiesis is a complex process that involves many steps, including the differentiation of early erythroid progenitors (burst-forming units-erythroid, BFU-E) via late erythroid progenitors (colony-forming units-erythroid, CFU-E), and finally morphologically recognizable erythroid precursors. 11 Nuclear condensation is a key event in the late stages of erythropoiesis, and enucleation is the final step in the development of mature erythrocytes, although the molecular and cellular mechanisms involved in these processes are poorly understood.Here we describe an efficient method to generate functional erythroid cells from hESCs under conditions suitable for scale-up. The cells possess oxygen-transporting capacity comparable with normal RBCs and respond to changes in pH and 2,3-diphosphoglycerate. We also show that they undergo a progressive decrea...
Liver dysfunction in patients with IBD treated with immunosuppressants is more frequent and severe in those with HBV than in HCV carriers and is associated with combined immunosuppression.
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. The differentiated colonies that formed contained clusters of hematopoietic-like cells, as well as structures similar in appearance to embryonic blood islands. 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 primary hematopoietic clusters containing clonogenic precursors (expandable
Gene expression patterns of CD34 ؉ CD38 ؊ cells derived from human embryonic stem cells (ESCs) were compared with those of cells isolated from adult human bone marrow (BM) using microarrays; 1692 and 1494 genes were expressed at levels at least 3-fold above background in cells from BM and ESCs, respectively. Of these, 494 showed similar levels of expression in cells from both sources, 791 genes were overexpressed in cells from BM (BM versus ESCs, at least 2-fold), and 803 genes were preferentially expressed in cells from ESCs (ESCs versus BM, at least 2-fold). The message of the flt-3 gene was markedly decreased in cells from ESCs, whereas there was substantial flt-3 expression in cells from BM. High levels of embryonic ⑀-globin expression were observed-but no adult -globin message-in CD34 ؉ CD38 ؊ cells from ESCs, whereas high levels of -globin expression-but no embryonic ⑀-globin message-could be detected in cells from BM. Furthermore, high levels of major histocompatibility complex (MHC) gene expression were demonstrated in cells from BM but very low levels of MHC message in corresponding cells from ESCs. These observations demonstrate that CD34 ؉ CD38 ؊ cells derived from ESCs correspond consistently to an early developmental stage at which the yolk sac and fetal liver are the primary sites of hematopoiesis.
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