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.

In vitro modeling of hematopoiesis: from pluripotency to blood

Pluripotent stem cells (PSCs) derived from the inner part of a blastocyst (embryonic stem cells, ESCs) or through reprogramming of terminally differentiated adult cells (induced pluripotent stem cells, iPSCs) are capable of self-renewal and differentiation into almost all cell types in the human body. Their differentiation capacities and proliferation potential make pluripotent stem cells a promising source of cells for various clinical applications including regenerative medicine.

fetal red blood cells resized 600Blood is considered to be a connective tissue both functionally and embryologically. It originates from the mesodermal layer, the same germ layer that gives rise to the other connective tissues such as bone, cartilage and muscle. Blood cells and blood vessels develop in parallel and form a functional circulatory system. Various studies have shown that hematopoietic differentiation of PSCs in vitro closely resembles early steps of blood development in the embryo and induces blood forming cell populations with mesodermal and hemato-endothelial properties [1]. Different types of mature blood cells were successfully generated from murine, primate and human pluripotent stem cells. Here, we will briefly review the major in vitro systems of hematopoietic differentiation from PSCs.

Embryoid Body formation

Hematopoietic differentiation of PSCs can be carried out in either a two-dimensional system (2D), where cells are attached to the plate during differentiation, or in a three-dimensional system (3D), where isolated cells are dispersed into a liquid or a semisolid medium to form embryoid bodies (EBs).

Embryoid bodies are spherical structures that are formed by embryonic bodies resized 600pluripotent stem cells grown in non-adherent culture conditions (3D system). Differentiation of PSCs in aggregates mimics three-dimensional embryonic development and yields the establishment of cell adhesion, paracrine signaling and a microenvironment similar to native tissue structures. Thus, EB formation is often used as a method for initiating spontaneous differentiation of PSCs towards all three germ lines.

Differentiation in the presence of growth factors specific for mesoderm (BMP4, FGF, activin A) and blood formation (VEGF, SCF, Flt3, IL-3, IL-6, G-SCF, TPO) promotes hematopoiesis within embryoid aggregates and may result in the appearance of tissue-like structures such as blood islands and early blood vessels. The combination of BMP4 with hematopoietic cytokines yields up to 20% of CD34+CD45+ cells that will give rise to erythroid, macrophage, granulocytic and megakaryocytic colonies [2].

To produce EBs of equal size and standardize differentiation, a certain number of cells can be used to form aggregates by a spin technique (centrifugation) or in a hanging drops method. Hanging drops are single 10-20μl droplets with known cell densities that are placed on a glass surface or into hanging drop plates. Several studies have shown improvements of this method that would allow it in practical application.

Coculture with stromal cells

This two-dimensional differentiation system is based on induction of hematopoiesis upon exposure to extrinsic signals from the feeder cells that underlie the PSCs in coculture. Stromal cells with the capacity to induce and support hematopoiesis can be isolated from a variety of anatomical sites associated with the hematopoietic development in vivo. A number of cell lines were established from mouse bone marrow (OP9, MS5 and S17), yolk sac endothelium (C166), fetal liver (mFLSC, EL08) and other sources. The genetically modified stromal cells, immortalized or expressing specific growth factors and signaling molecules, are widely used in hematopoietic coculture.

The standard coculture conditions comprise prolonged, up to 4 weeks, incubation of undifferentiated pluripotent stem cells on top of the stromal cells in the presence of fetal bovine serum (FBS) and/or hematopoietic cytokines. Both mouse and human pluripotent cells can be successfully differentiated into CD34+ multi-lineage blood progenitors in a coculture, though the efficiency of hematopoietic differentiation significantly varies between different stromal cell lines and compositions of differentiation media [3].

Defined feeder-free, serum-free systems

These systems are designed to avoid the use of undefined, animal-origin components such as FBS and stroma cells to achieve highly reproducible and efficient outputs. Thus, PSCs can be plated on matrix protein collagen IV and differentiated into primitive CD34+CD43+ hematopoietic progenitors by exposure to BMP4, bFGF and VEGF. This initial differentiation is more efficient when accompanied with the hypoxic conditions (5% oxygen tension) that resemble the environment of a developing embryo. A further incubation of blood progenitors with the various combinations of cytokines yields maturation of CD71+CD235a+ erythroid cells, CD41a+ CD42b+ megakaryocytes, HLA-DR+CD1a+ dendritic cells, CD14+CD68+ macrophages, CD45+CD117+ mast cells and CD15+CD66+ neutrophils [4].

Despite the great progress achieved in the in vitro modeling of hematopoiesis, blood production from PSCs is still a variable process.  The final goal of intensive research in this area – a consistent production of engraftable cells, capable of reconstituting all blood lineages in the body, remains a major challenge. Finding critical intrinsic and extrinsic factors that can recreate the unique properties of a hematopoietic stem cell niche in vitro could advance the generation and expansion of PSCs-derived hematopoietic stem cells in the future.



1. Moreno-Gimeno I, Ledran MH, Lako M. Hematopoietic differentiation from human ESCs as a model for developmental studies and future clinical translations. FEBS J. 2010 Dec;277(24):5014-25.Review.

2. Chadwick K, Wang L, Li L, Menendez P, Murdoch B, Rouleau A, Bhatia M.Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood. 2003 Aug 1;102(3):906-15.

3. Vodyanik MA, Bork JA, Thomson JA, Slukvin II. Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood. 2005 Jan 15;105(2):617-26.

4. Salvagiotto G, Burton S, Daigh CA, Rajesh D, Slukvin II, Seay NJ. A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs. PLoS One. 2011 Mar 18;6(3):e17829.