We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34 ؉ cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sacderived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I ( 2 ⑀ 2 ) and the orthochromatic erythroblasts hemoglobin Gower II (␣ 2 ⑀ 2 ). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34 ؉ cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult -globin mRNA. IntroductionIn humans, primitive erythropoiesis originates from the extraembryonic mesoderm, is first detectable in the yolk sac 14 to 19 days after conception, and persists in this organ until the ninth week of gestation. It has long been known that yolk sac-derived primitive erythrocytes undergo a partial hemoglobin (Hb) switch: At week 5, yolk sac erythroblasts synthesize primarily Hb Gower I ( 2 ⑀ 2 ), but at weeks 6 to 8, they also synthesize large amounts of Hb Gower II (␣ 2 ⑀ 2 ). 1 Definitive erythropoiesis, which originates from the aortagonado-mesonephros region of the embryo proper, 2 is first detectable in the fetal liver during the sixth week of development. Erythroblasts produced in this organ express , ⑀, ␣, ␥, and small amounts of -globin but the ⑀ and -globin genes are rapidly silenced while the ␣ and ␥-globin genes remain expressed at high level until around birth. At that point, bone marrow erythropoiesis, which is first detectable around the eleventh week of gestation, becomes the major site of erythropoiesis and expression of the -globin gene, which had slowly risen during gestation almost completely replaces ␥-globin expression. In addition to these differences in globin-expression patterns, yolk sac, fetal liver, and bone marrow erythrocytes differ in morphology because yolk sac erythroblasts are nucleated and megaloblastic, while both fetal liver and bone marrow erythrocytes are enucleated. Fetal and adult erythrocytes differ by size, with the fetal cells bigger than the adult ones.Human embryonic stem cells (hESCs) can self-renew indefinitely in culture while retaining the ca...
Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.
We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.
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