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Keywords:

  • red cell transfusion;
  • stem cells;
  • cellular therapy

Summary

  1. Top of page
  2. Summary
  3. Ontogeny of haematopoiesis
  4. Haematopoiesis: the importance of microenvironment
  5. Somatic haematopoietic stem and progenitor cells
  6. Embryonic stem cells
  7. Induced pluripotent stem cells
  8. Challenges in scale-up
  9. Concluding remarks
  10. References

Whilst red cell transfusion is a well established cellular therapy, the problems of insufficiency of supply, transfusion transmitted infections and the requirement for immunological matching persist. The possibility of generating large numbers of O Rh D negative red cells at Good Manufacturing Practice grade as a route to circumvent these issues is therefore an attractive proposition. Significant numbers of erythrocytes can be generated from somatic haematopoietic stem cells, but it seems unlikely that these can provide sufficient volumes for large scale manufacture. However, human embryonic stem cells (hESC) and, potentially, induced pluripotent stem cells (iPSC), may provide a route to this objective. Red cell transfusion is an attractive goal for pluripotent stem cell-derived therapeutics because it is a well-characterised single cell suspension that lacks nucleated cells and has a low expression of human leucocyte antigen molecules, but many challenges remain in translating this cellular therapy to the clinic.

It is just over 200 years since the modern era of red cell transfusion was initiated by the work of John Leacock and James Blundell. However, red cell transfusion remains problematic in a number of important respects. Sufficiency of supply remains challenging at times in developed economies and can be a very serious problem in some parts of the world due to a lack of infrastructure to support widespread non-remunerated donation. In Europe and the United States, around 30 million units of red cell concentrate are transfused to around 6 million patients with a total population of around 700 million, giving a rate of 30–50 donations per thousand population. Although the rate of red cell transfusion has declined recently, probably as a result of improved clinical education and audit, the ageing demography of Western populations is likely to reverse this trend over the next 40 years due to increased demand from medical intervention and diminished supply due to fewer younger donors (Robert-Bobee, 2006). In other countries the situation is much more serious, for example in Africa last year, just 4·2 million units were collected in a total of 27 countries, giving a donation rate of just 6·9 donations per thousand.

The impact of virus transmission by blood transfusion is well recognised, with severe outbreaks of transfusion-transmitted hepatitis B virus (HBV), human immunodeficiency virus (HIV) and hepatitis C virus (HCV) over the past few decades. However, bacterial contamination from the skin or blood of the donor remains the most common transfusion transmitted infection overall and many countries continue to experience problems with new and emerging infections, such as variant Creutzfeldt Jacob Disease, West Nile Virus and Chikungunya Fever. Immune incompatibility also remains a problem; blood transfusion surveillance data continues to show that ABO incompatible transfusion due to clinical or laboratory error remains the most widespread cause of transfusion related morbidity and mortality. Several groups have demonstrated that its is possible to enzymatically cleave A and B antigens to produce ‘universal’ red blood cells, but this has not as yet found widespread clinical application (Olsson & Clausen, 2008).

Finally, an accumulating body of clinical evidence suggests that ageing red cells themselves may impact negatively on patient outcomes (Basran et al, 2006; Koch et al, 2008). The cause of these clinical findings is unclear, though it is known that erythrocytes undergo a number of biochemical changes during storage including depletion in adenine triphosphate and 2,3-diphosphoglycerate, changes in haemoglobin, and loss of membrane elasticity and function, leading to progressive sphero-echinocytosis, reduction in deformability and increased osmotic fragility (Wolfe, 1985; Berezina et al, 2002). Indeed biphasic erythrocyte recovery and survival post-transfusion has been shown to deteriorate markedly with duration of storage both in animal models and in man (Gilson et al, 2009).

The large scale generation of erythrocytes in vitro therefore represents an attractive though challenging proposition. In particular, it could help to overcome the issues relating to sufficiency of supply and the risk of microbiological contamination, notably from new and emerging infections. The preferential manufacture of group O RhD negative red cells would reduce the need for immunological matching and associated risks, for the majority of patients. Production of a cohort of newly matured red cells could improve red cell recovery and extend survival, affording greater therapeutic impact with less adverse effects. The realisation of this vision requires us to revisit our understanding of the ontogeny and in vivo control of erythropoiesis, and use this to address issues relating to control of stem cell proliferation, differentiation and maturation in an in vitro environment.

Ontogeny of haematopoiesis

  1. Top of page
  2. Summary
  3. Ontogeny of haematopoiesis
  4. Haematopoiesis: the importance of microenvironment
  5. Somatic haematopoietic stem and progenitor cells
  6. Embryonic stem cells
  7. Induced pluripotent stem cells
  8. Challenges in scale-up
  9. Concluding remarks
  10. References

In humans, primitive haematopoietic stem and progenitor cells (HSPC) of limited developmental potential are generated in the blood islands of the extra-embryonic yolk sack in the 21-day-old embryo (Tavian et al, 1999) (Fig 1). Erythroid cells are megaloblastic and nucleated and express predominantly the embryonic haemoglobins Gower 1 (ζ2ε2), Gower 2 (α2ε2) and Portland (ζ2γ2) (Palis & Segel, 1998). Embryonic haemoglobins have a higher oxygen affinity compared with adult haemoglobins with less cooperativity and pH sensitivity (Bohr effect). The earliest intra-embryonic CD34+ CD45+ cells have been identified in the pre-umbilical region of the dorsal aorta – the Aortic-Gonado-Mesonephros (AGM) region from day 27 human embryos (Medvinski & Dzierzak, 1996; Tavian et al, 1996, 1999, 2001; Taoudi et al, 2008). The aortic and vitelline endothelium gives rise to multi-lineage myelo-lymphoid progenitors (Oberlin et al, 2002) and to definitive haematopoiesis characterised by normoblastic enucleated erythrocytes synthesising foetal haemoglobin (α2γ2). HSPC have been detected in the embryonic liver from day 32 of development, probably as a result of colonisation from the AGM region (Tavian et al, 1999, 2001) and the focus of haematopoiesis changes to the liver and spleen at approximately 6 weeks gestation and thereafter, to the bone marrow from 20 weeks onwards, giving rise to definitive haematopoiesis characterised by expression of adult haemoglobins (α2β2 and α2δ2).

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Figure 1.  Generation of primitive and definitive haematopoiesis from the extra- and intra-embryonic mesoderm respectively.

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There is experimental evidence to suggest that the self-renewal potential of stem cells themselves diminishes with advancing gestational age (Lansdorp et al, 1993).

The haematopoietic ontogenic pathway therefore involves a complex temporal and spatial choreography between stem cells of various degrees of pluri- or oligopotency and their microenvironment(s).

Haematopoiesis: the importance of microenvironment

  1. Top of page
  2. Summary
  3. Ontogeny of haematopoiesis
  4. Haematopoiesis: the importance of microenvironment
  5. Somatic haematopoietic stem and progenitor cells
  6. Embryonic stem cells
  7. Induced pluripotent stem cells
  8. Challenges in scale-up
  9. Concluding remarks
  10. References

Haematopoiesis is conceptualised as a hierarchical scheme in which haematopoietic stem cells, comprising 1/104–106 of the nucleated cells in the marrow, are capable of self-renewal and multi-lineage differentiation to produce the progenitors, precursors and terminally differentiated cells of the blood and immune systems. In man, identification of haematopoietic stem cells themselves has proved challenging because it is not possible to carry out sequential repopulation studies. Immunophenotypically, CD34 is widely used as a marker of human HSPC, but this population is itself heterogeneous, containing early progenitors of a variety of different lineages and also endothelial cells. It is likely that the more primitive haematopoietic stem cells lie within the CD34 bright lineage negative population (CD34hi/Lin-). Several in vitro assays have been proposed including the human blast-colony forming assay, the high proliferative potential colony forming assay and the long-term culture initiating cell assay. It is likely that the most stringent test of human haematopoietic stem cell potential is offered by serial transplantation in immune deficient mice (Lapidot et al, 1993).

Gregory and Eaves (1978) described three stages of erythroid progenitor differentiation distinguished by physical and biological properties: day 8 (late) Erythroid Burst-Forming Unit(BFU-E), day 3 (early) BFU-E and Erythroid Colony-Forming Unit (CFU-E). Subsequently, erythroid progenitors mature through a series of erythroid precursors, the proerythroblast, basophilic erythroblast, polychromatic erythroblast and orthochromatic erythroblast before enucleation to form a reticulocyte and finally a mature erythrocyte. Loken et al (1987) and Okumura et al (1992) have described the changes in surface antigen expression during erythroid differentiation, with CD34lo BFU-E rapidly losing CD34 and CD45 expression, but continuing to express CD41 (glycoprotein IIb/IIIa) and human leucocyte antigen (HLA) for several population doublings, whilst acquiring expression of CD71 and increasing expression of CD36 (thrombospondin receptor), glycophorin A and ABO blood group antigens from the erythroblast stage onwards. Haemoglobin was expressed as expected, from the erythroblast onwards (Wada et al, 1990; Okumura et al, 1992; Nakahata & Okumura, 1994).

The haematopoietic microenvironment is highly structured, with a variety of non-haematopoietic cell populations including neural, vascular, adipocytic and fibroblastic elements (Lichtman, 1981). Several studies have suggested that in the bone marrow, primitive HSPC reside adjacent to the inner endosteal surface of the bone and migrate towards the blood vessels at the centre of the bone marrow cavity as they differentiate and mature (Lord, 1990; Garrett & Emerson, 2009). The regulation of HSPC proliferation and differentiation in vivo is controlled by the microenvironmental niche in which they reside, which is itself composed of a variety of cellular and extra-cellular matrix components (Spradling et al, 2001; Zhu & Emerson, 2004; Porter & Calvi, 2008). Taichman and Emerson (1994, 1998) first reported that osteoblasts are a key component of the HSPC niche and latterly, Calvi et al (2003) and Zhang et al (2003) have provided experimental evidence of this in vivo. Location is mediated by a range of cell adhesion molecules including members of the integrin (very late antigen (VLA)4, VLA5), immunoglobulin (intercellular adhesion molecule 1) and cadherin (E-cadherin) families. Manipulation of the niche in vivo or in vitro can be used as an indirect route to directing expansion and differentiation of HSPC. For example, Frisch et al (2009) have demonstrated that prostaglandin E2 preferentially expands HSPC through effects on both the cells themselves and their microenvironment.

A balance of positive and negative regulators are involved in the control of cell survival, proliferation and differentiation. For example, to differentiate into erythroblasts in semi-solid culture, BFU-E require stem cell factor (SCF), granulocyte-monocyte colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) and erythropoietin (EPO), whilst CFU-E require EPO alone. In contrast, tumour necrosis factor alpha (TNFα), Interferon-gamma (IFN-γ) and transforming growth factor-beta1 (TGF-β1) are powerful inhibitors of erythropoiesis (Zermati et al, 2000).

In vivo, developing erythroblasts from the proerythroblast onwards are found in close association with central resident macrophages – the so called erythroblastic island (Bessis et al, 1978). Several cell adhesion molecules have been described as mediating the attachment of erythroblasts to macrophages (Hanspal, 1997) including a divalent cation-dependent haemagglutinin (Morris et al, 1991), VLA4/vascular cell adhesion molecule 1 (Sadahira et al, 1995) and a 30 kD heparin binding erythroblast macrophage protein (Hanspal & Hanspal, 1994; Hanspal et al, 1998). After enucleation the nucleus is rapidly phagocytosed by macrophages through a novel divalent cation independent receptor (Qiu et al, 1995).

Oxygen tension also appears to play a role in haematopoiesis. Oxygen gradients exist within the in vivo bone marrow and HSPC are distributed in the bone marrow according to local oxygen levels (Parmar et al, 2007). In vitro, Cipolleschi et al (1997) have shown that severe hypoxia (1% oxygen) enhances the maintenance of more primitive BFU-E but inhibits the terminal expansion and maturation of erythroid clones, whilst more moderate hypoxia (3·5–7% oxygen) has been shown to increase the number of BFU-E without interfering with their terminal differentiation (Lu & Broxmeyer, 1985).

It seems clear, therefore, that the microenvironment required to support HSPC differs from that required to support erythroid precursor proliferation and terminal differentiation, an insight which has important implications for the optimisation of ex vivo culture systems.

Somatic haematopoietic stem and progenitor cells

  1. Top of page
  2. Summary
  3. Ontogeny of haematopoiesis
  4. Haematopoiesis: the importance of microenvironment
  5. Somatic haematopoietic stem and progenitor cells
  6. Embryonic stem cells
  7. Induced pluripotent stem cells
  8. Challenges in scale-up
  9. Concluding remarks
  10. References

Most early studies of haematopoiesis were based on the use of colony assays in semi-solid media followed in some cases by short term liquid culture. In this kind of experimental system, HSPC will develop into discrete colonies representing a limited number of cell divisions followed by terminal differentiation. The quantity of cells obtained is usually too low to allow further propagation and analysis, and the system represents a one-stage continuous culture with limited flexibility to sequentially manipulate the culture conditions.

These limitations can be overcome in liquid culture systems, which can be tailored to selectively support proliferation and differentiation of different cell types in temporal sequence. For example, Fibach et al (1989, 1991) described the generation of erythroid cells from peripheral blood mononuclear cells in a two phase liquid culture system. The first phase culture in the presence of conditioned medium from human bladder carcinoma cells resulted in an expansion of erythroid colony-forming cells (BFU-E and CFU-E). The lymphocytes were removed from the non-adherent fraction by monoclonal antibody-mediated complement lysis or cyclosporin A. The second phase culture, undertaken in the presence of EPO, a high concentration of foetal calf serum, albumin, 2-mercaptoethanol, glutamine and dexamethasone, resulted in a very high yield of orthochromatic normoblasts and enucleated erythrocytes. Wada et al (1990) used a modification of this approach to examine the sequential expression of various blood group antigens including those of the ABH, MN, P and Lewis systems.

Most groups have moved on from heterogeneous cell populations and poorly defined conditioned media to defined combinations of cytokines in order to achieve more precise control over cell proliferation and differentiation. Malik et al (1998) described the generation of human erythroid precursors in vitro from enriched CD34+ cells from bone marrow, cord blood and peripheral blood using a liquid culture cocktail of low concentration IL-3, GM-CSF and high concentration EPO. After 18–21 d in culture erythroblastic islands developed followed by enucleation of 10–40% of cells. The enucleated cells had the characteristics of reticulocytes and expressed γ globins (cord blood) and β globin (adult bone marrow and peripheral blood).

A higher level of expansion was reported by Panzenbock et al (1998), who cultured CD34+ cells from cord blood and granulocyte colony-stimulating factor (G-CSF) mobilised peripheral blood in culture medium supplemented by EPO, SCF, insulin-like growth factor 1 (IGF-1), dexamethasone and oestradiol, resulting in a 105-fold expansion in erythroid progenitors by days 15–18. Erythroid progenitors were recovered and cultured further in EPO and insulin to induce terminal differentiation to erythrocytes.

Freyssinier et al (1999) cultured CD34+ cells from cord blood or G-CSF mobilised apheresis collections for 7 d in serum-free conditions with SCF, IL-3 and IL-6, resulting in a 30-fold expansion in cell numbers and the appearance of high numbers of CD36+ cells. CD36 is an early marker of erythroid progenitors (and a late marker of monocytic and megakaryocytic cells) and, as expected, contained an enriched population of BFU-E and CFU-E. The CD36+ cells purified by immunomagnetic separation were further cultured in the same medium and cytokines with the addition of EPO for an additional 3 d, leading to a dramatic cell expansion and maturation to glycophorin A-positive erythroblasts and a small proportion of enucleated red cells. Overall, from 106 CD34+ cells input, the first phase culture generated in the order of 107 CD36+ cells at day 7 and 1·5 × 108 erythroid precursors after a further 3 d of secondary culture. Unlike Malik et al (1998), enucleation did not seem to be dependent on the presence of macrophages in this system.

Similarly, Miharada et al (2006) developed a method to produce enucleated erythrocytes from cord blood CD34+ cells in a liquid culture system including vascular endothelial growth factor (VEGF), IGF-II and mifepristone (a glucocorticoid antagonist). This system did not make use of feeder cells or macrophages but was capable of generating 4 × 1012 erythroid cells from a single donation (5 × 106 CD34+ cells). Maggakis-Kelemen et al (2003) also used a similar culture system to that of Malik et al (1998) but with the addition of dimethylsulphoxide (DMSO), ferrous citrate and transferrin. They found that the latter dramatically enhanced haemoglobin synthesis but that whilst precursor cells showed a high degree of deformability, the cultured reticulocytes and erythrocytes showed reduced shear modulus compared to controls.

Functional analysis of the generated erythroid cells was extended in the study of Douay and colleagues who developed a protocol for differentiation of cord blood CD34+ HSPC in defined media, using a 3-step protocol: stimulation of CD34+ HSPC using SCF, Flt3-ligand and thrombopoietin (TPO); followed by expansion of erythroid progenitors using SCF, EPO and IGF-1; and terminal erythroid differentiation using EPO and IGF-1 (Douay, 2001; Neildez-Nguyen et al, 2002). The system produced 2 × 105-fold amplification with dominant erythroid rather than myeloid differentiation. They have subsequently modified this protocol for application also to adult blood and bone marrow (Giarratana et al, 2005; Douay & Andreu, 2007) again involving three steps: culture in liquid medium in the presence of SCF, IL-3 and EPO for 8 d; culture with EPO in the presence of a murine stromal cell line (MS5) or human mesenchymal cells for 3 d; followed by the stromal cells alone without growth factors for up to a further 10 d. Using this 15-day protocol they obtained a mean amplification of CD34+ cells of 20 000-fold from bone marrow or blood, 30 000-fold from G-CSF mobilised peripheral blood and 200 000-fold from cord blood (Douay et al, 2009). Commitment towards the erythroid lineage was morphologically apparent from day 8, with terminal differentiation to 65–80% reticulocytes by day 15 and maturation to erythrocytes from days 15–18 evidenced by loss of transferrin receptor (CD71) expression, 90–100% enucleation and typical morphological characteristics. These cells also showed normal glucose 6 phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) enzyme levels, membrane deformability and oxygen dissociation characteristics and similar survival to native red cells following carboxyfluorescein diacetate succinimidyl ester (CFSE) labelling and intraperitoneal infusion into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice.

This group have additionally reported that the degree of cell expansion is related to the duration of culture during the first step of their protocol and when this was prolonged for an additional 3 d, the level of expansion increased up to 106-fold for cord blood or mobilised peripheral blood and 2 × 105-fold for bone marrow. Taking into account an estimate of 1–2 × 108 CD34+ cells in an apheresis donation and 1 × 107 in a cord blood donation, this suggests that up to 5–10 units of red cell concentrate could be produced from a single donation. These findings are very encouraging, however, they required a return to the use of a supportive stromal layer particularly in the later stages of maturation (Douay & Giarratana, 2009).

Fujimi et al (2008) have used a 4-phase protocol to culture cord blood CD34+ cells on hTERT transduced human stromal cells in serum-free medium containing SCF, Flt-3 ligand and TPO, which expanded the CD34+ cells approximately 1000-fold and the total cells 10 000-fold. In the second phase these were cultured in SCF, IL-3 and EPO to expand them a further 100-fold and differentiate them into erythroblasts. In a parallel culture system, expanded cells from the first phase were cultured with GM-CSF, monocyte colony-stimulating factor (M-CSF), IL-3 and SCF to generate monocytes, which were incubated with serum and M-CSF to fully differentiate them to macrophages. In the third phase, erythroblasts were combined in co-culture with the macrophages in the presence of EPO to expand and differentiate them. Erythrocytes were then separated from nucleated cells using a leucodepletion filter. The authors reported that from one unit of cord blood it was possible to generate 6 × 1012 red cells with normal surface marker expression, haemoglobin content, oxygen dissociation and in vivo clearance. Hence, it seems that in order to achieve a high degree of enucleation and normal functionality some interaction with stroma or macrophages is required in most of the differentiation systems described so far (Kawano et al, 2003).

The stromal cells that have been shown to be useful are often of animal origin, poorly characterised and can be variable, and as such are unlikely to be acceptable for any eventual clinical applications. In an attempt to eliminate this need to use stroma, Baek et al (2008, 2009) have developed a protocol for production of clinical grade erythrocytes from cord blood CD34+ cells by replacing stroma with poloxamer 188, a polymer known to be cytoprotective against hydrodynamic stress. Their studies suggest that a key factor for production of viable erythrocytes in vitro is the membrane stability at the time of enucleation, when fragile cells are haemolysed. Cells cultured in poloxamer 188 showed enhanced survival, with 95% enucleation and improved erythrocyte morphology and resistance to osmotic challenge.

Thus overall, although it is clear that whilst bone marrow, cord blood and mobilised peripheral blood can be used as sources of HSPC to generate sufficient functional red cells for perhaps as many as 5–10 units per donation, truly large scale manufacture is not currently possible because of the limited replication capacity of unmodified somatic stem cells.

Embryonic stem cells

  1. Top of page
  2. Summary
  3. Ontogeny of haematopoiesis
  4. Haematopoiesis: the importance of microenvironment
  5. Somatic haematopoietic stem and progenitor cells
  6. Embryonic stem cells
  7. Induced pluripotent stem cells
  8. Challenges in scale-up
  9. Concluding remarks
  10. References

Human embryonic stem cells (hESC) were first isolated in 1998 (Thomson et al, 1998). They are derived from the inner cell mass of 3–5 d blastocysts by immunosurgery and grown out on feeder cells. hESC are undifferentiated pluripotent cell lines with a normal karyotype, which can be maintained indefinitely in culture due to high telomerase activity. Pluripotency is maintained by culture in basic fibroblast growth factor (bFGF) and either feeder cells or conditioned media. They can be differentiated into cells and tissue of all three embryonic germ lineages (ectoderm, mesoderm and endoderm) (Itskovitz-Eldor et al, 2000), thereby opening the possibility of providing a new source of all cell types for cellular therapy (Keller, 2005). They express cell surface markers, such as stage-specific embryonic antigen (SSEA) 3, SSEA-4, tumour rejection antigen (Tra)-1–60 and Tra-1–81 in addition to the transcription factors Oct 3/4, Nanog and Sox2. hESC also express some cell surface markers associated with HSPC, including CD117 (c-kit), CD133 (AC133) and CD90 (Thy-1) and LMO2, AML1 and c-MYB, as well as KDR (Flk1) and FGFR1 (Flt2) genes, characteristic of the endothelial pathway. Attention has been drawn to the extent to which the haematopoietic differentiation of hESC recapitulates the ontogeny of the haematopoietic system (Snodgrass et al, 1992; Daley, 2003; Kyba & Daley, 2003). Several studies have described the development of haemangioblast–like cells with both haematopoietic and endothelial potential, which define the onset of haematopoiesis in hESC differentiation culture, reminiscent of the ontogeny of haematopoiesis (vide supra) (Wang et al, 2004; Zambidis et al, 2005, 2008; Kennedy et al, 2007).

Several groups have demonstrated haematopoietic differentiation of hESC using two different approaches (Fig 2).

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Figure 2.  Schematic representation of the in vitro differentiation of human embryonic stem cells into haematopoietic stem cells showing the two main options used by different research teams and the subsequent differentiation into erythroid cells.

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The first approach entails the removal of hESC from feeder cells and culture in suspension, which leads to the formation of embryoid bodies (EB), 3-dimensional partially structured aggregates of differentiating cells. Chadwick et al (2003) demonstrated that bone morphogenetic protein 4 (BMP4), a ventral mesoderm inducer, strongly promotes haematopoietic differentiation in the context of a combination of haematopoietic cytokines (SCF, Flt-3 ligand, IL-3, IL-6 and G-CSF). It has also been demonstrated that addition of VEGF-A165, which is normally produced in response to hypoxia, further promotes erythropoiesis, particularly when augmented by EPO (Cerdan Rouleau & Bhatia, 2004).

Chang et al (2006) have described the generation of erythroid cells from hESC by subsequent processing of cells present at either early or late stages of EB formation, whereas Zambidis et al (2005) have more closely defined the progress of differentiation in EB through haematoendothelial, primary and definitive stages of haematopoiesis. Much of the seminal work characterising haematopoiesis in EB has come from the Keller group who have demonstrated that at 3–4 d of EB differentiation give rise to cell colonies expressing both endothelial and haematopoietic markers (KDR (FLK1) and CD117) (Kennedy et al, 2007). Other groups have also shown the appearance of CD34+ cells from 5 d of culture, peaking at day 12–15 with evidence of primitive erythropoiesis between 7 and 12 d and definitive erythropoiesis emerging from days 12–20 (Chadwick et al, 2003; Cerdan, Rouleau & Bhatia, 2004; Zambidis et al, 2005).

The use of EBs in the initial stages of haemopoietic differentiation of hESC is somewhat limited by the poor efficiency of commitment and the low number of cells that can be generated by this method (Keller et al, 1993). One of the most successful protocols for haematopoietic commitment was described by the Stanley/Elefanty group (Ng et al, 2005) who used the spin-EB method to force the aggregation of hESC into EBs in serum-free conditions. This method generated up to 20% CD34+ cells after 11 d of suspension culture in EBs; further analysis showed that the method was highly reproducible and determined that the frequency of HSPC was approximately 1 in 500 input cells. These outputs are significantly better than other EB-based methods, particularly in serum-free conditions (Tian et al, 2004).

One of the more clinically relevant published studies is that of Lu et al (2008), who have demonstrated the differentiation of hESC into functional erythrocytes on a large scale using a 4-step procedure. The first step involved the formation of embryoid bodies by culturing hESC in serum-free medium, BMP4, VEGF and bFGF with the addition of SCF, TPO and FLT-3 ligand after 48 h. Step 2 involved dissociation of the EBs with trypsin and culture of the single cell suspension in blast colony growth medium, bFGF and recombinant tPTD-HoxB4 fusion protein for 10 d. The 3rd step consisted of further culture with EPO for 5 d and thereafter with SCF, EPO and methylcellulose. Finally the erythroid cells were enriched through a plastic adherence step. Starting with one 6-well plate of hESC, they generated 1011–1012 nucleated erythroid cells with comparable oxygen dissociation curves, response to pH (Bohr effect) and 2·3 diphosphoglycerate (DPG) activity to normal adult red cells, despite expressing mainly embryonic and foetal globins. However, after 28 d of culture only 16% of the cells expressed β globin and the majority maintained expression of γ-globin, and just 10–30% of the cells became enucleated. Also, in order to achieve this impressive expansion in cell numbers during differentiation, it was necessary to treat the cells with a recombinant HoxB4 protein. HOXB4 is a homeobox gene, the protein product of which functions as a transcriptional regulator and which was shown to be involved in the proliferation and self-renewal of HSPC (Klump et al, 2005). HoxB4 has also been shown to enhance haematopoiesis from embryonic stem cells (Bowles et al, 2006) although it remains unclear whether it increases the proportion of cells committed to haemopoietic lineages (Kyba et al, 2002; Lee et al, 2008; Unger et al, 2008) or expands already committed HSPC (Wang et al, 2005a).

Thus, whilst it seems unlikely that undirected embryoid body culture will provide a route to generation of pure populations of red cells, given the relatively poor efficiency of haemopoietic commitment and the need for greater expansion, it does have the advantage of recapitulating the early stages of haematopoietic development (Chang et al, 2006).

The second approach entails co-culturing hESC with murine stromal lines, such as S17 or OP9 (Vodyanik et al, 2005), or the yolk sac endothelial cell line C166 (Kaufman et al, 2001) to promote or support the commitment of pluripotent hESC specifically to the haematopoietic lineage.

OP9 stroma has been shown to support the production of up to 10 times more HSPC compared with other murine bone marrow stromal cell lines (Vodyanik et al, 2005). This group demonstrated production of up to 20% CD34+ cells and isolated up to 107 CD34+ cells from a similar number of hESC. They used CD43 (leukosialin) as a marker to separate the haematopoietic from the endothelial (CD34+, CD43, CD31+, KDR+) and mesenchymal (CD34+, CD43, CD31, KDR) stem cell populations and further showed that CD43 was also expressed on more mature CD34 erythroid progenitors (Vodyanik et al, 2006).

In an attempt to recapitulate the microenvironment of developing haematopoietic cells in the embryo, Ledran et al (2008) showed that co-culture of hESC with monolayers of stromal cells derived from murine AGM, foetal liver, or other developmental niches enhanced early differentiation of CD34+ HSPC. These HSPC were capable of primary and secondary engraftment into immunodeficient mice and also generated both erythroid and myeloid cells in colony-forming assays. Interestingly, the AGM-derived AM20·1B4 line resulted in the best reconstituting capacity suggesting, perhaps logically, that hESC may be better supported by stroma from the sites where definitive haematopoiesis first occurs, rather than sites such as the bone marrow that is more active in the adult.

Several groups have now described the derivation and culture of hESC in animal-free conditions, using human foreskin fibroblasts as feeder cells in media containing serum replacement and basic FGF (Amit et al, 2003; Hovatta et al, 2003; Koivisto et al, 2004). Genbacev et al (2005) have derived and propagated hESC in serum–free conditions on human early-gestation placental fibroblasts and subsequently adapted them for growth on human placental laminin substrate in a defined medium. Amit et al (2004) have further described a feeder-free and serum-free system involving culture of hESC on fibronectin with TGF-β, bFGF and leukaemia inhibitory factor (LIF).

The Bouhassira group have shown that hESC culture on hTERT-transformed human foetal liver cell lines can generate 2–10% CD34+ cells, which can then be differentiated into an erythroid population using a 4-stage liquid culture protocol (Qiu et al, 2005; Olivier et al, 2006). Using this system, they have further reported that increasing the duration of culture on the FH-hTERT cell line from 14 to 21 or 35 d lead to a 5000-fold increase in cell number of erythroid cells with differentiation of the predominant population of megaloblastic nucleated erythroblasts to a small population of normoblastic enucleated red cells with an increase in α/ζ and γ/ε chain ratios measured at the protein level by High Pressure Liquid Chromatography (HPLC), recapitulating in vitro the shift from primitive to definitive haematopoiesis (Olivier et al, 2006; Olsen et al, 2006; Qiu et al, 2008.

These two methods of inducing haematopoietic differentiation have also been combined; Wang et al (2005b) demonstrated that the treatment of hESC during EB formation with a combination of low dose cytokines and human bone marrow stroma significantly increased haematopoietic differentiation. In a very promising study, Ma et al (2008) demonstrated that hESC co-cultured with murine foetal liver-derived stromal cells after an initial EB stage, produced large numbers of erythroid progenitors that underwent definitive differentiation with a shift from ε-globin expression to β-globin expression and production of enucleated erythrocytes. Comparative analysis against cord blood-derived progenitors showed similar expression of glycophorin A, CD71 and CD81, glucose 6 phosphate dehydrogenase activity and oxygen carrying capacity. However, they were only able to achieve a 100-fold expansion from hESC to mature erythrocytes; clearly, this is insufficient for clinical application production and significant improvements of scale need to be made.

The key to improving the process and making it possible to efficiently achieve haematopoiesis and erythroid maturation at scale and in a manner that is current good manufacturing practice (cGMP)-compliant, will be to identify and reconstruct the array of signals delivered by soluble factors and cell to cell contact from stroma. A number of these have already been identified for hESC interactions and include TGFβ1 and 3 (Ledran et al, 2008) and both canonical (Woll et al, 2008) and non-canonical wnt signalling (Vijayaragavan et al, 2009). However, other pathways including hedgehog, notch and adhesion molecule signalling are likely to be involved and more evidence can be gleaned from other sources, including the elegant studies of the adult haematopoietic niche of Wilson and Trumpp (2006). Additional important information can be gleaned from studies of haemato-endothelial or haemangioblast development, which have already highlighted the importance of angiotensin converting enzyme (ACE/CD143) (Zambidis et al, 2008), prostaglandins (North et al, 2007) and nitric oxide (North et al, 2009).

Induced pluripotent stem cells

  1. Top of page
  2. Summary
  3. Ontogeny of haematopoiesis
  4. Haematopoiesis: the importance of microenvironment
  5. Somatic haematopoietic stem and progenitor cells
  6. Embryonic stem cells
  7. Induced pluripotent stem cells
  8. Challenges in scale-up
  9. Concluding remarks
  10. References

Considerable excitement has arisen over the past 2–3 years around the possibility of inducing pluripotency in tissue cells though transfection with a combination of transcription factors, POU5F1, KLF4, SOX2 and MYC (Takahashi & Yamanaka, 2006). Murine fibroblasts transfected with these genes have been shown to acquire an embryonic stem cell phenotype, form teratomas in immunodeficient mice, contribute to adult tissues derived from all three germ layers after injection into blastocysts and undergo germline transmission (Okita et al, 2007; Wernig et al, 2007). This approach has also been explored in human cells, Takahashi et al (2007) used these four genes to transform neonatal and adult human fibroblasts into induced pluripotent stem cells (iPSC). Yu et al (2007) have shown that lentiviral transfection of Oct4, Sox2 in combination with NANOG and LIN28 can also generate human iPSC, thereby avoiding the oncogenic potential of c-Myc. Subsequently iPS cells generated from human sources have been shown to generate haematopoietic cells using methods similar to those for hESC (Park et al, 2008; Lengerke et al, 2009; Seifinejad et al, 2009). Methods to create iPSC are quickly improving and it is already possible to produce them without insertion of genetic material by either ectopic expression of the four factors (Stadtfeld et al, 2008), the use of an excisable transposon vector (ePiggyBac, Lacoste et al, 2009) or even by providing the four factors in the form of recombinant protein coupled to arginine-rich cell penetrating peptide (Zhou et al, 2009). It is clearly still very early in the development of this approach, but this progress, linked to the unravelling of the potential of small molecules to replace the transcription factors (Lin et al, 2009), should promote the use of iPSC especially if the observation that they retain some differentiation characteristic of the cells they originate from is confirmed (Marchetto et al, 2009).

Challenges in scale-up

  1. Top of page
  2. Summary
  3. Ontogeny of haematopoiesis
  4. Haematopoiesis: the importance of microenvironment
  5. Somatic haematopoietic stem and progenitor cells
  6. Embryonic stem cells
  7. Induced pluripotent stem cells
  8. Challenges in scale-up
  9. Concluding remarks
  10. References

Given that each red cell unit contains around 2·5 × 1012 erythrocytes and around 2·2 million red cell transfusion are administered in the UK each year (an estimated 80 million per annum world wide), large scale production to replace part or most of the existing blood supply would require production in the order of between 1017–1020 erythrocytes per annum. Cell production in these orders of magnitude will require substantive advances in the management of both cell proliferation and cell density in vitro.

Increased cell proliferation and density can be achieved in long term bone marrow cultures in which a stromal layer develops maintains the interaction between HSPC and the bone marrow microenvironment in vitro (Dexter et al, 1977; Gartner & Kaplan, 1980). A bioreactor perfusion system in a small culture chamber has been developed to study the production and consumption of growth factors and the effects of varying oxygen tension in long term bone marrow (Koller & Palsson, 1993; Koller et al, 1993, 1995, 1996, 1998; Palsson et al, 1993). The authors have shown that frequent media exchanges enabled an increase in cell density within the culture from 7·5 × 104/cm2 to 6·0 × 105/cm2 and lead to an 8- to 10-fold increase in cell, 10- to 30-fold increase in CFU-GM and 10-fold increase in Long Term Culture Initiating Cell (LTCIC) numbers, dependent on oxygen tension, seeding density and time of harvest. LTCIC output continued to increase up to a cell density of 1·2 × 106/cm2. The group has extended this work to mobilised peripheral blood and cord blood showing 50-, 80- and 20-fold expansion in total cells, CFU-GM and LTCIC respectively, over 14 d (Van Zant et al, 1994). At higher cell inoculum, expansions were equivalent with or without stroma. Optimisation of a clinical scale automated production system demonstrated the production of >107 CFU-GM from 1 × 108 nucleated cord blood cells, a 300-fold expansion. It has become clear that different measures of culture performance are optimised under different culture conditions. For example, systematic variation of inoculum density, medium exchange interval and preformed stroma showed that all three interacted strongly, differentially influenced culture performance and could be manipulated to achieve the optimisation of different performance criteria. This work reinforces the point that the performance of ex vivo haematopoietic cell cultures can be measured by more than one criterion and different measures of performance are optimised under different conditions (Koller et al, 1996).

More reproducible immortalised human stromal cell lines have been developed from bone marrow and cord blood by De Angeli et al (2004) and have been shown to be capable of replacing the use of bone marrow feeder layers.

However, Douay (2001) has drawn attention to the need for completely defined and standardised culture media, the impact of variation in cytokine mix and concentration on differential expansion of different cell subsets, and the need to change the culture environment, as well as the impact of cell density and oxygen tension. This has been extended by several groups, which have experimented with perfusion systems to prevent depletion of metabolic substrates, cytokines and oxygen in the medium, alteration of pH and accumulation of waste products (Koller et al, 1993; Van Zant et al, 1994; Sandstrom et al, 1995).

Concluding remarks

  1. Top of page
  2. Summary
  3. Ontogeny of haematopoiesis
  4. Haematopoiesis: the importance of microenvironment
  5. Somatic haematopoietic stem and progenitor cells
  6. Embryonic stem cells
  7. Induced pluripotent stem cells
  8. Challenges in scale-up
  9. Concluding remarks
  10. References

Considerable progress has been made over the past decade in demonstrating that it is possible to generate large numbers of erythrocytes from stem cells in vitro. Whilst HSPC derived from somatic sources produce functional erythrocytes and an amplification of up to five red cell transfusion unit equivalents, large scale manufacture is likely to require pluripotent stem cell lines. Here, the problem is that of replicating or shortcutting the ontogenic steps of haematopoiesis in order to produce functional adult erythrocytes. Regardless of the source, there are still many challenges to overcome in terms of scale-up to produce large numbers of erythrocytes (Table I). It is likely that a broad transformation in in vitro culture and bioreactor design will be required to realise the prospect of manufacturing red cells for transfusion.

Table I.   Comparison of prospective clinical utility of red cells derived from somatic, embryonic and induced pluripotent stem cells.
 Somatic stem cellsEmbryonic stem cellsInduced pluripotent stem cells
  1. cGMP, current good manufacturing practice; hESC, human embryonic stem cells; QC, quality control.

Self-renewal capacityLimitedIndefiniteIndefinite
Haematopoietic differentiationDemonstratedDemonstratedDemonstrated
Complexity of ex vivo culture protocolModerate: within the scope of current technologyChallenging: requires further innovationChallenging: requires further innovation
ScalabilityLimitedPotentially unlimitedPotentially unlimited
Immune compatibilitySimilar to current problem with blood supplyPotential for cGMP ‘universal donor’ cell linesCould target ‘universal donors’ with specific antigenic profiles.
Risk of infectionRelated to sterility of harvest, duration and complexity of culture and number of recipientsHigher due to increased duration and complexity of culture and number of recipientsHigher due to increased duration and complexity of culture and number of recipients
Risk of neoplasiaLowUncertain: genetic instability seen is some hESC linesPotentially high if oncogenes and/or insertion vectors used
Quality controlWithin the scope of current technologyRequires further innovation in QCRequires further innovation in QC
Regulatory complianceWithin the scope of current regulatory frameworkRequires further development of regulatory frameworkRequires further development of regulatory framework

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  2. Summary
  3. Ontogeny of haematopoiesis
  4. Haematopoiesis: the importance of microenvironment
  5. Somatic haematopoietic stem and progenitor cells
  6. Embryonic stem cells
  7. Induced pluripotent stem cells
  8. Challenges in scale-up
  9. Concluding remarks
  10. References
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