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.2008 Dec 1;112(12):4475-84.
doi: 10.1182/blood-2008-05-157198. Epub 2008 Aug 19.

Biologic properties and enucleation of red blood cells from human embryonic stem cells

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Biologic properties and enucleation of red blood cells from human embryonic stem cells

Shi-Jiang Lu et al. Blood..

Abstract

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 hESC-derived 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 beta-globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of beta-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 mum. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale.

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Figures

Figure 1
Figure 1
Large-scale production of erythroid cells from hESCs. (A) Erythroid cells (pellet) derived from 2 × 106 human ESCs. (B) Erythroid cells from panel A were resuspended in equivalent hematocrit of human whole blood. (C,D) Morphology of erythroid cells derived from human ESCs (C: original magnification ×200; D: original magnification ×1000). (E) Electrospray ionization mass spectra of globin chains in hemoglobins from hESC-derived erythroid cells, confirming the presence of α, ζ, ϵ, and γ globins. The observed molecular weight for each of the globins is shown. (F) Flow cytometric analysis of hESC-derived erythroid cells. Erythroid cells derived from hESCs were labeled with specific antibodies conjugated with phycoerythrin and analyzed on a FacScan flow cytometer (BD Biosciences) with the CellQuest program. Corresponding unspecific isotype antibodies conjugated with the same dyes were used as negative controls.
Figure 2
Figure 2
Functional characterization of hESC-derived erythroid cells. (A) Oxygen equilibrium curves of normal human RBCs and human ESC-derived erythroid cells. The 2 curves are virtually indistinguishable at their midpoints, whereas the curve of human ESC-derived erythroid cells is leftward shifted at low (→) and high (▾) oxygen saturation percentages. (B) The Bohr effect. (C) Effects of 2,3-DPG depletion. The solid lines represent the normal RBC control; dashed lines, the human ESC-derived erythroid cells. For each pair, the line on the right represents the fresh cells and the one to the left is the curve from cells depleted of 2,3-DPG.
Figure 3
Figure 3
Characterization of Rh(D) and ABO genotype of hESC lines by PCR. (A) Genotyping of RhD locus: Specific primers were designed for the Rh locus that when Rh(D) positive DNA was used, 1200-bp (weak) and 600-bp PCR products were amplified; whereas DNA from RhD-negative cells generated only the 1200-bp fragment. (B,C) Genotyping of the ABO locus: 2 pairs of primers were designed to amplify 2 regions of the ABO locus. The PCR products were digested with restriction enzymes to distinguish ABO types. Vertical white lines have been inserted in panels B and C to indicate repositioned gel lanes. ABO and Rh(D) genotypes are as follows: WA01, O( + ); MA99, B(−); MA133, A(−); WA07 and MA09, B( + ); and WA09 and MA01, A( + ). (D) RhD antigen expression analysis on erythroid cells derived from MA01 and MA99 hESCs by FACS. Erythroid cells generated from MA01 and MA99 hESCs were stained with phycoerythrin-labeled monoclonal anti-RhD antibody and analyzed by FACS. (E) ABO type characterization of hESC-derived erythroid cells. (i) Cells stained with monoclonal antibody against A-antigen (original magnification ×400). (ii) Cells stained with monoclonal antibody against B-antigen (original magnification ×400).
Figure 4
Figure 4
Enucleation of hESC-derived erythroid cells in vitro. (A) Diameter decreases with time in culture. Data for each day represent diameters of nucleated cells, except “27e” represents diameters of enucleated cells at 27 days. Enucleated cells decrease to less than half the original diameter on day 8. (B) Nuclear-to-cytoplasm ratio decreases with time in culture. Samples significantly different from day 8: *P < .05, **P < .001, #P < .002. (C,E) Erythroid cells derived from human ESCs were cultured in vitro for 4 weeks in Stemline II media with supplements and cocultured with OP9 stromal cells on day 36. On day 42, cells were cytospun and stained with Wright-Giemsa dye (C: original magnification ×200; E: original magnification ×1000). (D,F) Red blood cells from human blood were also cytospun and stained with Wright-Giemsa and compared with hESC-derived erythroid cells (D: original magnification ×200; F: original magnification ×1000). Scale bar represents 10 μm.
Figure 5
Figure 5
Maturation of hESC-derived erythroid cells mimic erythroid development. (A) Expression of CD235a, a mature erythrocyte marker, increases with time; and CD71, an immature RBC marker, shows a decrease in expression over time. (B) Expression of β-globin chain in hESC-derived erythroid cells. Cytospin samples of hESC-derived erythroid cells collected from day17 and day 28 differentiation and maturation cultures were stained with human β-globin chain specific antibody. (C) Progressive maturation of hESC-derived erythroid cells in vitro. Progressive morphologic changes from blast cells to erythroblasts, and eventually matured erythrocytes are accompanied by significant increase of hemoglobin and decrease in size during their in vitro differentiation and maturation. Cells were stained with both Wright-Giemsa and benzidine (A,B: original magnification ×200).
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Comment in

  • Trisomy 21 tilts the balance.
    Izraeli S.Izraeli S.Blood. 2008 Dec 1;112(12):4361-2. doi: 10.1182/blood-2008-09-176719.Blood. 2008.PMID:19029447No abstract available.

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