Erythroid production

Abstract

This invention provides method and media suitable for inducing and supporting the differentiation of stem cells into erythroid cells.

Claims

1. A method of inducing the differentiation of embryonic stem cells or induced pluripotent stem cells into erythroid cells, said method comprising: contacting the embryonic stem cells or induced pluripotent stem cells with BMP4, VEGF, Wnt3A, ActivinA and a GSK3 inhibitor under conditions which induce the formation of embryoid bodies; contacting the embryoid bodies with BMP4, VEGF, Wnt3A, ActivinA, FGF-, SCF, -estradiol and a GSK3 inhibitor; and dissociating the embryoid bodies to form dissociated embryoid bodies; and contacting the dissociated embryoid bodies with BMP4, VEGF, FGF-, SCF, IGF2, TPO, heparin, -estradiol and IBMX and then with hydrocortisone, SCF, Flt3L, BMP4, IL3, IL11, EPO and IBMX, to yield erythroid cells, wherein the GSK3 inhibitor is Inhibitor VIII or CHIR99021.

2. The method of claim 1, wherein the erythroid cells are characterised by expression of one or more haematopoietic/erythroid markers selected from the group consisting of: CD31; CD34; CD36; CD41a; CD43; CD45; CD71; and CD235a.

3. The method of claim 1, wherein the method is free of feeder cells.

4. The method of claim 1, wherein the step of contacting the embryonic stem cells or induced pluripotent stem cells with BMP4, VEGF, Wnt3A, ActivinA and a GSK3 inhibitor under conditions which induce the formation of embryoid bodies is carried out for 1, 2 or 3 days.

5. The method of claim 1, wherein the step of contacting the embryonic stem cells or induced pluripotent stem cells with BMP4, VEGF, Wnt3A, ActivinA and a GSK3 inhibitor under conditions which induce the formation of embryoid bodies is carried out for 2 days.

6. The method of claim 1, wherein the step of contacting the embryoid bodies with BMP4, VEGF, Wnt3A, ActivinA, FGF-, SCF, -estradiol and a GSK3 inhibitor is carried out for 0.5, 1 or 2 days.

7. The method of claim 1, wherein the step of contacting the embryoid bodies with BMP4, VEGF, Wnt3A, ActivinA, FGF-, SCF, -estradiol and a GSK3 inhibitor is carried out for 1 day.

8. The method of claim 1, wherein the step of contacting the dissociated embryoid bodies with BMP4, VEGF, FGF-, SCF, IGF2, TPO, heparin, -estradiol and IBMX is carried out for 1, 2 or 3 days.

9. The method of claim 1, wherein the step of contacting the dissociated embryoid bodies with BMP4, VEGF, FGF-, SCF, IGF2, TPO, heparin, -estradiol and IBMX is carried out for 2 days.

10. The method of claim 1, wherein the step of contacting the dissociated embryoid bodies with hydrocortisone, SCF, Flt3L, BMP4, IL3, IL11, EPO and IBMX is carried out for 4, 5, 6, 6.5, 7, 7.5, 8, 9, 10, 10.5, 11, 11.5, 12, 13 or 14 days.

11. The method of claim 1, wherein the step of contacting the dissociated embryoid bodies with hydrocortisone, SCF, Flt3L, BMP4, IL3, IL11, EPO and IBMX is carried out for 7 days.

12. The method of claim 1, wherein, in the step of contacting the embryonic stem cells or induced pluripotent stem cells with BMP4, VEGF, Wnt3A, ActivinA and a GSK3 inhibitor under conditions which induce the formation of embryoid bodies, the embryonic stem cells or induced pluripotent stem cells are contacted with: BMP4 at a concentration of 10 ng/ml; VEGF at a concentration of 10 ng/ml; Wnt3A at a concentration of 10 ng/ml; ActivinA at a concentration of 5 ng/ml; and the GSK3 inhibitor at a concentration of 2 M.

13. The method of claim 1, wherein, in the step of contacting the embryoid bodies with BMP4, VEGF, Wnt3A, ActivinA, FGF-, SCF, -estradiol and a GSK3 inhibitor, the embryoid bodies with: BMP4 at a concentration of 20 ng/ml; VEGF at a concentration of 30 ng/ml; Wnt3A at a concentration of 10 ng/ml; ActivinA at a concentration of 5 ng/ml; FGF- at a concentration of 10 ng/ml; SCF at a concentration of 20 ng/ml; -estradiol at a concentration of 0.4 ng/ml; and the GSK3 inhibitor at a concentration of 2 M.

14. The method of claim 1, wherein, in the step of contacting the dissociated embryoid bodies with BMP4, VEGF, FGF-, SCF, IGF2, TPO, heparin, -estradiol and IBMX, the dissociated embryoid bodies with: BMP4 at a concentration of 20 ng/ml; VEGF at a concentration of 30 ng/ml; FGF- at a concentration of 10 ng/ml; SCF at a concentration of 30 ng/ml; IGF2 at a concentration of 10 ng/ml; TPO at a concentration of 10 ng/ml; heparin at a concentration of 5 g/ml; -estradiol at a concentration of 0.4 ng/ml; and IBMX at a concentration of 50 M.

15. The method of claim 1, wherein, in the step of contacting the dissociated embryoid bodies with hydrocortisone, SCF, Flt3L, BMP4, IL3, IL11, EPO and IBMX, the dissociated embryoid bodies with: hydrocortisone at a concentration of 1 M; SCF at a concentration of 50 ng/ml; Flt3L at a concentration of 16.7 ng/ml; BMP4 at a concentration of 6.7 ng/ml; IL3 at a concentration of 6.7 ng/ml; IL11 at a concentration of 6.7 ng/ml; EPO at a concentration of 1.3 U/ml; and IBMX at a concentration of 50 M.

16. The method of claim 1, further comprising a step of contacting the yielded erythroid cells with hydrocortisone, SCF, IGF1, IL3, Il11 and EPO.

17. The method of claim 16, wherein the step of contacting the yielded erythroid cells with hydrocortisone, SCF, IGF1, IL3, Il11 and EPO is carried out for 5, 6, 6.5, 7, 7.5 or 8 days.

18. The method of claim 16, wherein the step of contacting the yielded erythroid cells with hydrocortisone, SCF, IGF1, IL3, Il11 and EPO is carried out for 7 days.

19. The method of claim 16, wherein, in the step of contacting the yielded erythroid cells with hydrocortisone, SCF, IGF1, IL3, Il11 and EPO, the yielded erythroid cells are contacted with: hydrocortisone at a concentration of 1 M; SCF at a concentration of 20 ng/ml; IGF1 at a concentration of 20 ng/ml; IL3 at a concentration of 6.7 ng/ml; IL11 at a concentration of 6.7 ng/ml; and EPO at a concentration of 2 U/ml.

Description

DETAILED DESCRIPTION

(1) The present invention will now be described in detail with reference to the following figures which show:

(2) FIG. 1: Adherent culture (d3-10) is not required for expansion & differentiation. Histogram representative of total fold amplification of erythroid differentiation culture of hESC line H1 cultivated in the presence of Inhibitor VIII on normal tissue culture treated surface or on ultra low adherence surface between day 3 and day 10.

(3) FIG. 2: Effect of GSK inhibitor on phenotype at d10. Flow cytometry analysis of differentiating hPSCs at day 10 of erythroid culture. All the cells tested show a high positivity for the pan hematopoietic CD43 antigen. The presence of CD34, CD31, CD41 and CD235a indicate that the analyzed cells are either at an hemangioblastic or shortly post hemangioblastic stage. The differences observed between H1 and iPSC reflect the differential kinetics of differentiation intrinsic to these cell lines.

(4) FIGS. 3A and 3B: >90% CD235a (GlyA+) erythroid cells at d24. FIG. 3A. Flow cytometry analysis of differentiated hPSCs at day 28 in erythroid culture conditions. The presence of transferring receptor (CD71) on more of 80% of the cells analysed show that the cells are still in expansion phase and the presence of glycophorin A (CD235a) on more than 95% of the cells analysed while CD31, CD34 and CD41 have disappeared (data not shown) is a good indicator of the erythroid character of the cells. FIG. 3B. Rapid Romanovski staining of a cytospin preparation of hiPSC differentiated into erythroid cells at day 28, the cells are mainly orthochromatic normoblasts.

(5) FIG. 4: GFs and GSK inhibitors are additive. Histogram representative of cumulative fold amplification during of erythroid differentiation culture of iPS line in absence or presence of Activin A.

(6) FIGS. 5A and 5B: InhibVIII increases cell numbers in multiple lines. FIG. 5A. Histogram representative of total fold amplification of erythroid differentiation culture of hESC lines RC9 and H1 in absence or presence of inhibitor VIII, FIG. 5B. Histogram representative of total fold amplification of erythroid differentiation culture of iPS line in absence or presence of inhibitor VIII, both conditions included IBMX.

(7) FIGS. 6A and 6B: IBMX further increases expansion. FIG. 6A. Histogram representative of cumulative fold amplification over time of erythroid differentiation culture of H1 (hESC) or hiPSC without any small molecules or with combinations of GSK3 inhibitors and IBMX using the standard option of 7 days in cytokine mix A. FIG. 6B. Histogram representative of cumulative fold amplification over time of erythroid differentiation culture of H1 (hESC) or hiPSC without any small molecule or with combinations of GSK3 inhibitors and IBMX using the prolonged period of 11 days in cytokine mix A.

(8) FIG. 7: InhVIII+IBMX Increase key molecular markers in iPSC. Histograms representative of mRNA expression for a set of genes involved in erythropoiesis. Expression was determined by RTqPCR of differentiating hPSCs +/ Inhibitor VIII alone or Inhibitor VIII+IBMX at day 0, 10, 17 and 24 of erythroid culture.

(9) FIGS. 8A-8D: Globins are almost exclusively fetal not embryonic. HPLC analysis of the globins produced by the differentiated hPSCs. The disappearance of haemoglobin Gower 1 (2 2 chains) is noticeable in erythroid cells derived from iPSCs (FIG. 8B) and hESCs H1 (FIG. 8C), compared with FIG. 8A, which exhibit a mixed expression of embryonic and fetal globins. The hPSC derived cells exhibit an HPLC profile similar to that obtained from control cells of fetal origin (FIG. 8D). The difference observed in globin chain may be due to some residual haemoglobin Portland (2 2 chains).

(10) FIG. 9: Comparison of cell numbers with wnt3a vs. wnt5a.

(11) FIGS. 10A and 10B: Effect of SC1+SR1 on iPSC (FIG. 10 A) or hESC (FIG. 10B) amplification during differentiation.

(12) FIG. 11: Comparison of cells at day 30 after culture +/SR1 and SC1.

(13) Methods

(14) The human pluripotent stem cells (hPSC) are maintained undifferentiated in Stempro medium (Life Technologies) on Cellstart (Life Technologies) and passaged approximately every 7 days, depending on their confluence, using the EZpassage tool (Life Technologies).

(15) For differentiation, confluent hPSC are cut into squares with the EZpassage tool (Life Technologies) and plated at 50010.sup.3/well on Ultra low adherence six well plates (Corning) in 3 ml/well of Stemline II (Sigma) to allow them to form embryoid bodies (EBs).

(16) On day 0 in order to induce differentiation, the following cytokines are added: Bone Morphogenic Protein 4 (BMP4) (10 ng/ml), Vascular Endothelium Growth Factor 165 (VEGF) (10 ng/ml), Wnt3A (and/or Wnt5A) (10 ng/ml), ActivinA (5 ng/ml) and Inhibitor VIII (2 M). GSK3 Inhibitor VIII is a specific name for the Merck product (361549), it is also called AR-A014418 or N-(4-Methoxybenzyl)-N-(5-nitro-1,3-thiazol-2-yl)urea

(17) On day 2 of differentiation (48 hours old EBs), a new set of cytokines is added in 0.5 ml/well of Stemline II; BMP4 (20 ng/ml), VEGF (30 ng/ml), Wnt3A (and/or Wnt5A) (10 ng/ml), ActivinA (5 ng/ml), Inhibitor VIII (2 M), Fibroblast Growth Factor (FGF) (10 ng/ml), Stem Cell Factor (SCF) (20 ng/ml) and -Estradiol (0.4 ng/ml).

(18) NB: Given cytokine concentrations are always the final concentrations of freshly added cytokinese.g. when cells are fed rather than undergoing complete media change, we add 0.5 ml of 6 cytokines to 2.5 ml already in well to give final total volume of 3 ml with 1 cytokines.

(19) On day 3 of differentiation the EBs are washed in PBS then dissociated using 1 ml/well TrypleSelect 10 for 10 minutes at 37 C. After addition of 10 ml of PBS, cells are centrifuged for 3 minutes at 1200 rpm, the supernatant is discarded and the cells are resuspended in 3 ml fresh Stemline II and replated at 20010.sup.3/well of a regular six well tissue culture plate with the following cytokines: BMP4 (20 ng/ml), VEGF (30 ng/ml), FGF (10 ng/ml), SCF (30 ng/ml), Insulin-like Growth Factor 2 (IGF2) (10 ng/ml), Thrombopoietin (TPO) (10 ng/ml), Heparin (5 ug/ml), Iso Butyl Methyl Xanthine (IBMX) 50 M and -Estradiol (0.4 ng/ml).

(20) On day 5 of differentiation a fresh set of cytokines identical to day 3 is added in 0.5 ml/well of Stemline II such that final concentrations of fresh cytokines are those stated for day 3.

(21) On day 7 of differentiation the cells are harvested, centrifuged 3 minutes at 1200 rpm and resuspended in fresh Stemline II medium supplemented with the same set of cytokines as day 3. If the number of cells is above 50010.sup.3, it is advisable to split the culture as any drastic depletion or major pH increase of the medium can have major repercussion on the differentiation efficiency.

(22) From day 7 to day 10 of differentiation the cell density should be closely monitored in order to maintain the cell number under 10.sup.6 per ml by adding fully supplemented media and splitting into additional wells if required.

(23) On day 9 of differentiation a half dose of day 3 cytokines is added in 0.5 ml/well of Stemline II such that final concentrations of fresh cytokines are half those stated for day 3.

(24) On day 10 of differentiation, the cells are centrifuged 3 minutes at 1200 rpm then re-plated in erythroid liquid culture conditions. i.e. the cells are plated at a density of 10010.sup.3 cells/well in 3 ml/well of Stemline II supplemented with the following cytokines: Hydrocortisone 10.sup.6M, SCF (50 ng/ml), Flt3-Ligand (Flt3L) (16.7 ng/ml), BMP4 (6.7 ng/ml), Interleukin 3 (IL3) (6.7 ng/ml), IL11 (6.7 ng/ml), IBMX (50 M) and Erythropoietin (EPO) 1.3 U/ml.

(25) From day 10 to day 17 (short protocol) or 21 (extended A phase protocol), the above cytokines are renewed every 2 days, added in 0.5 ml of Stemline II, such that final concentrations of fresh cytokines are those stated for day 10.

(26) On day 17 (short protocol) or 21 (extended A phase protocol) of differentiation the cells are centrifuged 3 minutes at 1200 rpm then re-plated at a density of 50010.sup.3 to 110.sup.6 cells/well in 3 ml/well of IBIT medium (composed of Incomplete Iscove's Medium with stable glutamine (Biochrom AG), 1% Bovine Serum Albumin (Life Lechnologies or Sigma), 10 ug/ml Insulin, 200 ug/ml Transferrin (both from Sigma) and xeno free component lipid mixture solution 200 (Peprotech)) supplemented with the following cytokines: Hydrocortisone 10.sup.6M, SCF (20 ng/ml), Insulin Growth Factor 1 (IGF1) (20 ng/ml), Interleukin 3 (IL3) (6.7 ng/ml), IL11 (6.7 ng/ml) and Erythropoietin (EPO) 2 U/ml.

(27) From day 17 or 21 to day 24 or 28, a full dose of the above cytokines are renewed every 2 days, added in 0.5 ml of Stemline II. such that final concentrations of fresh cytokines are those stated for day 17 or 21.

(28) On day 24 or 28 of differentiation the cells are centrifuged 3 minutes at 1200 rpm then re-plated at a density of 500-100010.sup.3 in fresh IBIT supplemented with 4 U/ml of EPO for 2 days, followed by 5 to 10 days in IBIT medium alone. The culture medium is refreshed every 2 days by addition of fresh IBIT medium.

(29) Analysis and Characterization

(30) At day 10, 17 or 21 and 24 or 28, cells were analysed by flow cytometry to evaluate their hematopoietic and erythroid characteristics. The antibodies used were directed against CD31, CD34, CD36, CD41a, CD43, CD45, CD71 and CD235a (also known as glycophorin A) (BD Biosciences and eBioscience) and the cells were analysed with a BD FACSCalibur flow cytometer (BD Biosciences).

(31) At day 17 onward, the erythroid stage of the cells was determined assessment of morphology after Rapid Romanovski staining of cytospin preparations.

(32) At days 0, 10, 17 and 24 the expression of selected genes of interest by the differentiating cells was monitored by qRT-PCR (Taqman)

(33) The gene panel was selected to comprise genes known to be expressed at various stages of erythropoiesis in order to evaluate the degree of differentiation of the hPSCs.

(34) At day 24, 28 or onward the globin protein expression profile was analysed by High Pressure Liquid Chromatography to evaluate the level of switch from primitive to definitive hematopoiesis.

(35) Results

(36) The generation of RBCs from hPSC described herein is a sequential differentiation process aimed at mimicking in vivo erythroid development, in order to obtain a final product similar to and consistent with the biological functions of in vivo derived RBCs.

(37) Firstly the hPSCs are encouraged to form EBs and are directed towards mesodermal germ layer specification through a balanced cocktail of BMP4, VEGF, Wnt3A (Wnt5A) and Activin A. Several dosages were tested in order to determine the best combination of cytokines to optimize conversion efficiency. Secondly, at day 2 of the EBs stage, hematopoietic lineage differentiation is primed through the increase of BMP4 and VEGF and the addition of SCF, FGF and -Estradiol.

(38) After the EBs dissociation the cells are further directed towards hematopoietic differentiation through the addition on day 3 of a cytokines mix designed to favour the emergence and multiplication of hematopoietic stem cells (HSC) rather than other mesodermal lineages. At this stage, dispersed EBs will adhere to culture surface if permitted to do so, however if this adherence is inhibited by using ultra low adherence surface culture plastic (which do not support cell adherence) there is no detrimental effect on cell numbers (FIG. 1), thus this method can be executed completely in suspension culture.

(39) HSCs are characterised in part by expression of the CD34 antigen and the maximum of CD34+ cells is generally reached between days 7 and 10, as shown in FIG. 2, CD34 can be detected in 30-80% of iPSC or hESC derived cells. Further confirmation of hematopoietic identity is provided by analysis of the CD43 antigen [Ref: Vodyanik M A, Thomson J A, Slukvin II, Blood, 2006 15; 108(6):2095-105], as shown in FIG. 2, at day 10 flow cytometry analysis shows that 50-100% of cells express this important marker. From the panel of antigens detected on the d10 cells, and notably the simultaneous presence of CD31, CD41a, CD43 and CD235a, it seems that the majority of the cells are at the hemangioblastic or post hemangioblastic stage. (FIG. 2) [Ref: Salvagiotto G et al, Exp Hematol. 2008 36(10):1377-89]. These cells at d10 are also capable of forming colonies comprising all myeloid lineages in CFU assays.

(40) At day 17 or 21 the antigen expression profile, as well as the rapid Romanovsky staining show that the large majority of the cells are clearly erythroid, with most of them being either pro or basophilic normoblasts. At this stage the basal medium is switched to IBIT as Stemline II does not support erythroblast maturation. The corresponding cytokines cocktail has been refined to produce cells which display the highest levels of erythrocytic markers.

(41) At day 24 or 28, 95% to 100% of the cells are erythroid as shown by the flow cytometry analysis of CD235a expression (FIG. 3). Cytospins of d24 or d28 cells show a variable distribution between the basophilic, polychromatic and orthochromatic subclasses of erythroblast, depending on the differentiation condition tested and the origin of the hPSCs used (FIGS. 3 A and B). From this point, when left in the culture conditions described in the methods, the differentiating cells evolve toward an almost homogenous population of orthochromatic normoblasts, with a small percentage of cells undergoing spontaneous enucleation.

(42) In order to reduce the use of costly recombinant protein growth factors we investigated whether recombinant Wnt3A could be replaced with small molecule Glycogen Synthase Kinase 3 (GSK3) inhibitors. These drugs were postulated as possible replacements for recombinant Wnt3A because of their ability to mimic sustained Wnt signalling by preventing the phosphorylation of -catenin by GSK3 and thereby allowing the release and accumulation of active -catenin.

(43) The addition of GSK3 inhibitors such as Inhibitor VIII (A-A014418) or CHIR99021 (but not the less specific inhibitor BiO) during day 0 to day 3 of the differentiation protocol could not reproducibly replace Wnt3a. However, when used along with Wnt3a, these inhibitors unexpectedly caused a marked improvement in the quality and quantity of erythrocytic cells produced by our differentiation protocol as shown in FIGS. 3B and 4. Preliminary results indicated that these GSK3 inhibitors prompt a differential response from different pluripotent cell lines. Therefore, we tested multiple combinations of cytokines and inhibitors administered during different phases of the culture protocol in order to establish the best conditions for each line and importantly, the best generally applicable conditions. As shown in FIG. 3, the combination given in the method above shows a marked improvement of amplification and cell robustness at the end of the liquid culture and is applicable for both iPSC and hESC.

(44) In H1 ESC differentiation Inhibitor VIII can replace Activin A but this effect wasn't observed in RC9 and iPSC G cell lines where the absence of Activin A markedly hinders the differentiation efficiency (FIG. 4). In order to define a method that is most generally applicable we use the combination of a GSK3 inhibitor, Wnt3A and Activin A in the early stage of differentiation which results in consistent results with all hPSC lines tested (FIGS. 5A and B). The second GSK3 inhibitor tested, CHIR99021 is more potent (0.2 M instead of 2 M for Inhibitor VIII) and has a stronger effect on hESC than hiPSC lines compared to Inhibitor VIII (Table 1). Additionally, increasing the period of treatment with InhVIII (d0 to d5) also increases the expansion in cell number.

(45) cAMP and its principal target, the cAMP-dependent protein kinase A (PKA) play important roles in many biological processes including proliferation and differentiation in wide variety of cell types and can stimulate cell proliferation by activating ERKs in dividing cells through Ras-mediated activation of either B-Raf or Raf-1. Here we have shown that 3-isobutyl-1-methylxanthine (IBMX), non specific inhibitor of cAMP and cGMP phosphodiesterases which regulate the degradation of intracellular cAMP can increase cell numbers in the culture and when tested with the GSK3 inhibitor, IBMX has a synergetic effect on cell amplification (FIGS. 6A and B, Table 1). IBMX has been tested at various stages of the differentiation protocol and was found to be most effective in inducing maximum amplification when added to culture medium through out the period day 3 and 17 (Table 1).

(46) As the cells treated with the combination of GSK3 inhibitor and IBMX appear morphologically more intact and robust, we pushed the capacity for amplification by increasing the length of the differentiation period in cytokines A mix, normally 7d between d10-17, to a total of 11d as shown in FIG. 6B the extra time allow for an additional amplification and suggests that this phase may be extended further with consequent increases in cell number.

(47) The cells were also investigated at the molecular level at different timepoints using real time quantitative PCR to evaluate the differences of level of expression of a set of genes involved in hematopoiesis and more specifically, erythropoiesis (FIG. 7). Results from either hiPSC or hESC show that small molecules Inhibitor VIII and IBMX increase expression of globin genes and other markers characteristic of definitive hematopoiesis (HBA, HBG, HBB, Runx1, Gata2, HoxB4) as well as genes specific of different stage of erythropoiesis (HOXA9, CD36 and NFE2) and do not affect the necessary reduction in pluripotent markers.

(48) The addition of IBMX and GSK3 inhibitors did not have any negative effect on the cells morphology and maturation compared to the control culture conditions and as small molecules are highly compatible with cGMP compliant production of in vitro generated red blood cells. The hPSCs which underwent the full differentiation process in presence of both GSK3 inhibitor and IBMX exhibit a HPLC globin profile almost similar to the cells of fetal origin with very little embryonic globins left (FIG. 8) which is a good indicator that the vast majority of the cells produced are typical of definitive hematopoiesis. However, only a small % of the cells undergo spontaneous enucleation in the final stages of culture.

(49) Inclusion of 2 additional small molecules improves quality and amplification of cells. On day 5 of differentiation a fresh set of cytokines identical to day 3 is added in 0.5 ml/well of Stemline II. The small molecule StemRegenin1 (SR1) (Cellagen Technology) at a final concentration of 1 M is added along the cytokines. On days 14 the small molecule Pluripotin (SC1) (Stemgent) is added along the cytokines for a final concentration of 500 nM. On days 16 the small molecule Pluripotin (SC1) (Stemgent) is added along the cytokines for a final concentration of 250 nM The addition of SR1 at day 5 and SC1 at days 14 and 16 allow for a greater rate of cell amplification in the late stage of the method (FIG. 2). The cells are also sturdier and less prone to lysis (FIG. 3). These small molecules were tested because of their published properties on CD34+ amplification (SR1) or maintenance of pluripotency (SC1) (refs below) but they have not previously been implicated in erythroid development. Their effect is strongly dependant of the timing of administration.

(50) Summary

(51) Here we present a differentiation protocol which uses suspension based liquid culture throughout and is therefore scalable, it also achieves a degree of efficiency high enough to avoid the need for any purification step (>80% HPC at d10 and >90% erythroid series by d24). The method supports a considerable amplification of cell numbers as they differentiate to RBCs (up to 350,000 fold d0-24) which is in excess of previously reported methods, even those using HoxB4 as an amplifying agent [REFS]. The method is suitable for either human induced pluripotent stem cells (iPSC) or embryonic stem cells (hESC) referred to together as human pluripotent stem cells (hESC), including hESC lines that have been derived under fully GMP compliant and licensed conditions. Furthermore, the cells reach the orthochromatic normoblast stage of erythropoiesis and display characteristics of definitive hematopoiesis (including the shut off of embryonic globins and expression of A globin). As another step towards scale-up of the process we have also ensured that the protocol starts with hPSC that have been maintained for many passages in feeder-free culture using GMP-compliant reagents amenable to large scale mechanised production. This differentiation method can be used for the efficient differentiation of human pluripotent SC including hiPSC and hESC. Actvin A is required for efficient differentiation of iPSC An increased dose of Activin A doesn't have any beneficial effect (data not shown) A prolonged pulse of Inhibitor VIII (d0-5) increases the yield of differentiated cells (iPSC or hESC) The combination of Inhibitor VIII or CHIR99021 and IBMX is synergistic, but BiO, a less specific GSK3 inhibitor does not have this effect. A prolonged culture in cytokine mix A (d10-21) allows for an increased yield of differentiated cells Combination of the tested parameters has enabled us to maximize the yield erythroid cells from hPSC to the point where we have achieved 35010e3 erythroid cells per hPSC. This expansion is equivalent to 1 unit of RBC concentrate (210e12 cells) per 610e6 iPSC which could be harvested from less than 3 wells of a 6 well plate. This protocol has been optimized using PSC that had previously been maintained on GMP grade cell free substrate (CellStart, Life Technologies) in GMP-grade serum free medium (StemPro, Life Technologies) and unlike many other protocols which use feeder maintained PSC, it is fully compatible with GMP-grade manufacturing.

(52) TABLE-US-00001 TABLE 1 table summarizing results obtained when comparing side by side the effect of the addition of GSK3 inhibitors, IBMX or both on the overall amplification of differentiating erythropoietic cultures of hPSCs. The side by side comparison within each experiment allow the assessment of the direct effect of the compound tested without interference from other parameters like quality of initial hPSCs culture, hPCSs passage number, experimenter, activity of cytokines used or other equipment inconsistency. Results consistently show a positive effect of Inhibitor VIII and IBMX. The gain provided by a prolonged period in cytokine mix A is indicated in the bold italicised columns.