Embryonic to Fetal Globin Switch Is Associated with Erythroid Cell Maturation in Primitive Hematopoiesis Derived from hESCs.

In humans, the first erythroblasts appear 14–19 days post-fertilization. At week 5, yolk sac erythroblasts synthesize primarily ζ and ε globins, but at weeks 6 and 7, these primitive erythroblasts also synthesize large amounts of α-globin and moderate amounts of γ-globin. Therefore yolk-sac-derived primitive erythrocytes undergo a partial hemoglobin switch. Cells derived from the yolk sac do not synthesize β-globin. At 6 weeks of gestation, erythropoiesis also takes place in the fetal liver, and express ζ, α, ε, γ and small amounts of β-globin. In this organ, the ζ and ε-globin genes are rapidly silenced while the γ genes remain expressed at high level until birth. Little is known about early globin switches in human because experimental material is difficult to obtain. Here we took advantage of a recently developed method (see accompanying abstract) of production of large numbers of human primitive erythroid cells in liquid culture to characterized globin switching in human primitive cells. This method is a 5 steps procedure that leads from hESCs to virtually pure populations of orthochromatic erythroblasts. At step 3, 90% pure populations of hemoglobinized immature polychromatic erythroblasts (hemoglobin content 6pg/10⁶ cells) were analyzed by HPLC for globin expression. Results revealed that these cells express mostly ζ and ε globins and only traces of α and γ globins with α/ζ<0.05. At the end of step 5 the cells had matured to orthochromatic erythroblasts (hemoglobin content 20 pg/10⁶ cells) and expressed much larger amounts of α and γ-globin with α/ζ and γ/ε ratios of 3.74±1.59 and 0.52±0.08 demonstrating a switch in globin expression during the culture. Since differentiation of these erythroid cells is not completely synchronized, the globin switches could happen either gradually during the erythroid maturation process or could be due to a late developing population of progenitors remaining in the third step culture. To discriminate these possibilities we sorted 3^rd step cells with α/ζ and γ/ε < 0.05 according to their CD45 and CD235a expression into immature erythroblasts (CD45⁻/235a^low) and polychromatophilic erythroblasts (CD45⁻/CD235^high) and place them back in culture. After 7 days of culture the sorted populations of CD45^−/CD235a⁺ cells had switched and respectively exhibited α/ζ ratios of 1.40 and 1.71, and γ/ε ratios of 0.59 and 0.53. These results suggested that these globin switches are associated with terminal erythroblastic maturation rather than with the production of successive waves of progenitors expressing different globin expression programs. To determine if the switches could be observed in clonally derived population of cells, we developed an assay in which individual CD34⁺ cells derived from hESCs (step 1 of our procedure) were expanded in liquid culture and analyzed by real-time RT-PCR for globin expression. These experiments revealed that all 8 clonally derived populations of cells switched from producing ζ to α globin with mean α/ζ ratio to 0.11±0.10 at step 2 and 159.80±219.71 at step 5. Only traces of β-globin expression could be detected in all the cells tested by HPLC or PCR. We conclude that the switches in globin expression that occur in the yolk sac during early human development are very different from the switch that occur later in life since they are caused by differential expression of the ζ, α, ε, and γ globin genes during late erythroid maturation rather than to production of progenitors with different expression programs.