Generation of Red Blood Cells from Human Pluripotent Stem Cells

After the brief review of the in vitro systems for hematopoietic differentiation of pluripotent stem cells (PSCs), I would now like to take a closer look at the functional properties of PSCs-derived blood cells and discuss their potential for clinical application.

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Erythrocytes or red blood cells (RBCs) are the most abundant cell population comprising of ~45% of the total blood volume, whose main function is to deliver oxygen to the body tissues. Red blood cells lack a nucleus and most organelles to provide maximum space for hemoglobin – a complex metalloprotein containing heme groups whose iron atoms temporarily bind to oxygen molecules and release them throughout the body.

Mammalian erythroid progenitors originate from a megakaryocyte-erythroid progenitor (MEP) and undergo the gradual process toward terminal differentiation. Two globin gene switches occur during development: the embryonic to fetal globin switch, which coincides with the transition from embryonic (yolk sac) to definitive (fetal liver) hematopoiesis; and fetal to adult switch, which occurs during the perinatal period. During erythroblast differentiation, the chromatin condenses while the hemoglobin concentration increases. Chromatin condensation involves histone deacetylation and unknown signals that activate the Rac-GTPases-mDia2 pathway, which is required for the formation of a contractile actin ring and subsequent enucleation, the process in which the nucleus is rapidly squeezed out of the cell.

In vivo, erythroid precursors proliferate, differentiate, and enucleate within specialized niches called erythroblastic islands. These hematopoietic compartments are composed of erythroblasts surrounding a central macrophage. The central macrophage communicates with erythroblasts through a number of signaling molecules and phagocytizes their nuclei after enucleation.


In 1977, the American biochemist Eugene Goldwasser isolated the human protein erythropoietin (EPO), which stimulates red blood cell production. EPO became a blockbuster product that changed the lives of millions of patients suffering from anemia. Although EPO with other specific additives allow red blood cells to mature in vitro without a supportive role of macrophages, the resulting proliferation and enucleation efficiency of red blood cells is lower than their capacities in vivo, suggesting the importance of a niche microenvironment.

Blood transfusions are a common treatment for severe anemia and massive blood loss due to trauma. A type O-negative red blood cell can be transfused to patients of all blood types and is always in great demand. Thus, the derivation of (O)Rh-negative RBCs from PSCs could be an effective way to overcome shortages in donated red blood cells.

Red blood cells can be produced from human pluripotent stem cells (hESCs and hiPSCs) through various differentiation systems, such as an embryoid body (EB) formation and coculturing hPSCs on top of stromal feeder cells. In general, the existing methods are sufficient for a large-scale production of hPSC-derived red blood cells, whose in vitro expansion capacity is greater than the expansion potential of the bone marrow, peripheral blood, or even cord blood-derived erythroid progenitors. Despite the large amounts of RBCs obtained in many studies, the majority of the resulting RBCs expresses embryonic ε- and fetal γ-globins with low levels of detectable adult β-globin. Although no differences were observed between hiPSC and hESC lines in terms of erythroid commitment and expression of erythroid markers, iPSC-derived red blood cells have lower proliferation activity and produce less enucleated cells.

Robert Lanza’s group suggested the idea of developing an early hemato-endothelial progenitor, a hemangioblast, which can be expanded and cryopreserved.This study, published in Nature Methods in 2007, demonstrated the regenerative properties of blast cells that differentiate into multiple hematopoietic lineages as well as into endothelial cells. The extended coculture of these cells on OP9 feeders facilitated enucleation in up to 65% of cells and the expression of β-globin in up to 15% of the cells.

Lapillonne and colleagues employed a feeder free, two-step differentiating system to produce mature blood cells from hESCs and  iPSCs. In the first step, researchers initiated erythropoiesis by conditioning embryoid bodies in the presence of cytokines. To obtain mature erythrocytes, they further cultured cells in the presence of EPO, SCF, IL3 and 10% of human plasma for another 25 days. The resulting population contained up to 10% of enucleated cultured RBC from hiPSC, and 66% of enucleated RBC from hESC. The vast majority (~93%) of PSCs-derived red blood cells expressed the tetrameric form of fetal hemoglobin HbF (α2γ2). The CO-rebinding kinetics of hemoglobin from hESC- and hiPSC-derived erythroid cells was almost identical to those of cord blood cells suggesting that the HbF in these erythrocytes is functional.

Several studies have shown a time-dependent increase in β-globin expression, the oxygen dissociation curve and G6PD activities similar to normal RBCs. Nevertheless, significant progress is needed in the production of terminally differentiated/enucleated erythrocytes. Thus, at least two major steps are required for future therapeutic use of in vitro generated RBCs: (i) finding a cost-effective method for generating fully maturated, enucleated erythrocytes, and (ii) the evaluation of their biophysical parameters such as membrane surface potential, pliability, half-life in vivo, hemoglobin packing, gas exchange properties, and immunogenicity.


Further Reading:

1. Peng Ji, Maki Murata-Hori, Harvey F. Lodish Formation of mammalian erythrocytes: Chromatin condensation and enucleation Trends Cell Biol. 2011 July; 21(7): 409–415.

2. Joel Anne Chasis, Narla Mohandas Erythroblastic islands: niches for erythropoiesis Blood. 2008 August 1; 112(3): 470–478.

3. Lu SJ, Feng Q, Caballero S, Chen Y, Moore MA, Grant MB, Lanza R. Generation of functional hemangioblasts from human embryonic stem cells. Nat Methods. 2007 Jun;4(6):501-9.

4. Hélène Lapillonne, Ladan Kobari, Christelle Mazurier et al. Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine Haematologica. 2010 October; 95(10): 1651–1659.

5. Dias J, Gumenyuk M, Kang H, Vodyanik M, Yu J, Thomson JA, Slukvin II. Generation of red blood cells from human induced pluripotent stem cells. Stem Cells Dev. 2011 Sep;20(9):1639-47.

6. Chang KH, Bonig H, Papayannopoulou T. Generation and characterization of erythroid cells from human embryonic stem cells and induced pluripotent stem cells: an overview. Stem Cells Int. 2011;2011:791604.



1. Scanning electron microscope (SEM) image of a single red blood cell on the tip of a needle:

2. Confocal immunofluorescence image of an island reconstituted from freshly harvested mouse bone marrow cells stained with erythroid-specific marker (red), macrophage marker (green) and DNA probe (blue). Central macrophage is indicated by an arrow and a multilobulated reticulocyte by an arrowhead. Joel Anne Chasis, Narla Mohandas Erythroblastic islands: niches for erythropoiesis Blood. 2008 August 1; 112(3): 470–478.

About Irina Elcheva

Irina Elcheva is a Associate Researcher at Wisconsin National Primate Research Center, Madison, WI. She received her Ph.D at N.N. Blokhin Moscow Cancer Research Center. Her main research interests include direct differentiation of pluripotent stem cells into blood cells and hematopoietic stem cell development and specification.