WO2016116769A1

WO2016116769A1 - Cell maturation process

Info

Publication number: WO2016116769A1
Application number: PCT/GB2016/050146
Authority: WO
Inventors: Robert Thomas; Forhad AHMED; Rachel BAYLEY; Joanne Mountford; Katie GLEN
Original assignee: Scottish National Blood Transfusion Service; University Court Of The University Of Glasgow; Loughborough University Innovations Limited
Priority date: 2015-01-23
Filing date: 2016-01-22
Publication date: 2016-07-28
Also published as: EP3247790A1; GB201501159D0

Abstract

Disclosed are methods and procedures which may be applied to cells (including differentiating (or partially differentiated) cells, cells in the terminal stages of differentiation and/or differentiated cells) to facilitate late stage maturation towards mature erythrocyte lineages – in particular towards reticulocytes and/or enucleated (anucleate) mature erythrocytes. The disclosure provides a method in which mature enucleated erythrocytes and/or reticulocytes are produced maintaining and/or culturing stem cells (for example differentiating or differentiated stem cells) under one or more maturation conditions in which the pH, level of (dissolved) oxygen; and level of mechanical stress is controlled.

Description

CELL MATURATION PROCESS

FIELD OF THE INVENTION

The present invention provides processes for achieving the expansion, differentiation and maturation of differentiated stem cells to cells of the myeloid lineages in particular, reticulocytes and/or enucleated mature erythrocytes. Specifically, the invention provides scalable methods for the late stage maturation of erythrocyte precursors into a relatively pure and terminally differentiated state.

BACKGROUND OF THE INVENTION

The production of red blood cells from stem cells (in particular human pluripotent stem cells (hPSC)) has been the subject of growing interest in the scientific community during recent years. This is in part, driven by the increased difficulty in obtaining sufficient clinically safe blood from donors to sustain global transfusion requirements.

Despite the increased interest in generating stem cell derived red blood cells there has been little progress made to take the early research discoveries towards the clinic and several major obstacles to RBC differentiation remain. Specifically, these are i) the efficiency of stem cell differentiation, ii) production of definitive as opposed to primitive erythroid cells, iii) sustained amplification throughout the differentiation process and iv) enucleation and terminal maturation of differentiated cells.

The present invention provides methods which may be used in protocols for the production of red blood cells (erythrocytes) from stem cell sources (including induced pluripotent stem cells) to facilitate late stage maturation and in particular enucleation and terminal differentiation processes.

Lindsey & Papoutsakis (201 1 ; Stem Cells: From Mechanics to Technologies: World Scientific Publishing and Imperial College) describe the importance of physiologically impaired physiochemical parameters in haematopoietic stem cell maintenance and lineage- specific differentiation in ex vivo cultures. The data presented in this document is derived from experiments conducted on cells from non-pluripotent sources and focusses on the effect of a variety of maturation conditions during stages of haematopoietic differentiation. The invention concerns the effect pH, oxygen level and/or mechanical stress on differentiated and differentiating stem cells and in particular the enucleation phenomenon. Endo et al (1994: Leuk Res 18: 49-54) investigated the effect of pH on human erythroid cell lined KU-812 and K562 and found that a significantly greater proportion of cells acquire a mature erythroid phenotype when cultured at pH7.6 that when cultured at pH7.4. As such, while the prior art has noted a link between the pH of cell culture conditions and effects on cell differentiation, there has been no specific disclosure of scalable methods which exploit a combination of maturation conditions/factors to facilitate the late stage maturation of l erythrocyte precursor into relatively pure and terminally differentiated reticulocytes and/or enucleated mature erythrocytes.

SUMMARY OF THE INVENTION

The present invention provides methods and procedures which may be applied to cells (including differentiating (or partially differentiated) cells, cells in the terminal stages of differentiation and/or differentiated cells) to facilitate the late stage maturation of those cells towards mature erythrocyte lineages - in particular towards reticulocytes and/or enucleated (anucleate) mature erythrocytes. The inventors have discovered that the methods described herein are not only scalable but may be used to generate populations of reticulocytes and/or enucleated, mature erythrocytes of improved quality and/or or high purity.

Specifically, the methods provided by this invention may be applied to cells differentiated (or in the process of differentiating) from stem cells. The term stem cells may encompass any type of totipotent, multipotent or pluripotent cell including, for example human embryonic stem cells (- including (hESC) stem cell lines that have been derived under fully GMP compliant and licensed conditions), induced pluripotent stem (iPS) cells, stem cells derived from umbilical cord blood and/or stem cells derived from peripheral blood and/or haematopoietic/haematological tissue sources (such as bone marrow, foetal liver, menstrual blood and the like).

Throughout the specification, the term "differentiated cells" or "differentiated stem cells" is used. It should be understood that the term "differentiated" embraces cells which are in the process of differentiating and which may have one or more additional differentiation stages to progress through before reaching a desired or predetermined differentiation status or before becoming what is generally known as terminally differentiated. For example the term "differentiated stem cell" may relate to a cell which has been differentiated from a stem cell but which is not yet, itself, terminally differentiated. Thus, the methods provided by this invention may be applied to differentiating or partially differentiated (stem) cells. Differentiating cells may be in the process of being differentiated towards or into one or more differentiated or terminally differentiated (adult) cell types.

More specifically, stem cells from which the cells subjected to the methods of this invention may be derived or differentiated may include any cell which is able to self-renew and indefinitely divide - cells of this type may be described as "immortal". In addition, when cultured under suitable conditions and/or contacted with, or exposed to, particular compounds and/or conditions, stem cells may differentiate into one or more of the specialised cell types which form embryonic and/or adult tissues.

Stem cells may be totipotent in nature and one of skill will appreciate that totipotent cells may be capable of generating a complete viable organism as well as any given specialised cell type. Stem cells may be pluripotent - cells of this type are not capable of generating a complete viable organism, but are able to differentiate to one or more (sometimes any) specialised cell type. As such, the present invention may be applied to cells differentiated or derived from any type of totipotent and/or pluripotent stem cell.

The term "stem cells" may encompass embryonic, foetal, adult and/or induced pluripotent (iPS) stem cells. The term "stem cells" may further encompass progenitor cells of any type. In one embodiment, the stem cells mentioned herein may be mammalian cells; for example, the term "stem cells" may be applied to human and/or non-human stem cells of all types. By way of example the methods of this invention may be applied to cells derived or differentiated from stem cells derived or obtained from, or provided by, primates, ungulates, ruminants and/or rodents (specifically, sheep, pigs, cattle, goats, horses, rats and mice).

Stem cells may be characterised by the presence of one or more markers selected from the group consisting of: ABCG2; ACE; ALCAM; Alkaline Phosphatase; beta-Ill Tubulin; BMP-2; BMPR-IA/ALK-3; BMPR-IB/ALK-6; BMPR-II ; E-Cadherin; CCR4; CD9; CD71 ; CD90; CD90/Thy1 ; Cripto; CXCR4; DPPA5/ESG1 ; Endoglin/CD105; FABP1 ; FABP2; FGF-4; FGF R4; FoxD3; FoxP3; Frizzled-9;GAD1/GAD67; GATA-4; GATA-6; GDF-3; Glutl ; HNF-3 beta; Integrin alpha 6/CD49f; Integrin beta 1/CD29; Lefty; MAP2; Musashi-1 ; Nanog; NCAM-L1 ; Nectin-2/CD1 12; Nestin; NeuroDI ; Nodal; Noggin; NF-L; NF-M; Nucleostemin; Otx2; Oct ¾; PAX6; Podocalyxin; Prominin 2; ROB03; Sca-I; SCF R/c-kit; SHH; SOX2; SOX7; SOX17; SPARC; SSEA-1 ; SSEA-3; SSEA-4; STAT3; STRO-1 ; TP63/TP73L; Tyrosine Hydroxylase; gamma-Secretase; alpha-Secretase; beta-Secretase; beta-Ill tubulin; alpha-Fetoprotein; beta-Catenin; Vimentin and VCAM-1 . Collectively, these markers may each be referred to as stem cell markers and references in this specification to one or more "stem cell markers", may therefore encompass one or more of the abovementioned markers. One of skill will appreciate that to identify or detect a stem cell, a cell may be probed (using, for example antibodies or other agents capable of binding one or more of the listed stem cell markers) for the presence of one or more of the stem cell markers listed above.

It should be understood that the methods of this invention may be applied to cells differentiating, differentiated or derived from any of the stem cells described above. A more detailed description of some specific types of stem cell is provided below.

This invention may be exploited to generate reticulocytes and/or enucleated mature erythrocytes from cells differentiated or derived from embryonic stem cells (ESC), for example, mammalian and/or human embryonic stem cells (hESC). ESCs may be derived from early stage embryos and in particular from the inner cell mass of the developing morula or blastocyst. Embryonic stem cells, for example those derived from embryos in the stages immediately following conception (and for a short time thereafter), may be totipotent (capable of generating a complete viable organism as well as any given specialised cell type). Embryonic stem cells derived from later stage embryos (i.e. from the inner cell mass of a developing blastocyst) may be pluripotent (not capable of generating a complete viable organism, but capable of differentiating to any specialised cell type). As such, the present invention may be applied to cells derived or differentiated from embryonic (i.e. totipotent and/or pluripotent) stem cells.

One of skill will appreciate that hESCs and other cell lines for use as source material for cells to be subject to the methods described herein may be obtained from an embryo without destruction of the embryo, as described, for example, in Chung et al (Cell Stem Cell, vol 2, issue 2, 1 13-1 17, 2008). Stem cells may also be generated using the methods described by Chung et al., (2006) which methods involve taking a blastomere cell from an early stage embryo prior to formation of the blastocyst (at approximately the 8-cell stage) and co-culturing this cell with established stem cell lines to generate a fully competent stem cell line. Stem cells obtained by the methods described by Chung et al (2006, 2008) andTachibana et al (2013, Cell 153 (6), p1228-1238 and Cell 154 (2), p465-466) may be used to establish stem cell lines which themselves may serve as sources of stem cells for use in providing cells to be subject to the methods of this invention.

Markers of embryonic stem cells may include, for example, ABCG2, Alkaline Phosphatase, E-Cadherin, CCR4, CD9, Cripto, DPPA5/ESG1 , FGF-4, FGF R4, FoxD3, FoxP3, GDF-3, Integrin alpha 6/CD49f, Integrin beta 1/CD29, Lefty, Nanog, Oct ¾, Podocalyxin, SOX2, SPARC, SSEA-1 , SSEA-3, SSEA-4 and STAT3.

The term "stem cells" may also be taken to refer to the pluripotent cells derived from any of the three primary germ layers (ectoderm, mesoderm and endoderm) which develop during the process of gastrulation. Cells derived from these layers may express one or more markers which may be used as a means of identification. By way of example, ectoderm germ layer may express markers, including, for example, Otx2, Nestin, TP63/TP73L, beta-Ill Tubulin, SHH, and PAX6. Ectoderm has the potential to form cell types such as neurons and early neuronal lineage markers include ACE, ALCAM, CD90/Thy1 , GAD1/GAD67, Glutl , MAP2, NCAM-L1 , Nectin-2/CD1 12, NeuroDI , NF-L, NF-M, ROB03, gamma- Secretase, alpha-Secretase, beta-Secretase, beta-Ill tubulin, Tyrosine Hydroxylase. Neural stem cell markers include ABCG2, CXCR4, FGF R4, Frizzled-9, Musashi-1 , Nestin, Noggin, Nucleostemin, Prominin 2, SOX2, Vimentin. Markers of early endodermal cells include, for example, FABP1 , FABP2, GATA-4, HNF-3 beta (collectively referred to as definitive endodermal stem cells markers) as well as those markers for primitive endoderm such as alpha-Fetoprotein, beta-Catenin, GATA-4, SOX17 and SOX7.

The invention may also be applied to cells differentiating, differentiated or derived from "adult" stem cells - cells of this type may be taken to be stem cells obtained from adult animals and or adult (or developed/differentiated) tissue (including adult humans and/or human (adult) tissue). However, it should be understood that the term "adult" also includes stem cells derived from neonatal, infant, juvenile and/or adolescent animals. Adult stem cells may be sourced from any suitable tissue, including bone marrow and/or specialised structures such as, for example hair follicles, skin, teeth and the like.

Stem cells from which cells to be subjected to the methods of this invention may be differentiated or derived, may be obtained from a variety of sources including, for example, embryonic animals (including human embryos), said embryos being either aborted or created as part of a fertility program. Alternatively, it may be possible to obtain stem cells from established stem cell lines and thus avoiding the use of mammalian, particularly human, embryos. By way of example, stem cells for use as source material for differentiated cells suitable for use in the methods of this invention may be obtained from the H1 and/or RC9/1 1/12/13 cell lines.

Alternatively, the methods of Meissner & Jaenisch (2006) may be used to obtain stem cells from which suitable differentiated cells may be obtained. In these methods, the cdx2 gene is silenced in the donor nucleus during the process of nuclear transfer to prepare a reconstructed embryo from which a line of embryonic stem cells is derived. The cdx2 gene is turned back on in the isolated blastocyst cell taken from the embryo which is used to prepare the cell line. This is an example of, so-called, "alternative nuclear transfer" where the embryo is not capable of implantation but the stem cell line derived therefrom is fully competent.

The term "stem cells" may also encompass cells otherwise known as induced pluripotent stem cells (iPS). These are re-programmed adult somatic cells which have been modified to express certain factors (such as transcription regulators) and, as a consequence, become pluripotent and thus capable of differentiating to any other specialised cell type. As such, iPS cells, particularly mammalian, for example rodent iPS stem cells, may be used as sources of cells which are suitable for application in the methods of this invention.

Thus any of the stem cells described and defined herein, may be used as a source of (differentiating or differentiated) cells to be subject to the methods of this invention.

The invention is based on the finding that cells (in the process of) differentiating or differentiated from stem cells may be exposed to or cultured or maintained under a set of conditions (referred to hereinafter as "maturation conditions") so as to ensure further differentiation of the differentiated stem cells to mature enucleated erythrocytes and/or reticulocytes. Indeed, the inventors have noted that the method of this invention represents an improvement over prior art methods as the instant methods yield populations of cells having greater numbers of quality reticulocytes or enucleated erythrocytes than population prepared or produced by prior art methods. Moreover, cell populations provided by methods according to this invention often exhibit a high purity of reticulocytes or enucleated erythrocytes. Throughout this specification we make use of the terms "maintained" and/or "cultured" - for example in the context of cells being "maintained" or "cultured" under certain maturation conditions. Cells which are "maintained" or "cultured" may be retained in a specific stage for a predetermined or defined period of time. For example, maintained differentiated (or differentiating) stem cells, may be retained or held at or in a particular state of differentiation for a period or prolonged period of time and/or over a number of passages. For example, maintained cells may remain a particular phenotype throughout the period of maintenance. Cells which are "cultured" under certain conditions may proliferate, expand and/or differentiate towards one or more cell lineages.

Throughout this specification the term "comprise" is used to denote that aspects and embodiments of this disclosure "comprise" a particular feature or features; however, the term "comprising" may also encompass aspects and/or embodiments which "consist essentially of" or "consist of" the relevant feature or features.

The maturation conditions of the method of this invention may comprise one or more modulated physiochemical and/or environmental conditions including, for example, predetermined or modulated pH conditions, predetermined or modulated oxygen conditions and/or exposure of the cells to agitation and/or mechanical stress. The maturation conditions may comprise conditions in which the differentiated (or differentiating cells) are maintained or cultured under a specific and/or predetermined pH or in which the pH under which the differentiated (or differentiating) cells are maintained or cultured is reduced from a first pH to a second different pH. Likewise, the maturation conditions of this invention may comprise the use of a specific or predetermined level of oxygen or conditions in (or under) which the level of oxygen is modulated from a first level to a second (different) level. Further, the maturation conditions may comprise subjecting cells to some form of agitation and/or mechanical stress.

Thus, in a first aspect, the present invention provides a method of providing mature enucleated erythrocytes and/or reticulocytes, said method comprising maintaining and/or culturing differentiated stem cells under one or more maturation conditions, wherein said maturation conditions are selected from the group consisting of:

(i) a predetermined pH;

(ii) a predetermined or specific level of (dissolved) oxygen; and

(iii) mechanical stress.

A method according to the first aspect may exploit one, two or all three of the specified maturation conditions. The conditions may be applied concurrently (together) or separately and at different and/or overlapping times. The maturation conditions may be applied continuously or continually throughout a culture protocol or at one or more specific and/or predetermined times. For example, a pH and oxygen level may be set so as to bring cells (for example differentiated and/or differentiating stem cells) to a "state of readiness" for enucleation and maturation conditions comprising mechanical stress used to affect the process of enucleation.

The differentiated stem cells may comprise stem cell derived haematopoietic progenitor cells or erythroid progenitor cells. Thus the invention provides a method in which the provision of mature enucleated erythrocytes and/or reticulocytes, is achieved by maintaining and/or culturing haematopoietic progenitor cells and/or erythroid progenitor cells under one or more maturation conditions, wherein said maturation conditions are selected from the group consisting of:

(i) a predetermined pH;

(ii) a predetermined or specific level of (dissolved) oxygen; and

(iii) mechanical stress.

The differentiated stem cells, for example the haematopoietic progenitor cells and/or erythroid progenitor cells for use in this invention may be provided or obtainable by any suitable method including, for example, those methods disclosed in PCT/GB2013/051917 (the entire contents of which is incorporated herein by reference). For example, the differentiated stem cells may be provided by methods which involve contacting stem cells with a GSK3 inhibitor and/or a phosphodiesterase inhibitor. It should be noted that the methods described in PCT/GB2013/051917 provide for the differentiation of stem cells into erythroid cells and as such, the cells for use in this invention may be those derived from only part of the procedures described therein - from example, cell obtained from those procedures executed through to about day 10 of the methods of PCT/GB2013/051917. The methods used to generate differentiate stem cells for use in this invention may comprise a first phase (phase 1 ) in which stem cells or embryoid bodies are contacted with a GSK3 inhibitor (for example Inhibitor VIII and/or CHIR99021 ) and a second phase (phase 2) in which the cells are contacted with a phosphodiesterase inhibitor (such as IBMX). One of skill will appreciate that once contacted with a GSK3 inhibitor, stem cells may undergo a degree of differentiation (initially towards cells of the mesodermal germ layer specification and later towards cells of the haematopoietic lineage); as such, cells subjected to the second phase of the methods described herein may be referred to as partially differentiated stem cells or differentiated cells. One of skill will appreciate that the purpose of the second phase of the methods described herein is to differentiate cells produced by the first phase through haematopoietic lineages towards erythroid cells.

Phase 1 of the methods provided by this invention may comprise the step of contacting stem cells (or embryoid bodies formed therefrom) with a GSK3 inhibitor and one or more supplementary compounds. The supplementary compounds may be selected from the group consisting of:

(i) Bone Morphogenic Protein 4 (BMP4); (ϋ) Vascular Endothelial Growth Factor 165 (VEGF);

(iii) Wnt3A and/or Wnt5a;

(iv) ActivinA;

(v) Fibroblast Growth Factor a (FGF );

(vi) Stem Cell Factor (SCF); and

(νϋ) β-estradiol.

Phase 1 may comprise the step of contacting stem cells with culture media supplemented with a GSK3 inhibitor and one or more of the supplementary compounds noted above.

Phase 2 of the methods provided by this invention may comprise the step of contacting cells produced or generated by the phase one methods with a phosphodiesterase inhibitor and one or more supplementary compounds. The one or more supplementary compounds may be selected from the group consisting of:

(i) Bone Morphogenic Protein 4 (BMP4);

(ii) Vascular Endothelial Growth Factor 165 (VEGF);

(iii) Fibroblast Growth Factor a (FGFa);

(iv) Stem Cell Factor (SCF);

(v) β-estradiol.

(vi) Insulin-like Growth Factor 2 (IGF2);

(νϋ) Thrombopoietin (TPO);

(viii) Heparin;

(ix) Hydrocortisone;

(x) Flt3-Ligand;

(xi) Interleukin 3 (IL3);

(xii) IL1 1 ;

(xii) Erythropoietin (EPO);

(xiv) Insulin Growth Factor 1 (IGF1 );

(XV) StemRegeninl (SR1 ); and

(xvi) Pluripotin (SC1 )

Phase 2 may comprise the step of contacting stem cells with culture media supplemented with a phosphodiesterase inhibitor and one or more of the supplementary compounds noted above as (i)-(xvi). As stated, in PCT/GB2013/051917 the phase 2 methods are executed in order to provide erythroid cells - however, the methods of this invention may exploit cells which have been subjected only to part of the complete phase 2 method as described in PCT7GB2013/051917. The complete phase 2 process may comprise a number of sub-phases - provided as sub-phases 2a-2g, wherein the product of sub-phase 2g is an erythroid cell. The methods of this invention may exploit cells which are the product of sub-phase 2d

Thus, differentiated stem cells for use in this invention may be provided by the following 2-phase method.

Phase 1 of the 2-phase method may comprise a first sub phase (referred to herein after as "sub-phase 1 a"). Sub-phase 1 a may comprise the step of contacting stem cells (or embryoid bodies) with one or more compounds selected from the group consisting of:

(i) a GSK3 inhibitor;

(ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Vascular Endothelial Growth Factor 165 (VEGF);

(iv) Wnt3A and/or Wnt5A; and

(v) ActivinA.

Phase 1 of the methods may further comprise a second sub-phase (referred to hereinafter as "sub-phase 1 b") executed after sub-phase 1 a. Sub-phase 1 b may comprise the step of contacting the stem cells (or embryoid bodies) with one or more compounds selected from the group consisting of:

(i) a GSK3 inhibitor;

(ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Vascular Endothelial Growth Factor 165 (VEGF);

(iv) Wnt3A and/or Wnt5A;

(v) ActivinA;

(vi) Fibroblast Growth Factor a (FGF );

(νϋ) Stem Cell Factor (SCF); and

(viii) β-estradiol.

Optionally and before execution of phase 2 of the methods described herein, cells or embryoid bodies provided or produced by phase 1 (and in particular sub-phases 1 a and 1 b) of the method may be subjected to a dissociation protocol. For example, cells or embryoid bodies provided or produced by phase 1 of the method may be mechanically or chemically dissociated, and harvested by, for example centrifugation. Harvested cells may (after re- suspension) be subjected to phase 2. Harvested cells may be re-suspended in suitable medium such as, for example, Stemline II. Additionally, and prior to execution of phase 2, cells may be plated out at around 200x10³ cells/per well.

Phase 2 of the 2-phase method may comprise a first sub phase (referred to hereinafter sub-phase 2a). Sub-phase 2a may comprise the step of contacting the cells or embryoid bodies provided or produced by the first phase (and specifically sub-phases 1 a and 1 b), which cells or embryoid bodies may have been subjected to dissociation and harvesting protocols, with one or more compounds selected from the group consisting of:

(i) BMP4;

(ii) VEGF;

(iii) FGFa;

(iv) SCF;

(v) Insulin-like Growth Factor 2 (IGF2);

(vi) Thrombopoietin (TPO);

(vii) Heparin;

(viii) A phosphodiesterase inhibitor (for example IBMX); and

(ix) β-estradiol.

Phase 2 may further comprise a second sub-phase (referred to hereinafter as "sub- phase 2b). Sub-phase 2b may comprise repeating the method of sub-phase 2a - in other words, sub-phase 2b comprises the step of contacting cells subjected to sub-phase 2a with one or more of the compounds listed as (i)-(ix) immediately above. Optionally, the cytokines used in sub-phase 2b (compounds (i)-(ix) listed immediately above) may be supplemented with a quantity of StemRegeninl (SR1 ). The inventors have discovered that the optional addition of SR1 during sub-phase 2b (at day 5) enhances or increases the rate of cell amplification - in particular in later stages of the protocols of this invention. Moreover, the inventors noted that cells cultured using methods which exploit StemRegeninl (SR1 ) are more "sturdy" and less prone to lysis.

Phase 2 of the methods described herein may further comprise a third sub-phase (referred to hereinafter as "sub-phase 2c"). Sub-phase 2c may replicate the method of sub- phase 2a and/or 2b. Optionally, before executing sub-phase 2c, the cells produced by sub- phase 2b may first be harvested by, for example centrifugation. Moreover, if the total number of cells after sub-phase 2b exceeds 500x10³, the cells may be split before executing sub- phase 2c. Additionally, throughout sub-phase 2c, the total cell number may be kept under 1 x10⁶ per ml.

Phase 2 of the methods described herein may comprise a fourth sub-phase (referred to herein after as sub-phase 2d). Sub-phase 2d may replicate the method of sub-phase 2a. Sub-phase 2d comprises the step of contacting cells provided or produced by sub-phase 2c with one or more of the compounds selected from the group consisting of:

(i) BMP4;

(ii) VEGF;

(iii) FGFa;

(iv) SCF; (v) Insulin-like Growth Factor 2 (IGF2);

(vi) Thrombopoietin (TPO);

(vii) Heparin;

(viii) A phosphodiesterase inhibitor (for example IBMX); and

(ix) β-estradiol.

The concentration of the one or more compounds used in sub-phase 2d may be half that used in sub-phase 2a.

The product of the method of sub-phase 2d may be differentiated stem cells which are suitable for application in the methods of this invention.

The complete disclosure of PCT/GB2013/051917 is reproduced in this specification

(see below) and it should be understood that all or part of the methods described therein (in particular phases 1 -2d (as summarised above)) may be applied to stem cells in order to generate differentiated (differentiating or partially differentiated) stem cells and/or haematopoietic progenitor and/or erythroid progenitor cells suitable for use in the methods of the instant invention.

As stated, the culture conditions applied to the differentiated stem cells may be referred to as maturation conditions. The maturation conditions may comprise a predetermined pH - in other words, the methods of this invention may involve culturing or maintaining differentiated (or differentiating) stem cells in (or under conditions) which comprise a predetermined pH. For example, the predetermined pH may be selected from a pH of about 4.0 to about 7.9. For example the maturation conditions may comprise a pH of about pH4.5, about pH5.0, about pH5.5, about pH6.0, about pH6.1 , about pH6.2, about pH6.3, about pH6.4, about pH6.5, about pH6.6, about pH6.7, about pH6.8, about pH6.9, about pH7.0, about pH7.1 , about pH7.2, about pH7.3, about pH7.4, about pH7.5 about pH7.6, about pH7.7, about pH7.8 or about pH7.9. The maturation conditions may comprise the use of a pH above about pH7.4; for example at about pH7.5. The maturation conditions of this invention might comprise one or more of the above listed pH values; for example, the maturation conditions may modulate between different pH levels - for example a first pH and a second pH.

It should be noted that the term "about" as used herein with reference to the pH of the maturation conditions, may encompass a ± 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09 or 0.095 variation in any given pH value.

As such, this invention sets out a method of providing mature enucleated erythrocytes and/or reticulocytes, said method comprising culturing differentiated (or differentiating) stem cells at a pH above about pH7.4, for example at about pH 7.5. The methods of this invention may comprise subjecting cells generated or obtainable by a method described in PCT/GB2013/051917 (for example the method defined by phases 1 -2d thereof) to culture conditions which comprise a pH above about pH7.4, for example at about pH 7.5.

The invention further provides a method of providing mature enucleated erythrocytes and/or reticulocytes, said method comprising subjecting cells generated or obtainable by a method described in PCT/GB2013/051917 (for example the method defined by phases 1 -2d thereof) to culture conditions in which the pH is set at a pH above about pH7.4; for example at about pH7.5 .

The inventors have discovered that when cultured at a pH above about 7.4, for example at about pH7.5, differentiated stem cells (for example cells which are the produce of the day 10 stage (phase 2d) of the methods described in PCT/GB2013/051917) develop a (very) mature, terminally differentiated phenotype, characteristic of mature reticulocytes and/or enucleated erythrocytes. Moreover, the methods provided by this invention are scalable in that they can be practiced on small and large volumes of cells to provide the required number of mature, terminally differentiated reticulocytes and/or enucleated erythrocytes.

While prior art methods have shown some success in maturing reticulocytes from adult or cord blood derived progenitor cells (using for example wave culture and/or stirred tank apparatus), these methods may not achieved proper late stage maturation of erythrocytes. Without wishing to be bound by any particular theory, the inventors suggest that use of a pH above about 7.4 (for example a pH of about 7.5) greatly speeds up the terminal maturation of red cells and/or strongly selects for erythroid commitment. It is also possible that a pH of about 7.5 suppresses and/or destroys a cell population (either alternate lineage or different maturity same lineage) and/or prevents the persistence of non-erythroid lineage cells that might otherwise inhibit the terminal maturation of red cell progenitors.

Mature, terminally differentiated cells of this invention may, for example, take the form of erythrocyte sized haemoglobin containing cells which are anucleate (without nuclei). The mature, terminally differentiated cells of this invention may be visualised using, for example, stains such as, benzidine-giemsa stain which stains haemoglobin as red/brown and other, nucleated cells as blue. Additionally or alternatively, the mature, terminally differentiated cells may be detected through the presence or absence of certain reticulocyte and/or erythrocyte markers. For example, the presence or absence of the cell surface marker protein Glycophorin A may be used as the basis of the detection of mature terminally differentiated reticulocytes and/or erythrocytes (mature, terminally differentiated cells expressing Glycophorin A).

Differentiated stem cells suitable for application in this invention may be directly subjected to culture conditions which comprise a pH above about pH 7.4, for example about pH 7.5. For example, upon attaining a suitable level or stage of differentiation, a differentiated stem cell may be immediately subjected to culture conditions which comprise a pH above about pH 7.4 (for example about 7.0). For example, cells which are the product of the day 10 protocol disclosed in PCT/GB2013/051917 (that is a method comprising phase 1 to at least sub-phase 2d of the methods disclosed in PCT/GB2013/051917) may be immediately (or without substantial delay) subjected to, or contacted with, maturation conditions which comprise a pH above about pH7.4 or set at about 7.5. Alternatively, the differentiated stem cells may be maintained or cultured for a period of time before being subjected to maturation conditions which comprise a pH above about pH 7.4 or set at about pH 7.5.

The period of time may be anywhere from about 0 to about 1 1 days, for example 0,

1 , 2, 3, 4, 5, 6, 7 ,8 ,9, 10 or 1 1 days.

Cells subjected to the methods of this invention may be cultured or maintained under conditions which comprise a pH above about pH 7.4 (or set at about pH 7.5) for the duration of cell culture protocol or alternatively, for example, in the final 1 -10, for example 2-9, 3-8 or

4-5, 6 or 7 days of maturation.

The methods of this invention may be conducted at any suitable temperature - including, for example, at temperatures of about 30^SC, about 32 ^SC, about 34 ^SC, about 35

^SC, about 36 ^SC, about 37 ^SC, about 38 ^SC, about 40 ^SC or about 42^SC. In this regard, the term "about" may encompass a variation of ± 0.1 ^SC, 0.15^SC, 0.2^SC, 0.25^SC, 0.3^SC, 0.35^SC,

0.4^SC, 0.45^SC, 0.5^SC, 0.55^SC, 0.6^SC, 0.65^SC, 0.7^SC, 0.75 ^SC, 0.8^SC, 0.85^SC, 0.9^SC, 0.95^SC in any of the temperature values recited herein.

The methods of this invention may be conducted in a bioreactor system. For example a stirred tank bioreactor system. For example, the methods may be conducted in a stirred tank culture array. Suitable bioreactor systems will be known to those skilled in this field but may include, for example the "AMBR" systems developed by TAP Biosystems. These systems mimic the characteristics of classical bioreactors at microscale. These systems are particularly useful as they permit precise control of certain physiochemical parameters including, for example, pH and oxygen. Bioreactor systems for use in this invention may take the form of agitated/shaken vessels (for example agitated bags). As explained in more detail below, the bioreactor system may be used as a means to impart mechanical stress to differentiated and/or differentiating stem cells.

Thus, cells suitable for application in the methods of this invention (namely, differentiated stem cells of the type described herein) may be cultured or maintained in a bioreactor system at a predetermined pH - for example a pH above about pH 7.4 or a pH set at about pH 7.5. Prior to use, or seeding with cells, bioreactors for use in this invention may be primed or prepared for use. For example, suitable media (for example any of media A or B as described below) may be added and stabilised to a temperature of about 37^SC. The oxygen level within the bioreactor may also be set (perhaps to a predetermined level) as too may be the pH of the added medium. The priming of the bioreactor system may further include the addition of antifoaming agents - for example, antifoam C additions may be made periodically (for example every 12 or 24 hours). Volumes, 5μΙ_, 10μΙ_, 15μΙ_, 20μΙ_ or 25μΙ_ of a 1 % antifoam C solution may be added. Moreover, the priming may involve the addition of sodium bicarbonate; for example 5μΙ_, 10μΙ_, 15μΙ_, 20μΙ_ or 25μΙ_ of a 1 Μ solution may be added.

The pH of the culture systems and/or methods of this invention may be controlled or set using techniques familiar to one of skill in this field. For example, Carbon dioxide gas and/or sodium bicarbonate may be applied to the culture system in order to set/prime, control and/or modulate the pH both during the methods of this invention and/or prior to cell seeding of a culture system.

The bioreactor system may be set to stir or mix the reaction components (cells + (optionally supplements) media). For example the impeller of the bioreactor may be set to rotate at a speed of about 10, 50, 100, 200, 300, 400, 450, 500, 600 rpm. For example, the impeller may be set to rotate at about 450 rpm. One of skill will appreciate that depending on the size of the reactor and volume of fluid to be stirred the speed of rotation may vary. As explained below, the speed of rotation may be set or adjusted in order to impart some level or levels of mechanical stress to differentiated and/or differentiating stem cells.

The methods of this invention may further involve predetermined oxygen conditions or modulating oxygen conditions. For example, as an alternative or in addition to culturing differentiated stem cells under conditions comprising a predetermined pH (for example a pH above about pH 7.4 or at about pH 7.5), the cells may be further cultured in the presence of a specific level of (dissolved) oxygen.

For example, the maturation conditions exploited by the method of this invention may comprise anywhere between about 1 % and about 30% oxygen (0₂). It should be noted that all references to "oxygen levels" in this specification refer to the amount of dissolved oxygen in the culture media. Thus the "%" figure describes the percentage of oxygen saturation of the culture media that exists relative to the oxygen content of a hypothetical culture at equilibrium under a pure oxygen atmosphere.

The maturation conditions may exploit a level of oxygen which is less than about 90%, 80%, 70%, 60%, 50%, 40% or 30% of atmospheric oxygen. For example, the methods of this invention may exploit a level of oxygen which is less than about 50% of atmospheric oxygen. The maturation conditions may exploit a level of dissolved oxygen (i.e. oxygen dissolved in the culture medium) of about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 1 1%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28% or about 29%. For example the level of dissolved oxygen may be set at about 1 1 %.

It should be noted that the term "about" as used herein with reference to the oxygen level of the maturation conditions, may encompass a ± 0.1 %, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9% or 0.95% variation in any given 0₂ level.

In view of the above, the methods of this invention may provide a method of generating or providing mature enucleated erythrocytes and/or reticulocytes, said method comprising culturing differentiated stem cells:

at (i) a predetermined pH and/or

at (iii) a predetermined oxygen level.

The methods of this invention exploit modulated oxygen levels, where the oxygen level is modulated from a first oxygen level to a second oxygen level. For example the first and/or second oxygen levels may be different with the second oxygen level being higher or lower than the first oxygen level. For example, the first and second oxygen levels may be selected from the group consisting of about 1 % oxygen, about 2% oxygen, about 3% oxygen, about 4% oxygen, about 5% oxygen, about 6% oxygen, about 7% oxygen, about 8% oxygen, about 9% oxygen, about 10% oxygen, about 1 1 % oxygen, about 12% oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen, about 20% oxygen, about 21 % oxygen, about 22% oxygen, about 23% oxygen, about 24% oxygen, about 25% oxygen, about 26% oxygen, about 27% oxygen, about 28% oxygen, about 29% oxygen and about 30% oxygen.

The oxygen levels of the methods of this invention may be modulated over a predetermined period of time. For example, the differentiated stem cells may initially be cultured or maintained under conditions comprising a first oxygen level. After a predetermined period of time, the oxygen level may then be modulated towards a second oxygen level. The modulation may occur in a stepwise fashion or may take place directly -i.e. without intermediate steps. It should be understood that methods which involve modulated oxygen levels may exploit a plurality of different oxygen levels. For example the method may exploit one or more high (perhaps about 30%) oxygen level(s), one or more medium/intermediate (perhaps about 7% to about 13%, about 14%, about 15%, about 16% or about 17%, 19% or 25%) oxygen level(s) and one or more low (perhaps about 1 % or about 5%) oxygen level(s). For example, the methods may require the culture of cells at a predetermined (for example intermediate) oxygen level and a predetermined pH greater than about pH7.0 (but perhaps not higher than about pH7.9) before (perhaps after about 1 , 2, 3, 4 or 5 days) culturing the cells under lower or higher oxygen level and at about pH7.0 or lower.

Differentiated (or differentiating) stem cells may be subject or exposed to a predetermined oxygen level at any time during any given protocol (for example a protocol designed to yield mature erythrocyte cell lineages). The predetermined oxygen level(s) may be applied continuously or continually throughout a protocol or at one or more certain specific time points. The predetermined oxygen level(s) may be applied for any suitable length of time. For example, a suitable length of time may depend on the nature (and duration) of the culture protocol but may be from about 1 minute to about 30 days. For example, the time during which a predetermined oxygen level or levels may be applied may be about 15 min, about 30 min, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days about 8 days, about 9 days, about 10 days, about 15 days, about 20 days, about 25 days or about 30 days.

The inventors have also noted that mechanical stress (alone or in combination with any of the maturation conditions detailed above) can be used as a means to achieve and/or improve or enhance enucleation in, for example erythroid progenitor cells. Cells - for example differentiated or differentiating stem cells, may be subjected to variable (or modulating) levels of mechanical stress or some constant and predetermined level of mechanical stress. For example the cells may be subjected to some predetermined high level of mechanical stress or a predetermined low level of mechanical stress.

One of skill in this field will be familiar with those techniques which may be used to impart a level of mechanical stress to a cell culture and any of these techniques may be used here. For example, a level or levels of mechanical stress may be imparted to a cell culture by forcing or passing the culture (medium + cells) through a flow device, pump, tubing or the like. Additionally or alternatively, mechanical stress may be created in a cell culture within a bioreactor. Some bioreactors are formed and adapted such that the movement of an impeller within the bioreactor creates the mechanical stress. Without wishing to be bound by theory, the movement of an impeller through a cell culture medium may create currents, eddies, shear forces and/or fluid pinch points which contribute to a level of mechanical stress experienced by any cells within the medium.

A level of mechanical stress may be created controlling the speed at which a bioreactor impeller tip moves through a cell culture. Impeller tip speeds of 50-500 rpm, for example speeds of about 100-450 rpm, 150-300 rpm or 200-250 rpm may be used. As stated, the level of mechanical stress used may modulate between two or more predetermined levels of mechanical stress. Under such circumstances, and where a bioreactor is used to impart the necessary mechanical stress, variable or modulating mechanical stress used may be created via the selection (and use) of one or more different impeller speeds.

Cells (for example differentiated stem cell or differentiating stem cells) to be enucleated, may be subjected to a level or levels of mechanical stress at any point during a culture protocol. For example mechanical stress may be applied continuously or continually throughout a culture protocol or at the beginning and/or end (terminal stages of) a culture protocol. Differentiated (or differentiating) stem cells may be subjected to mechanical stress during the terminal stages of any culture protocol designed to yield mature (enucleated) erythrocyte cell lineages (including, for example enucleated erythroid progenitor cells).

A level or levels of mechanical stress may be applied for any suitable period of time. For example, a level or levels of mechanical stress may be applied for about 1 minute to about 10 days. For example, a level or levels of mechanical stress may be applied continuously or continually throughout or during a period spanning about 1 minute to about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, 24 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days or about 9 days.

While the invention may provide methods which exploit one of the abovementioned maturation conditions, the methods may further exploit any combination of maturation conditions detailed herein. That is, the methods may, for example exploit combinations of a predetermined pH and predetermined oxygen level, pH and mechanical stress, oxygen level and mechanical stress and/or a combination of all three of the stated maturation conditions.

The maturation conditions may be used concurrently (that is together) or separately and at different or overlapping times. The maturation conditions may be applied at one or more time points during a cell culture protocol. For example the conditions may each (independently) be applied continuously or continually throughout the culture protocol or at some predetermined time points during the same.

This invention may provide a method of providing mature enucleated erythrocytes and/or reticulocytes, said method comprising maintaining and/or culturing differentiated (or differentiating) stem cells at a pH of about pH 7.5 and at 1 1 % dissolved oxygen. Optionally, the method may be supplemented with the application of some level or levels of mechanical stress. The differentiated or differentiating stem cells may be exposed to a pH of about 7.5 and 1 1 % dissolved oxygen for the duration of the culture protocol.

The maturation conditions of the methods of this invention may further comprise certain types of cell culture media optionally supplemented with one or more cytokines. For example the methods of this invention may use Iscove's Modified Dulbecco's Media (IMDM) or Dulbecco's Modified Eagle Medium based medium. Media of this type include, for example, one or more selected from the group consisting of:

(i) Stemline^® I and II (Sigma)

(ii) Stemspan^® (Stem Cell Technologies)

(iii) X-vivo 10, 15 or 20 (BioWhittaker/Lonza)

(iv) Stem Pro^® 34 (Life Technologies)

(v) Poietics HPGM (Lonza)

(vi) APEL (Stem Cell Technologies)

(vii) IBIT medium

(viii) Other custom or "home-made" medium based on IMDM or DMEM + factors (similar to BIT).

For example, the methods of this invention may use Stemline II media. As stated, the medium (or media) for use in this invention may be supplemented with one or more supplementary compounds selected from the group consisting of:

(i) Stem cell factor (SCF);

(ii) Flt3-Ligand;

(iii) Bone morphogenic protein 4 (BMP4);

(iv) Interleukin 3 (IL3);

(v) Interleukin 1 1 (IL1 1 );

(vi) Erythropoeitin (EPO);

(vii) Hydrocortisone; and

(viii) Phosphodiesterase inhibitor (for example, IBMX).

For convenience, media (for example Stemline II media) supplemented with one or more of the cytokines listed as (i)-(viii) above shall be referred to as media A.

The methods may further exploit IBIT medium, which medium comprises incomplete Iscove's medium supplemented with stable glutamine, bovine serum albumin, insulin, transferring and xeno-free component lipid mixture solution. The IBIT medium may be further supplemented with one or more supplementary compounds selected from the group consisting of:

(i) Stem cell factor (SCF);

(ii) Insulin Growth Factor I (IGF1

(iii) IL3;

(iv) IL1 1 ; and

(v) EPO.

For convenience, media (For example IBIT medium) supplemented with one or more of the cytokines listed as (i)-(v) above shall be referred to as media B. Medium A and/or medium B may further comprise quantities (volumes) of antifoam compounds (for example antifoam C), sodium bicarbonate and/or C0₂. These components may be automatically added at regular or predetermined intervals and/or time points. The amounts or volumes to be added may vary and approximate quantities and volumes of these compounds are noted above with reference to the priming of the bioreactor systems used in this invention.

The exact quantity of supplementary compound to be added to the various media for use in this invention may vary. In this regard, it should be understood that the various final concentrations and amounts of the supplementary compounds described herein may be the final concentrations and amounts of the supplementary compounds added to the base media.

SCF may be used at a final concentration of anywhere between about 10ng/ml and about 60ng/ml. For example, SCF may be used at a concentration of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 ng/ml. When used as a supplement for Stemline II media, SCF may be used at a final concentration of about 40, 45, 50 or about 55 ng/ml. For example Stemline II media may be supplemented with about 50 ng/ml SCF. When used as a supplement for IBIT medium, SCF may be used at a final concentration of about 15, 17, 20 or about 25 ng/ml. For example IBIT media may be supplemented with about 20 ng/ml SCF.

Bone Morphogenic Protein 4 (BMP4) may be used at a final concentration of about 1 , 5, 10, 15, 20 or 25 ng/ml. For example, BMP-4 may be used at a final concentration of about 6, 6.2, 6.4, 6.5, 6.6, 6.7, 6.8, 7 or 7.5 ng/ml. BMP4 may be used at a final concentration of about 6.7 ng/ml.

The phosphodiesterase inhibitor (for example IBMX) may be used at a final concentration of about 10, 20, 23, 24, 25, 26, 27, 30, 40, 45, 50, 55, 60, 70, 80, 90 or 100 μΜ. The phosphodiesterase inhibitor (for example IBMX) may be used at a final concentration of about 50 μΜ.

Hydrocortisone may be used at a final concentration of about 0.1 , 0.5, 1 , 1 .5 or 2μΜ Hydrocortisone may be used at a concentration of about 1 μΜ.

Flt3 ligand may be used at a final concentration of about 10, 12, 15, 16, 17 or 20ng/ml. Flt3 ligand may be used at a final concentration of about 16.1 , 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8 or 16.9 ng/ml. Flt3 ligand may be used at a final concentration of about 16.7 ng/ml.

IL3 may be used at a final concentration of about 6, 6.2, 6.4, 6.5, 6.6, 6.7, 6.8, 7 or 7.5 ng/ml. IL3 may be used at a final concentration of about 6.7 ng/ml.

IL1 1 may be used at a final concentration of about 6, 6.2, 6.4, 6.5, 6.6, 6.7, 6.8, 7 or

7.5 ng/ml. IL1 1 may be used at a final concentration of about 6.7 ng/ml. IGF1 may be used at a final concentration of about 5, 10, 15, 20, 25 or 30 ng/ml. IGF1 may be used at a final concentration of about 20 ng/ml.

EPO may be used at a final concentration of about 1 , 1 .5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 U/ml. EPO may be used at a final concentration of about 3 U/ml.

The methods of this invention may be executed over about 1 to about 15 days. For example, the methods may be executed over about 1 to about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or

14 days. Since the cells for use in this invention may already have been subjected to procedures lasting about 10, days, those cells may be cultured or maintained for a further 1 -

15 days (for example about 1 1 days) in accordance the protocols and methods of this invention.

The methods of this invention may comprise the use of both media A and/or B as noted above. For example, the methods of this invention may exploit only media A (that is, the protocol of this invention may be conducted using media A only). Additionally the methods may exploit only media B (that is the protocol of this invention may be conducted using media B only). Alternatively, the methods may exploit both media A and media B.

Differentiated stem cells may be maintained in media A for at least about 1 , 2, 3, 4 or 5 days. For example, the differentiated stem cells may be maintained in media A for at least about 4 days. Once the cells have been maintained or cultured in medium A for the required period of time (for example for about 4 days), the cells may be cultured or maintained in medium B for about 1 to about 10 days. For example, the cells may be maintained or cultured in medium B for at least about 1 , about 2, about 3, about 4, about 5, about 6 or about 7 days.

The selected supplementary compounds may be added to the media at day 1 (i.e. the first day of a protocol established by this invention) and replenished (or further added) on, for example, days 2 and 4. Once any necessary period of maintenance or culture in medium A has been completed (after, for example, about 4 days), the cells may be cultured in medium B for at least about 7 days. The supplementary compounds used in Medium B (noted above) may be added to the media at day 1 (i.e. day 5 of the protocol of this invention) and replenished (or further added) on at least days 5 and 7 (days 9 and 1 1 of the protocol of this invention).

In view of the above, the present invention provides a method of generating or providing mature enucleated erythrocytes and/or reticulocytes, said method comprising culturing or maintaining differentiated stem cells in culture medium A and then in culture medium B, wherein the pH of at least culture medium B is set at a pH higher than about pH 7.4, for example at about pH 7.5.

As noted above, the methods of this invention may be conducted using a (primed) bioreactor system. While maintained or cultured in any of the media described above, the differentiated (or differentiating) stem cells may be maintained or cultured under conditions which comprise a pH above about pH 7.4. For example the differentiated stem cells may be maintained or cultured under conditions which comprise a pH of about 7.5. Moreover, the cells may be maintained at a level of dissolved oxygen of about 10%, about 1 1 %, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20% or about 21 %. For example, the cells may be maintained or cultured at about pH 7.5 and at a level of dissolved oxygen of about 1 1 %.

Given the disclosure of this invention, one of skill will appreciate that numerous variations of the precise protocol are possible, with cells being maintained for longer or shorter period in each of media A and/or B and at different pHs and/or oxygen levels.

In view of the above, the present invention provides a method of generating or providing mature enucleated erythrocytes and/or reticulocytes, said method comprising culturing or maintaining differentiated stem cells in culture medium A and then in culture medium B, wherein medium A comprises a quantity of SCF, Flt3L, BMP4, IL3, IL1 1 , EPO, hydrocortisone and phosphodiesterase inhibitor; and

medium B comprises a quantity of SCF, IGF1 , IL3, IL1 1 and EPO;

wherein either or both of media A or B have a pH of greater than about pH 7.4 (for example a pH of about 7.5).

It should be understood that the pH of either medium A or B may be modulated from a first pH to a second different pH.

Differentiated cells for use in this invention may be seeded into medium A and/or into a bioreactor system comprising medium A, at a density of about 1 x10⁴, 1x10^5, 1x10⁶ 1 x10⁷ _, 1 x10⁸ 1 x10⁹ or 1x10¹⁰ cells/ml. For example, the differentiated cells may be seeded at a density of about 1 x10⁵ cells/ml. The cells may be allowed to expand through the remainder of their period of culture in medium A and once (and if) transferred to medium B may be re- suspended at a density of about x10⁴, 1 x10⁵' 1x10⁶ 1 x10⁷ _, 1 x10⁸ 1 x10⁹ or 1x10¹⁰ cells/ml. For example, the cells may be re-suspended at a density of about 1 x10⁶ cells/ml to about 1 x10⁸ cells/ml.

Using the methods of this invention, differentiated cells may adopt a suitable mature phenotype (i.e. they may appear as anucleate erythrocytes and/or reticulocytes) after about 10 days of culture (that is about 20 days after initial differentiation from stem cells).

The methods of this invention may be in vitro or ex vivo methods.

In view of the above, the invention provides a method of generating or providing mature enucleated erythrocytes and/or reticulocytes, said method comprising culturing or maintaining in a bioreactor system, differentiated stem cells in culture medium A for at least about 4 days, wherein culture medium A and/or B has a pH above about pH 7.4 or of about pH 7.5;

wherein medium A comprises a quantity of SCF, Flt3L, BMP4, IL3, IL1 1 , EPO, hydrocortisone and phosphodiesterase inhibitor; and

medium B comprises a quantity of SCF, IGF1 , IL3, IL1 1 and EPO and has a pH of about pH 7.0.

It should be noted that both phases of the methods of this invention - namely the phase involving culture of differentiated cells using medium A and then the subsequent culture of those cells using medium B, may occur in a bioreactor system of the type described above. The bioreactor may be used to impart a level or levels of mechanical stress (or agitation) to the differentiated cells. Further, the methods may be conducted in the presence of defined and/or predetermined (or modulating) oxygen levels as described herein.

In a further aspect, the present invention provides a reticulocyte and/or an erythrocyte or populations of either, produced or obtainable by the methods described herein.

Additionally, the invention provides media for use in the methods of this invention, said media comprising Iscove's Modified Dulbecco's Media (IMDM) or Dulbecco's Modified Eagle Medium based media. Suitable base media may include, for example, one or more selected from the group consisting of:

(i) Stemline^® I and II (Sigma)

(ii) Stemspan^® (Stem Cell Technologies)

(iii) X-vivo 10, 15 or 20 (BioWhittaker/Lonza)

(iv) Stem Pro^® 34 (Life Technologies)

(v) Poietics HPGM (Lonza)

(vi) APEL (Stem Cell Technologies)

(vii) IBIT medium

The media of this invention may be based on Stemline II media. The medium (or media) for use in this invention may be supplemented with one or more supplementary compounds selected from the group consisting of:

(i) Stem cell factor (SCF);

(ii) Flt3-Ligand;

(iii) Bone morphogenic protein 4 (BMP4);

(iv) Interleukin 3 (IL3);

(v) Interleukin 1 1 (IL1 1 ); (vi) Erythropoeitin (EPO);

(vii) Hydrocortisone; and

(viii) Phosphodiesterase inhibitor (for example, IBMX).

The invention may further provide an IBIT based medium, which medium comprises incomplete Iscove's medium supplemented with stable glutamine, bovine serum albumin, insulin, transferring and xeno-free component lipid mixture solution. The IBIT medium may be further supplemented with one or more supplementary compounds selected from the group consisting of:

(i) Stem cell factor (SCF);

(ii) Insulin Growth Factor I (IGF1 );

(iii) IL3;

(iv) IL1 1 ; and

(v) EPO.

For convenience, media (For example IBIT medium) supplemented with one or more of the cytokines listed as (i)-(v) above shall be referred to as media B.

Medium A and/or medium B may further comprise quantities (volumes) of antifoam compounds (for example antifoam C), sodium bicarbonate and/or C0₂. The amounts or volumes to be added may vary and approximate quantities and volumes of these compounds are noted above with reference to the priming of the bioreactor systems used in this invention.

In another aspect, the present invention provides methods and media as described above and in the detailed description and figures which follow.

As explained above, the disclosure of PCT/GB2013/051917 is reproduced below. At least those parts which provide a protocol for establishing haematopoietic progenitor cells (at day 10 of the described protocol encompassing at phases 1 -2d) are useful for providing cells to be used in the methods described herein. As stated, cells prepared using the methods of PCT/GB2013/051917 may be subjected to the (bioreactor based) methods of this invention so as to facilitate late stage maturation processes and to improve the yield and quality of reticulocytes and/or enucleated erythrocytes.

Described herein are feeder-free culture systems and media to induce and support the differentiation of stem cells into erythroid cells.

Described are methods of inducing differentiation of stem cells into erythroid cells, said method comprising the step of contacting stem cells with a GSK3 inhibitor and a phosphodiesterase inhibitor. The GSK3 inhibitor may comprise a GSK3-beta (GSK3- ) inhibitor. The GSK3 inhibitor may comprise a specific GSK3 inhibitor; for example the GSK3 inhibitor may be comprise a specific GSK3-P inhibitor. One of skill will appreciate that a specific GSK3 inhibitor may bind to and/or inhibit GSK3 but may not detectably bind and/or inhibit any other kinase. The GSK3 inhibitor may comprise, for example, N-(4-methoxybenzyl)-N'-(5-nitro-1 ,3- thiazol-2-yl)urea - otherwise known as Inhibitor VIII or AR-A014418. The GSK3 inhibitor may be a N-(4-methoxybenzyl)-N'-(5-nitro-1 ,3-thiazol-2-yl) derivative or analogue. Additionally or alternatively, the GSK3 inhibitor may comprise CHIR99021 or a derivative or analogue thereof.

As such, all references to GSK3 inhibitors should be understood as encompassing:

(i) specific GSK3 inhibitors; (ii) GSK3-P inhibitors; (iii) Inhibitor VIII and/or (iv) CHIR99021

The phosphodiesterase inhibitor may be 3-isobutyl-1 -methylxanthine (iso butyl methyl xanthine: (IBMX)). Compounds of this type may have the formula:

wherein is hydrogen, alkyl or methyl;

R₂ is hydrogen, alkyl, methyl or isobutyl;

R₃ is hydrogen, alkyl or methyl; and

R₄ is hydrogen, alkyl (lower or higher alkyl - for example linear or branched C Ces or C₇-C₁₀), phenyl, substituted phenyl, hydroxyl, methyl or (CH₂)n-0-R₅;

wherein R₅ is hydrogen, alkyl or methyl.

The methods described herein may exploit IBMX derivatives such as, for example, 8- MeO-IBMX - otherwise known as MMPX.

MMPX may have the formula:

The methods may exploit Inhibitor VIII and/or CHIR99021 and IBMX.

The inventors have discovered that the methods described herein may be used to obtain improved yields of erythroid cells from stem cells. Specifically, the inventors have discovered that methods which comprise exposing or contacting cells with the GSK3 inhibitors and phosphodiesterase inhibitors described herein, result in the generation of erythroid cells exhibiting improved quality and which are near clinical grade. Additionally, erythroid cells produced by the methods described herein have been found to be more robust than those made by prior art methods. Additionally, it should be understood that the methods described herein may exploit suspension based liquid culture systems and are thus scalable. Moreover, methods described herein achieve a degree of efficiency high enough to avoid the need for any purification step (>80% HPC at d10 and >90% erythroid series by d24). In addition, the methods described herein support a considerable amplification of cell numbers as they differentiate to RBCs (up to about 350,000 fold dO-24) - this represents a considerable improvement over prior art methods, including those exploiting HoxB4 as an amplifying agent. The methods described herein may be applied to human induced pluripotent stem cells (iPSC) or human embryonic stem cells - including (hESC) stem cell lines that have been derived under fully GMP compliant and licensed conditions. Cells of this type are generally referred to as "stem cells" in this specification (see the definition below). The inventors have further determined that stem cells subjected to the methods described herein reach the orthochromatic normoblast stage of erythropoiesis and display characteristics of definitive hematopoiesis (including the shut off of embryonic globins and expression of Αγ globin).

It should be understood that the term "stem cells" is defined above and the same definitions applies here - i.e. the term "stem cells" may be taken to refer to any cell which is able to self renew and indefinitely divide - cells of this type may be described as "immortal". In addition, when cultured under suitable conditions and/or contacted with, or exposed to, particular compounds and/or conditions, stem cells may differentiate into one or more of the specialised cell types which form embryonic and/or adult tissues. For the avoidance of doubt, the term stem cell includes induced pluripotent stem cell and stem cells obtained by methods which do not involve the destruction of human embryos.

An erythroid cell generated by the methods described may be characterised by expression of one or more haematopoietic/erythroid markers selected from the group consisting of: CD31 ; CD34; CD36; CD41 a; CD43; CD45; CD71 ; and CD235a

Stem cells to be used in the methods described herein may used directly from source. Additionally or alternatively, stem cells for use may comprise stem cells which have been maintained for a period of time.

Stem cells which are "maintained" may be retained in a proliferative and/or pluripotent state for a period or prolonged period of time and/or over a number of passages. Maintained stem cells may remain pluripotent or retain the pluripotent phenotype while at the same time being characterised by the expression of one or more of the stem cell markers described herein.

The stem cells for use in the methods described herein may be maintained by subjecting stem cells to a stem cell maintenance protocol. A stem cell maintenance protocol may comprise the use of media and/or substrates suitable for maintaining stem cells. For example, stem cells may be maintained in a stem cell medium such as, for example Stem Pro^®. Additionally, the stem cells may be maintained on a substrate such as, for example, a CELLstart™ substrate. Maintained stem cells may be passaged every 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

The methods described herein may include the additional step of inducing stem cells, for example stem cells which have been maintained for a period of time, to form embryoid bodies. One of skill will appreciate that stem cells may form embryoid bodies if subjected or exposed to specific conditions. By way of example, stem cells, for example those maintained using the maintenance protocols described herein, may be transferred to culture systems comprising low adherence substrates. When cultured on, or in the presence of, a low (for example ultra-low) adherence substrate, stem cells tend to form embryoid bodies. One of skill will appreciate that prior to being transferred to culture systems which induce the formation of embryoid bodies, confluent monolayers of stem cells may be cut or sectioned and clumps or sections of monolayer transferred to a culture system whereupon the sections and/or clumps may be induced to form embryoid bodies.

In view of the above, disclosed herein is a method of inducing differentiation of stem cells (hESC) into erythroid cells, said method comprising the steps of:

(a) inducing stem cells to form embryoid bodies; and

(b) contacting the embryoid bodies with a GSK3 inhibitor and a phosphodiesterase inhibitor. The methods described herein may comprise first and second phases. The first phase and second phases may comprise steps which progressively differentiate stem cells towards cells exhibiting characteristics of the mesodermal germ layer, cells of the haematopoietic lineage and ultimately erythroid cells.

The method may comprise a first phase (phase 1 ) in which stem cells or embryoid bodies are contacted with a GSK3 inhibitor (for example Inhibitor VIII and/or CHIR99021 ) and a second phase (phase 2) in which the cells are contacted with a phosphodiesterase inhibitor (such as IBMX). One of skill will appreciate that once contacted with a GSK3 inhibitor, stem cells may undergo a degree of differentiation (initially towards cells of the mesodermal germ layer specification and later towards cells of the haematopoietic lineage); as such, cells subjected to the second phase of the methods described herein may be referred to as partially differentiated stem cells or differentiated cells. One of skill will appreciate that the purpose of the second phase of the methods described herein is to differentiate cells produced by the first phase through haematopoietic lineages towards erythroid cells.

Phase 1 of the methods may comprise the step of contacting stem cells (or embryoid bodies formed therefrom) with a GSK3 inhibitor and one or more supplementary compounds. The supplementary compounds may be selected from the group consisting of:

(i) Bone Morphogenic Protein 4 (BMP4);

(ii) Vascular Endothelial Growth Factor 165 (VEGF);

(iii) Wnt3A and/or Wnt5a;

(viii) ActivinA;

(ix) Fibroblast Growth Factor a (FGF );

(x) Stem Cell Factor (SCF); and

(xi) β-estradiol.

Phase 2 of the methods may comprise the step of contacting cells produced or generated by the phase one methods with a phosphodiesterase inhibitor and one or more supplementary compounds. The one or more supplementary compounds may be selected from the group consisting of:

(i) Bone Morphogenic Protein 4 (BMP4);

(ii) Vascular Endothelial Growth Factor 165 (VEGF);

(iii) Fibroblast Growth Factor a (FGFa);

(iv) Stem Cell Factor (SCF); (v) β-estradiol.

(vi) Insulin-like Growth Factor 2 (IGF2);

(νϋ) Thrombopoietin (TPO);

(viii) Heparin;

(ix) Hydrocortisone;

(x) Flt3-Ligand;

(xi) Interleukin 3 (IL3);

(xii) IL1 1 ;

(xii) Erythropoietin (EPO);

(xvii) Insulin Growth Factor 1 (IGF1 );

(xviii) StemRegeninl (SR1 ); and

(xix) Pluripotin (SC1 )

Phase 2 may comprise the step of contacting stem cells with culture media supplemented with a phosphodiesterase inhibitor and one or more of the supplementary compounds noted above.

The latter stages of phase 2 may exploit methods in which stem cells or cells differentiated therefrom, are contacted with media supplemented with EPO alone.

Phase 1 of the methods described herein may comprise a first sub phase (referred to herein after as "sub-phase 1 a"). Sub-phase 1 a may comprise the step of contacting stem cells (or embryoid bodies) with one or more compounds selected from the group consisting of:

(i) a GSK3 inhibitor;

(ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Vascular Endothelial Growth Factor 165 (VEGF);

(iv) Wnt3A and/or Wnt5A; and

(v) ActivinA.

Phase 1 of the methods described herein may further comprise a second sub-phase (referred to hereinafter as "sub-phase 1 b") executed after sub-phase 1 a. Sub-phase 1 b may comprise the step of contacting the stem cells (or embryoid bodies) with one or more compounds selected from the group consisting of:

(i) a GSK3 inhibitor;

(ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Vascular Endothelial Growth Factor 165 (VEGF);

(iv) Wnt3A and/or Wnt5A;

(ix) ActivinA;

(x) Fibroblast Growth Factor a (FGF ); (xi) Stem Cell Factor (SCF); and

(xii) β-estradiol.

Phase 2 of the methods described here may comprise a first sub phase (referred to hereinafter sub-phase 2a). Sub-phase 2a may comprise the step of contacting the cells or embryoid bodies provided or produced by the first phase (and specifically sub-phases 1 a and 1 b), which cells or embryoid bodies may have been subjected to dissociation and harvesting protocols, with one or more compounds selected from the group consisting of:

) BMP4

i) VEGF

ii) FGFa

v) SCF;

(v) Insulin-like Growth Factor 2 (IGF2) ;

(vi) Thrombopoietin (TPO);

(vii) Heparin;

(viii) A phosphodiesterase inhibitor (for example IBMX) ; and

(ix) β-estradiol.

Phase 2 may further comprise a second sub-phase (referred to hereinafter as "sub- phase 2b). Sub-phase 2b may comprise repeating the method of sub-phase 2a - in other words, sub-phase 2b comprises the step of contacting cells subjected to sub-phase 2a with one or more of the compounds listed as (i)-(ix) immediately above. Optionally, the cytokines used in sub-phase 2b (compounds (i)-(ix) listed immediately above) may be supplemented with a quantity of StemRegeninl (SR1 ). The inventors have discovered that the optional addition of SR1 during sub-phase 2b (at day 5) enhances or increases the rate of cell amplification - in particular in later stages of the protocols described herein. Moreover, the inventors noted that cells cultured using methods which exploit StemRegeninl (SR1 ) are more "sturdy" and less prone to lysis.

Phase 2 of the methods described herein may further comprise a third sub-phase

(referred to hereinafter as "sub-phase 2c"). Sub-phase 2c may replicate the method of sub- phase 2a and/or 2b. Optionally, before executing sub-phase 2c, the cells produced by sub- phase 2b may first be harvested by, for example centrifugation. Moreover, if the total number of cells after sub-phase 2b exceeds 500x10³, the cells may be split before executing sub- phase 2c. Additionally, throughout sub-phase 2c, the total cell number may be kept under 1 x10⁶ per ml.

(i) BMP4;

(ii) VEGF;

(iii) FGFa;

(iv) SCF;

(v) Insulin-like Growth Factor 2 (IGF2);

(vi) Thrombopoietin (TPO);

(vii) Heparin;

(viii) A phosphodiesterase inhibitor (for example IBMX); and

(ix) β-estradiol.

Phase 2 of the methods described herein may comprise a fifth sub-phase (referred to herein after as "sub-phase 2e"). Sub-phase 2e may comprise the step of contacting cells produced or provided by sub-phase 2d, with one or more compounds selected from the group consisting of:

(i) Hydrocortisone;

(ii) SCF;

(iii) Flt3-Ligand;

(iv) BMP4;

(v) Interleukin 3 (IL3);

(vi) IL1 1 ;

(vii) A phosphodiesterase inhibitor (for example IBMX); and

(viii) Erythropoietin (EPO).

Optionally, sub-phase 2e may be repeated 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times and the time between each repeat of sub-phase 2e may be 1 , 2 or 3 days. Preferably, sub-phase 2e may be repeated 3 or 5 times with the time between each repeat being approximately 2 days. During sub-phase 2e, the cells may be further or additionally contacted with a quantity of pluripotin (SC1 ). For example on about day 14 and about day 16, the cells may be contacted with pluripotin (SC1 ). Again, is has been discovered that the optional addition of pluripotin (SC1 ) during sub-phase 2e (at about, for example day 14 or 16) enhances or increases the rate of cell amplification in later stages of the protocols described herein. Moreover, it has been noted that cells cultured using methods involving the use of pluripotin (SC1 ) were more "sturdy" and less prone lysis.

Phase 2 may comprise a sixth sub-phase (referred to hereinafter as "sub-phase 2f"). Sub-phase 2f may comprise the step of contacting the cells produced or provided by sub- phase 2f with one or more compounds selected from the group consisting of:

(i) Hydrocortisone;

(ii) SCF;

(iii) Insulin Growth Factor 1 (IGF1

(iv) IL3;

(v) IL1 1 ; and

(vi) EPO.

Prior to executing sub-phase 2f, cells provide or produced by sub-phase 2e may be transferred to IBIT medium. One of skill will appreciate that IBIT medium may comprise, for example, Incomplete Iscove's Medium supplemented with stable glutamine, Albumin (for example human and/or (foetal) bovine serum albumin), Insulin, Transferrin and xeno-free component lipid mixture solution. Sub-phase 2f, may comprise the stem of using IBIT medium supplemented with the cytokines listed as (i) to (vi) above. Optionally and prior to executing sub-phase 2f, cells provided or produced by sub-phase 2e may be harvested (perhaps by centrifugation) and plated out at a density of between about 500 x 10³ to about 1 x 10⁶ per 3ml of IBIT medium.

Sub-phase 2f may be repeated 1 , 2, 3 or 4 times. Moreover, each time sub-phase 2f is repeated, fresh IBIT medium and fresh compounds may be used.

Phase 2 of the methods described herein may comprise a seventh sub-phase (referred to hereinafter as "sub-phase 2g"). Sub-phase 2g may comprise the step of harvesting (perhaps by centrifugation) cells produced or generated by sub-phase 2f . Sub-phase 2g may further comprise the step of maintaining (optionally harvested) cells generated or produced by sub-phase 2f in IBIT medium supplemented with EPO. Optionally harvested cells produced or generated by sub-phase 2f may be maintained in IBIT supplemented with EPO for about 1 , 1 .5, 2, 2.5 or 3 days. Sub-phase 2g may comprise the further step of, after maintaining cells in IBIT supplemented with EPO, maintaining the cells in IBIT alone. Cells may be maintained in IBIT medium for about 3, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 9.5, 10, 10.5, 1 1 , 12, 13 or 14 days. Described herein are methods of inducing differentiation of stem cells into erythroid cells, said methods comprising executing phase 1 as described herein and then phase 2 as described herein, wherein phase 1 comprises sub-phases 1 a and 1 b and phase 2 comprises sub-phases 2a-2g.

Disclosed herein is a method of inducing differentiation of stem cells into erythroid cells, said method comprising the steps of:

(a) maintaining stem cells and inducing the formation of embryoid bodies;

(b) subjecting the embryoid bodies to the sub-phase 1 a;

(c) subjecting the product of sub-phase 1 a to sub-phase 1 b;

(d) subjecting the product of sub-phase 1 b to sub-phase 2a;

(e) subjecting the product of sub-phase 2a to sub-phase 2b;

(f) subjecting the product of sub-phase 2b to sub-phase 2c;

(g) subjecting the product of sub-phase 2c to sub-phase 2d;

(h) subjecting the product of sub-phase 2e to sub-phase 2f; and

(i) subjecting the product of sub-phase 2f to sub-phase 2g;

wherein the product of sub-phase 2g is an erythroid cell.

The methods described herein may be executed over a number of days. Sub-phase 1 a may represent the first step of the methods described herein; as such, sub-phase 1 a may be executed on day 0. Sub-phase 1 a may last about 1 , 2 or 3 days. Sub-phase 1 a may last about 2 days.

Sub-phase 1 b may last about 0.5, 1 or 2 days. Sub-phase 1 b may last about 1 day.

Sub-phase 2a may last about 1 , 2 or 3 days. Sub-phase 2a may last about 2 days.

Sub-phase 2b may last about 1 , 2 or 3 days. Sub-phase 2b may last about 2 days.

Sub-phase 2c may last about 1 , 2 or 3 days. Sub-phase 2c may last about 2 days.

Sub-phase 2d may last about 0.5, 1 , 1 .5 or 2 days. Sub-phase 2d may last about 1 day.

Sub-phase 2e may last about 4, 5, 6, 6.5, 7, 7.5 8, 9, 10, 10.5, 1 1 , 1 1 .5, 12, 13, or 14 days. Sub-phase 2e may last about 7 days or about 1 1 days

Sub-phase 2f may last about 5, 6, 6.5, 7, 7.5 or 8 days. Sub-phase 2f may last about 7 days. Sub-phase 2g may last about 3, 4, 5, 6, 6.5, 7, 7.5, 8, 9, 10, 1 1 , 1 1 .5, 12, 12.5, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. Sub-phase 2g may last about 7 or about 12 days.

As stated, sub-phase 1 a may represent the first step of the methods described herein; as such, sub-phase 1 a may be executed on day 0 and sub-phase 1 b may be executed on about day 2. Sub-phase 2a may be executed on about day 3 and sub-phase 2b may be executed on about day 5. Sub-phase 2c may be executed on day 7 and sub-phase 2d may be executed on about day 9. Sub-phase 2e may be executed on about day 10 and sub- phase 2f may be executed on about day 17 or on about day 21 . Sub-phase 2g may be executed on about day 24 or on about day 28. GSK3 inhibitors for use may be used at final concentrations of about 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 1 .5, 2, 2.5, 3, 4 or 5 μΜ. For example, Inhibitor VIII may be used at a final concentration of about 2μΜ in both sub-phases 1 a and 1 b of the methods described herein. CHIR99021 may be used at a final concentration of about 0.2μΜ in sub-phases 1 a and 1 b of the methods outlined above.

Bone Morphogenic Protein 4 (BMP4) may be used at a final concentration of about 1 , 5, 10, 15, 20 or 25 ng/ml. For example, in sub-phase 1 a, BMP-4 may be used at a final concentration of about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 ng/ml. In sub-phase 1 a, BMP4 may be used at a final concentration of about 10 ng/ml. In sub-phases 1 b and 2a, 2b and 2c, BMP 4 may be used at a final concentration of about 15, 17.5, 20, 22.5 or 25 ng/ml. In sub-phases 1 b, 2a, 2b and 2c, BMP-4 may be used at a final concentration of about 20 ng/ml. In sub-phase 2d, BMP4 may be used at a final concentration of about 5, 7, 10, 12 or 15 ng/ml. In sub-phase 2d, BMP4 may be used at a final concentration of about 10 ng/ml. In sub-phase 2e, BMP4 may be used at a final concentration of about 6, 6.2, 6.4, 6.5, 6.6, 6.7, 6.8, 7 or 7.5 ng/ml. In sub-phase 2e, BMP4 may be used at a final concentration of about 6.7 ng/ml.

VEGF may be used at a final concentration of about 5, 10, 12.5, 15, 17.5, 20, 25, 30 or 35 ng/ml. In subphases 1 a and 1 b, VEGF may be used at a final concentration of about 10ng/ml whereas in sub-phase 2a, VEGF may be used at a final concentration of about 30ng/ml. In sub-phase 2d, VEGF may be used at a final concentration of about 15 ng/ml.

Wnt3A and/or Wnt5A may be used at a final concentration of about 5, 7.5, 10, 12.5 or 15 ng/ml. In sub-phases 1 a and 1 b, Wnt3A and/or Wnt5A may be used at a final concentration of about 10 ng/ml.

Activin A may be used at a final concentration of about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 ng/ml. In sub-phases 1 a and 1 b, ActivinA may be used at a final concentration of about 5 ng/ml.

FGFa may be used at a final concentration of about 5, 7.5, 10, 12.5 or 15 ng/ml. In sub-phases 1 b, 2a, 2b and 2c FGFa may be used at a final concentration of about 10 ng/ml. In sub-phase 2d, FGFa may be used at a final concentration of about 5 ng/ml.

SCF may be used at a final concentration of about 10, 14.5, 15, 15.5, 19.5, 20, 20.5 25, 29.5, 30 and 30.5 ng/ml. In sub-phases 2a, 2b and 2c, SCF may be used at a final concentration of about 30 ng/ml. In sub-phase 2d, SCF may be used at a final concentration of about 15 ng/ml. In sub-phase 2f, SCF may be used at a final concentration of about 20 ng/ml.

IGF2 may be used at a final concentration of about 1 , 2, 3, 4, 4.5, 5, 5.5, 7.5, 10, 12.5 or 15 ng/ml. In sub-phases, 2a, 2b and 2c, IGF2 may be used at a final concentration of about 10 ng/ml. In sub-phase 2d, IGF2 may be used at a final concentration of about 5 ng/ml.

TPO may be used at a final concentration of about 1 , 2, 3, 4, 4.5, 5, 5.5, 7.5, 10, 12.5 or 15 ng/ml. In sub-phases, 2a, 2b and 2c, TPO may be used at a final concentration of about 10 ng/ml. In sub-phase 2d, TPO may be used at a final concentration of about 5 ng/ml. Heparin may be used at a final concentration of about 1 , 1 .5, 2, 2.5, 3, 3.5 4, 4.5 or 5 ng/ml. In sub-phases, 2a, 2b and 2c, Heparin may be used at a final concentration of about 5 ng/ml. In sub-phase 2d, Heparin may be used at a final concentration of about 2.5 ng/ml.

The phosphodiesterase inhibitor (for example IBMX) may be used at a final concentration of about 10, 20, 23, 24, 25, 26, 27, 30, 40, 45, 50, 55, 60, 70, 80, 90 or 100 μΜ. In sub-phases, 2a, 2b, 2c and 2e the phosphodiesterase inhibitor (for example IBMX) may be used at a final concentration of about 50 μΜ. In sub-phase 2d, the phosphodiesterase inhibitor (for example IBMX) may be used at a final concentration of about 25 μΜ.

β-estradiol may be used at a final concentration of about 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ng/ml. In sub-phases, 1 b, 2a, 2b and 2c β-estradiol may be used at a final concentration of about 0.4 ng/ml. In sub-phase 2d, β-estradiol may be used at a final concentration of about 0.2 ng/ml.

Hydrocortisone may be used at a final concentration of about 0.1 x10^"6M, 0.2x10^"6M, 0.5x10^"6M, 1 x10^"6M, 2x10^"6M, 3x10^"6M, 4x10^"6M, 5x10^"6M, 6x10^"6M, 7x10^"6M, 8x10^"6M, 9x10^" ⁶M or 10x10^"6. Hydrocortisone may be used at a concentration of about 1 x10^"6M.

Flt3 ligand may be used at a final concentration of about 30, 40, 45, 50, 55 ng/ml. In sub-phase, 2e, Flt3 ligand may be used at a final concentration of about 50 ng/ml.

IL3 may be used at a final concentration of about 6, 6.2, 6.4, 6.5, 6.6, 6.7, 6.8, 7 or 7.5 ng/ml. In sub-phase 2e and 2f, IL3 may be used at a final concentration of about 6.7 ng/ml.

IL1 1 may be used at a final concentration of about 6, 6.2, 6.4, 6.5, 6.6, 6.7, 6.8, 7 or 7.5 ng/ml. In sub-phase 2e and 2f, IL1 1 may be used at a final concentration of about 6.7 ng/ml.

IGF1 may be used at a final concentration of about 5, 10, 15, 20, 25 or 30 ng/ml. In sub-phase 2f, IGF1 may be used at a final concentration of about 20 ng/ml.

EPO may be used at a final concentration of about 1 , 1 .5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 U/ml. In sub-phase 2f, EPO may be used at a final concentration of about 2 U/ml. In sub- phase 2g, EPO may be used at a final concentration of about 4 U/ml.

StemRegeninl (SR1 ) may be used at a final concentration of about 0.1 -10 μΜ. For example a final concentration of 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10μΜ may be used in sub-phase 2b. Typically, a final concentration of about 1 μΜ StemRegenin (SR1 ) is used.

Pluripotin (SC1 ) may be used at a final concentration of about 100-1000 nM. 0.1 -

10μΜ. For example a final concentration of 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 nM may be used in sub-phase 2e.

Typically, a final concentration of about 250 nM or about 500 nM Pluripotin (SC1 ) is used.

For example, on about day 14 of sub-phase 2e, about 500 nM Pluripotin (SC1 ) may be added. On about day 16, about 250 nM Pluripotin maybe added.

It should be understood that the concentrations and amounts of the various supplementary compounds indicated above, may be the final concentrations/amounts of each compound in a culture medium. Some suitable forms of culture media for use in the methods described herein discussed in more detail below, however any of sub-phases 1 a,

1 b, 2a, 2b, 2c, 2d and 2e described above may utilise any base culture media suitable for the maintenance and/or expansion/differentiation of stem cells. For example, these sub- phases may exploit volumes of an Iscove's Modified Dulbecco's Media (IMDM) or

Dulbecco's Modified Eagle Medium based medium. Media of this type include, for example, one or more selected from the group consisting of:

(i) Stemline^® I and II (Sigma)

(ii) Stemspan^® (Stem Cell Technologies)

(iii) X-vivo 10, 15 or 20 (BioWhittaker/Lonza)

(iv) Stem Pro^® 34 (Life Technologies)

(v) Poietics HPGM (Lonza)

(vi) APEL (Stem Cell Technologies)

(vii) Other custom or "homemade" medium based on IMDM or DMEM + factors (similar to BIT).

It should be understood that the various final concentrations and amounts of the supplementary compounds described herein may be the final concentrations and amounts of the supplementary compounds added to the base media.

Also described are media for inducing differentiation of stem cells into erythroid cells and/or for use in a method of inducing differentiation of stem cells into erythroid cells, said medium comprising a GSK3 inhibitor and/or a phosphodiesterase inhibitor and one or more supplementary compounds selected from the group consisting of:

(i) Bone Morphogenic Protein 4 (BMP4);

(ii) Vascular Endothelial Growth Factor 165 (VEGF);

(iii) Wnt3A and/or Wnt5A;

(iv) ActivinA. (v) Fibroblast Growth Factor a (FGF ) ;

(vi) Stem Cell Factor (SCF);

(νϋ) β-estradiol.

(viii) Insulin-like Growth Factor 2 (IGF2) ;

(ix) Thrombopoietin (TPO);

(x) Heparin;

(xi) Hydrocortisone;

(xii) Flt3-Ligand;

(xiii) Interleukin 3 (IL3);

(xiv) IL1 1 ;

(XV) Erythropoietin (EPO);

(xvi) Insulin Growth Factor 1 (IGF1 );

(xx) StemRegeninl (SR1 ) ; and

(xxi) Pluripotin (SC1 )

The medium may comprise a base medium suitable for the maintenance and/or expansion of stem cells. The base medium may comprise a medium suitable for the maintenance and/or expansion of stem cells. Media of this type may comprise, for example, compounds and molecules which facilitate the maintenance and/or expansion of stem cells. The base medium may comprise an Iscove's Modified Dulbecco's Media (IMDM) or Dulbecco's Modified Eagle Medium based medium. By way of example, the base medium may comprise one or more selected from the group consisting of:

(i) Stemline^® 1 and II (Sigma)

(ii) Stemspan^® (Stem Cell Technologies)

(iii) X-vivo 10, 15 and 20 (BioWhittaker/Lonza)

(iv) Stem Pro^® 34 (Life Technologies)

(v) Poietics HPGM (Lonza)

(vi) APEL (Stem Cell Technologies)

(νϋ) Other custom or "home made" medium based on IMDM or DMEM + factors (similar to BIT)

Suitable base media may be serum free.

The base medium may comprise, for example, Stemline^® II medium (Sigma-Aldrich Co. LLP).

The methods described herein may utilise any IMDM or DMEM based media but by way of example, the methods may utilise media comprising Stemline^® II. Media of this type may be suitable for use in any of sub-phases 1 a, 1 b, 2a, 2b, 2c, 2d and/or 2e. Where the methods require the maintenance or expansion of erythrocyte precursor cells - such as might occur in sub-phases 2f and/or 2g, the base medium may comprise IBIT medium itself comprises Incomplete Iscove's medium supplemented with stable glutamine, bovine serum albumin, insulin, transferring and xeno-free component lipid mixture solution.

A medium for inducing differentiation of stem cells into erythroid cells and/or for use in a method of inducing differentiation of stem cells into erythroid cells may comprise a GSK3 inhibitor and one or more compounds selected from the group consisting of:

(i) Bone Morphogenic Protein 4 (BMP4);

(ii) Vascular Endothelial Growth Factor 165 (VEGF);

(iii) Wnt3A and/or Wnt5A;

(iv) ActivinA.

(v) Fibroblast Growth Factor a (FGF );

(vi) Stem Cell Factor (SCF);

(νϋ) β-estradiol.

(viii) Insulin-like Growth Factor 2 (IGF2);

(ix) Thrombopoietin (TPO);

(x) Heparin;

(xi) Hydrocortisone;

(xii) Flt3-Ligand;

(xiii) Interleukin 3 (IL3);

(xiv) IL1 1 ;

(XV) Erythropoietin (EPO); and

(xvi) Insulin Growth Factor 1 (IGF1 ).

A medium for inducing differentiation of stem cells into erythroid cells and/or for use in a method of inducing differentiation of stem cells into erythroid cells may comprise:

(i) a GSK3 inhibitor

(ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Vascular Endothelial Growth Factor 165 (VEGF);

(iv) Wnt3A and/or Wnt5A;

(v) ActivinA.

(vi) Fibroblast Growth Factor a (FGFa);

(vii) Stem Cell Factor (SCF); and

(viii) β-estradiol.

(i) a GSK3 inhibitor (ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Vascular Endothelial Growth Factor 165 (VEGF);

(iv) Wnt3A and/or Wnt5A; and

(v) ActivinA.

A medium of the type described immediately above may be used in sub-phase 1 a of the methods described herein.

(i) a GSK3 inhibitor

(ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Vascular Endothelial Growth Factor 165 (VEGF);

(iv) Wnt3A and/or Wnt5A;

(v) ActivinA.

(vi) Fibroblast Growth Factor a (FGF );

(vii) Stem Cell Factor (SCF); and

(viii) β-estradiol.

A medium of the type described immediately above may be used in sub-phase 1 b of the methods described herein.

A medium for inducing differentiation of stem cells into erythroid cells and/or for use in a method of inducing differentiation of stem cells into erythroid cells may comprise one or more compounds selected from the group:

(i) A phosphodiesterase inhibitor;

(ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Vascular Endothelial Growth Factor 165 (VEGF);

(iv) Fibroblast Growth Factor a (FGFa);

(v) Stem Cell Factor (SCF);

(vi) Insulin-like Growth Factor 2 (IGF2);

(vii) Thrombopoietin (TPO);

(viii) Heparin;

(ix) β-estradiol

(x) Hydrocortisone;

(xi) Flt3-Ligand;

(xii) Interleukin 3 (IL3);

(xiii) IL1 1 ;

(xiv) Erythropoietin (EPO);

(XV) StemRegeninl (SR1 ); and (xvi) Pluripotin (SC1 )

A medium for inducing differentiation of stem cells into erythroid cells and/or for method of inducing differentiation of stem cells into erythroid cells may comprise

(i) A phosphodiesterase inhibitor;

(ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Vascular Endothelial Growth Factor 165 (VEGF);

(iv) Fibroblast Growth Factor a (FGF );

(v) Stem Cell Factor (SCF);

(vi) Insulin-like Growth Factor 2 (IGF2);

(vii) Thrombopoietin (TPO);

(viii) Heparin; and

(ix) β-estradiol

A medium of the type described immediately above may be used in sub-phases 2a, 2b, 2c and 2d of the methods described herein.

(i) A phosphodiesterase inhibitor

(ii) Bone Morphogenic Protein 4 (BMP4);

(iii) Hydrocortisone;

(iv) Flt3-Ligand;

(v) Interleukin 3 (IL3);

(vi) IL1 1 ;

(vii) Erythropoietin (EPO);

(viii) StemRegeninl (SR1 ); and

(ix) Pluripotin (SC1 )

A medium of the type described immediately above may be used in sub-phase 2e of the methods described herein.

The media described above and which are suitable for use in sub-phases 1 a, 1 b, 2a, 2b, 2c, 2d and 2e of the methods may further comprise Stemline^® ll.

(i) Stem Cell Factor (SCF);

(ii) Hydrocortisone;

(iii) Interleukin 3 (IL3);

(iv) IL1 1 ;

(v) Erythropoietin (EPO); and (vi) Insulin Growth Factor 1 (IGF1 ).

A medium of the type described immediately above may be used in sub-phase 2f of the methods described herein.

A medium for inducing differentiation of stem cells into erythroid cells and/or for use in a method of inducing differentiation of stem cells into erythroid cells may comprise Erythropoietin (EPO).

A medium of the type described immediately above may be used in sub-phase 2g of the methods described herein.

Media suitable for use in sub-phases 2f and 2g (such as the media described above) may further comprise IBIT medium as defined above.

It should be understood that the precise concentrations and amounts of various supplementary compounds described above may vary depending upon the application but in general, the media may contain concentrations and amounts of supplementary compounds substantially identical or similar to the concentration and amounts required in the methods described herein. Suitable concentrations and amounts of the various supplementary compounds are described in more detail above (see section discussing methods and sub- phases).

Also described herein is a kit for inducing differentiation of stem cells into erythroid cells and/or for use in a method of inducing differentiation of stem cells into erythroid cells, said kh comprising one or more components selected from the group consisting of:

(a) one or more of the media described herein;

(b) one or more compounds selected from the group consisting of:

(i) Bone Morphogenic Protein 4 (BMP4);

(ii) Vascular Endothelial Growth Factor 165 (VEGF);

(iii) Wnt3A and/or Wnt5A;

(iv) ActivinA.

(v) Fibroblast Growth Factor a (FGF );

(vi) Stem Cell Factor (SCF);

(νϋ) β-estradiol.

(viii) Insulin-like Growth Factor 2 (IGF2);

(ix) Thrombopoietin (TPO);

(x) Heparin;

(xi) Hydrocortisone;

(xii) Flt3-Ligand;

(xiii) Interleukin 3 (IL3);

(xiv) IL1 1 ; (xv) Erythropoietin (EPO);

(xvi) Insulin Growth Factor 1 (IGF1 ).

(xvii) StemRegeninl (SR1 ); and

(xviii) Pluripotin (SC1 )

(c) Optionally sterile receptacles for the culture and/or maintenance of stem cells, embryoid bodies and/or cells;

(d) Tools and/or implements for adding supplements to media (for example pipettes and/or syringes); and

(e) Instructions for use.

DETAILED DESCRIPTION

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

Figure 1 : High purity mature erythrocytes from Day 20 of a method performed in accordance with an embodiment of this invention: ejected nuclei and erythrocyte sized haemoglobin containing cells can be seen.

Figure 2: Low purity erythrocytes from a higher pH (7.5) culture.

Figure 3: Bioreactor Culture at pH 7.0 and 21 % 0₂ from the point the cells were received - the results show the production of highly mature cells after only 5 days.

Figure 4: A subsequent run employing a pH drop to 7.0 at Day 17 (7 days after receiving cells) where cells had been exposed to alternate oxygen conditions of 7%, 10%, 13%, 16% or 19% at pH 7.3 or 13% at pH 7.5, showed high quality red cell maturation upon exposure to 13%, 16% and 19% oxygen at day 21 (1 1 days after receiving cells). A short 12 hour exposure to pH 7.0 at day 8 followed by reversion to pH 7.4 up to day 21 did not replicate the effect as shown in the next images (all images day 21 ).

Figure 5: A subsequent experiment explored the effect of dropping pH to 7.0 after 15, 17, or 19 days (after 5, 7 or 9 days in the bioreactor after transfer). The pH drop at day 5 showed good red cell maturity at day 20 (day 20 shown in images, day 24 was also observed).

Note: Figures 6-16 were presented in PCT/GB2013/051917

Figure 6: 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.

Figure 7: 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.

Figure 8: >90% CD235a (GlyA+) erythroid cells at d24. A. 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. B. Rapid Romanovski staining of a cytospin preparationof hiPSC differentiated into erythroid cells at day 28, the cells are mainly orthochromatic normoblasts.

Figure 9: 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.

Figure 10: InhibVIII increases cell numbers in multiple lines. A-Histogram representative of total fold amplification of erythroid differentiation culture of hESC lines RC9 and H1 in absence or presence of inhibitor VIII : B-Histogram representative of total fold amplification of erythroid differentiation culture of iPS line in absence or presence of inhibitor VII I, both conditions included IBMX.

Figure 11 : A. IBMX further increases expansion. 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 GSK3P inhibitors and IBMX using the standard option of 7 days in cytokine mix A. B. 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 GSK3P inhibitors and IBMX using the prolonged period of 1 1 days in cytokine mix A.

Figure 12: Inh VIII + IBMX Increase key molecular markers in iPSC. Histograms representative of imRNA 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.

Figure 13: Globins are almost exclusively fetal not embryonic. HPLC analysis of the globins produced by the differentiated hPSCs. The disappearance of haemoglobin Gower 1 (ζ2 ε2 chains) is noticeable in erythroid cells derived from iPSCs (Panel 2: B) and hESCs H1 (Panel 3: C), compared with Panel 1 (A) 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 (panel 4: D). The difference observed in globin ζ chain may be due to some residual haemoglobin Portland (ζ2 γ2 chains).

Figure 14: Comparison of cell numbers with wnt3a vs wnt5a Figure 15: Effect of SC1 + SR1 on iPSC (A) or hESC (B) amplification during differentiation Figure 16: Comparison of cells at day 30 after culture +/- SR1 and SC1

Figure 17: Graph A shows the average effect of different oxygen levels across a pH range; robust maturation and enucleation is achieved at oxygen levels under 50% of atmospheric oxygen (or under 1 1 % dissolved Oxygen). This graph also shows culture at below pH 7.4 may lead to significantly lower enucleation. Under these lower pH conditions non-erythroid lineage cells can be seen to persist in the culture system consequently lowering the enucleated yield (data not shown). Graph B shows that these effects (those shown in graph A) are non-independent; a low oxygen level may reduce the sensitivity to the detrimental effects of a low pH, and a higher pH may reduce the sensitivity to the detrimental effects of higher Oxygen. Culturing cells above about pH 7.4 and beneath about 50% atmospheric Oxygen (1 1 % dissolved oxygen) may lead to robust enucleation.

Figure 18: Graph A shows the increase in peak enucleation observed when cells are cultured under high vs low mechanical stress. The stress in this case is created by a bioreactor impeller with a tip speed of 157mm/second (350rpm) or 236mm/second (450rpm). The majority of the mechanical effect is on the terminal enucleation event, rather than any accumulated effect through culture; this is shown in graph B where cells from a non- mechanically stressed system are transferred to the mechanically stressed system during the enucleation phase (after 19 days). Two days later a 17 percentage point improvement in enucleation is observed, and this advantage is maintained over the subsequent week. The effect of mechanical agitation may be relative to the underlying level of enucleation in the culture and the absolute effect is therefore not independent of pH and oxygen. Without wishing to be bound by theory, this may represent an economic advantage as it will enable greater control of population purity at harvest and enables peak enucleation levels to be achieved significantly closer to peak cell yield. A method of this invention which exploits a predetermined pH level (for example a pH greater than about pH 7.4 (for example about pH 7.5) and an oxygen level at or under 50% of atmospheric oxygen (or at or under about 1 1 % dissolved oxygen) may be supplemented with some exposure to a level or levels of mechanical stress. In combination with the control of the pH and 0₂ levels, this may further increasing the purity of an enucleated erythrocyte population produced from pH/02 control. Equipment

AMBR stands for Advanced Micro Bioreactor. The system is a proprietary platform from TAP Bio-systems designed to provide a scaled down model of the most common scaled up production system for biologic products: a stirred tank bioreactor.

Example 1

Previous work in the literature has shown the ability to mature to reticulocytes from adult or cord blood derived progenitors in formats such as wave culture bags or stirred tank apparatus. To our knowledge, late stage maturation of pluripotent derived cells to either reticulocytes or reasonably high purity enucleated mature erythrocytes in a scalable format has not been shown.

We have shown that pluripotent cell derived erythroid progenitor cells in a multi 10ml vessel, scaled down, stirred tank culture array (Advanced Micro Bioreactor: AMBR) held at pH 7.0 develop a very mature terminally differentiated phenotype. We have shown that this can happen if the pluripotent cell derived erythroid progenitor cells are immediately placed in the low pH 7.0 conditions, or if this pH drop is delayed for up to 7 days (to promote further growth).

In combination this suggests that the effect of the pH drop may be dependent on the maturation state of the cells, and potentially through this or alternate mechanism, interdependent with any alteration to the media composition used in the methods to generate differentiated stem cells for use.

Method

Differentiated stem cells obtained using the methods set out in PCT/GB2013/051917

(namely the product of the 10^th day of that protocol) were transported (in a cool box) at ambient temperature over approximately 5-15, for example 8 hours.

Bioreactor inoculation and maintenance - Prior to cell seeding, AMBR™ vessels (TAP Biosystems, Royston, UK) were filled with 14ml_ of medium and stabilised for temperature (37 °C), d0₂ (16%, 21 %) and pH (7.3). Automated antifoam C additions (20μΙ_ of 1 % solution, Sigma-Aldrich) were made every 24hr. Automated sodium bicarbonate additions (20μΙ_ of 1 M solution, Sigma-Aldrich) and C0₂ gassing maintained culture set pH (automatically monitored every 2hr and adjusted as required). An impeller speed of 450 rpm was used to maintain the cells in stirred suspension in the AMBR system.

Day 10 cells (i.e. received cells) were inoculated into the pre-conditioned AMBR™ vessels. Cells were cultured at an initial density of 1 x10⁵ cells/ml in Stemline II media (Sigma-Aldrich) supplemented with cytokine mix A: SCF (50ng/ml_, Flt3L (16.7ng/ml_, BMP4, IL3, IL1 1 (all 6.7ng/ml_), EPO (3 Units/mL and hydrocortisone 1 uM) and reagent-x (IBMX; 50 uM). Physicochemical conditions were maintained as above. Cultures were supplemented with cytokine mix A on days 2 and 4. On day 5 of culture pH was dropped and maintained at pH 7 (16% 0₂) for the remainder of the culture. On day 7 replacement of culture medium was conducted through centrifugation and re-suspension of cells at 1 E6/ml in fresh IBIT medium (Biochrome Ag, Germany) supplemented with cytokine mix C: SCF, IGF1 (both 20ng/ml_), IL3, IL1 1 (both 6.7 ng/mL) and Epo (3 Units/mL). Cultures were supplemented with cytokine mix C on days 19 and 21 (9 and 1 1 days after bioreactor inoculation). The most mature cell phenotype was observed on day 20 (10 days post inoculation) Staining

Cells were visualised by sampling from the bioreactors after about day 20 (i.e. 10 days after start of maturation protocol of this invention). Cell samples were processed onto slides with Cytospin and stained with benzidine-Giemsa stain (Haemaglobin shows red/brown other cells and nuclei show blue).

Discussion

Control of the pH is causing late stage mature red cells to emerge as this is a highly consistent theme. However, it is not clear how long the cells persist for, and whether some of the conditions that appeared not to mature (such as the day 7 pH drop above) was due to no optimum observation point (probably day 12). -4-5 days seems to have been the optimum timing for exposure to low pH in every successful condition. The exposure to oxygen tension throughout also appears to interact with the pH effect.

The slight inconsistency in the effects of timing could be due to variability in the day

10 input populations in each run.

Generally, earlier exposure to low pH seems to have produced more robust maturation, potentially providing a method to robust the process to variability in input cells.

However, this may come at the expense of proliferation.

A persistent low pH 7.0 after the red cells are observed leads to regrowth of a further non-haemaglobin containing population. We have not studied if this will mature with prolonged culture.

Table 1 : Outline of experimental conditions gx e io Pftme WftPh *!: (87-814}≠8ete hg$C celts density reset 02 Cefi ensity res

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7 m ¾ .. 21 Norte 7,60 2i

?.5S S, 2i

725 3

iOS¾Oi3 Fmfc S, IS 7.S 21

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EXAMPLE 2: DATA presented in PCT/GB2013/051917

Methods 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).

For differentiation, confluent hPSC are cut into squares with the EZpassage tool (Life Technologies) and plated at 500x10³/well on Ultra low adherence six well plates (Corning) in 3ml/well of Stemline II (Sigma) to allow them to form embryoid bodies (EBs).

On day 0 in order to induce differentiation, the following cytokines are added: Bone Morphogenic Protein 4 (BMP4) (10ng/ml), Vascular Endothelium Growth Factor 165 (VEGF) (10ng/ml), Wnt3A (and/or Wnt5A) (10ng/ml), ActivinA (5ng/ml) and Inhibitor VIII (2μΜ). 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

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

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

On day 3 of differentiation the EBs are washed in PBS then dissociated using

1 ml/well TrypleSelect 10X 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 3ml fresh Stemline II and replated at 200x10³/well of a regular six well tissue culture plate with the following cytokines: BMP4 (20ng/ml), VEGF (30ng/ml), FGFa

(10ng/ml), SCF (30ng/ml), Insulin-like Growth Factor 2 (IGF2) (10ng/ml), Thrombopoietin (TPO) (10ng/ml), Heparin (5ug/ml), Iso Butyl Methyl Xanthine (IBMX) 50μΜ and β-Estradiol (0.4ng/ml).

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.

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 500x10³, 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. From day 7 to day 10 of differentiation the cell density should be closely monitored in order to maintain the cell number under 10⁶ per ml by adding fully supplemented media and splitting into additional wells if required.

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.

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 100x10³ cells/well in 3ml/well of Stemline II supplemented with the following cytokines:

Hydrocortisone 10^"6M, SCF (50ng/ml), Flt3-Ligand (Flt3L) (16.7ng/ml), BMP4 (6.7ng/ml), Interleukin 3 (IL3) (6.7ng/ml), IL1 1 (6.7ng/ml), IBMX (50μΜ) and Erythropoietin (EPO) 1 .3U/ml.

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

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 500x10³ to 1 x10⁶ 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), 10ug/ml Insulin, 200ug/ml Transferrin (both from Sigma) and xeno free component lipid mixture solution 200x (Peprotech)) supplemented with the following cytokines: Hydrocortisone 10^"6M, SCF (20ng/ml), Insulin Growth Factor 1 (IGF1 ) (20ng/ml), Interleukin 3 (IL3) (6.7ng/ml), IL1 1 (6.7ng/ml) and Erythropoietin (EPO) 2U/ml. From day 17 or 21 to day 24 or 28, a full dose of the above cytokines are renewed every 2 days, added in 0.5ml of Stemline II. such that final concentrations of fresh cytokines are those stated for day 17 or 21.

On day 24 or 28 of differentiation the cells are centrifuged 3 minutes at 1200 rpm then re- plated at a density of 500-1000 x10³ in fresh IBIT supplemented with 4U/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.

Analysis and Characterization

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, CD41 a, 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).

At day 17 onward, the erythroid stage of the cells was determined assessment of morphology after Rapid Romanovski staining of cytospin preparations. At days 0, 10, 17 and 24 the expression of selected genes of interest by the differentiating cells was monitored by qRT-PCR (Taqman)

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.

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.

Results

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.

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, FGFa and β-Estradiol.

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 (Figure 6), thus this method can be executed completely in suspension culture.

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 Figure 7, 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 MA, Thomson JA, 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 , CD41 a, 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.

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.

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 (Fig 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..

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 3β (GSK3P) 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 β-catenin by GSK3P and thereby allowing the release and accumulation of active β-catenin.

The addition of GSK3P 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 Fig 3B and 4. Preliminary results indicated that these GSK3P 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 Figure 8, 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.

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 9). In order to define a method that is most generally applicable we use the combination of a GSK3P inhibitor, Wnt3A and Activin A in the early stage of differentiation which results in consistent results with all hPSC lines tested (Fig 10 Aand B). The secondGSK3p inhibitor tested, CHIR99021 is more potent (0.2μΜ instead of 2μΜ for Inhibitor VIII) and has a stronger effect on hESC than hiPSC lines compared to Inhibitor VIII (Table 2). Additionally, increasing the period of treatment with InhVIII (dO to d5) also increases the expansion in cell number.

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 (Fig 1 1 A and B, Table 2). 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 throughout the period day 3 and 17 (Table 2).

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 1 1 d as shown in Figure 1 1 B the extra time allow for an additional amplification and suggests that this phase may be extended further with consequent increases in cell number.

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 12). 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, Runxl , 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.

The addition of IBMX and GSK3P 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 13) 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.

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 0.5ml/well of Stemline II. The small molecule StemRegeninl (SR1 ) (Cellagen Technology) at a final concentration of 1 μΜ is added along the cytokines. On days 14 the small molecule Pluripotin (SC1 ) (Stemgent) is added along the cytokines for a final concentration of 500nM. On days 16 the small molecule Pluripotin (SC1 ) (Stemgent) is added along the cytokines for a final concentration of 250nM 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 (Figure 7). The cells are also sturdier and less prone to lysis (Figure 8). 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.

Summary

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 dO-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 Αγ 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 (dO-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 350x10e3 erythroid cells per hPSC. This expansion is equivalent to 1 unit of RBC concentrate (2x10e12 cells) per 6x10e6 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.

Table 2: table summarizing results obtained when comparing side by side the effect of the addition of GSK3P 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 coloured columns.

Claims

1 . A method of providing mature enucleated erythrocytes and/or reticulocytes, said method comprising maintaining and/or culturing differentiated stem cells under one or more maturation condition(s), wherein said maturation conditions are selected from the group consisting of:

(i) a predetermined pH;

(ii) a predetermined or specific level of (dissolved) oxygen; and

(iii) mechanical stress.

2. The method of claim 1 , wherein the differentiated stem cells comprise stem cell derived haematopoietic progenitor cells or erythroid progenitor cells.

3. The method of claim 1 or 2 wherein the differentiated stem cells are provided or obtainable by methods in which stem cells or embryoid bodies are first contacted with a GSK3 inhibitor and then with a phosphodiesterase inhibitor.

4. The method of any preceding claim, wherein the maturation conditions comprises a predetermined pH comprising:

(i) one or more predetermined pH values selected from a pH of about pH 4.0 to about pH 7.9;

(ii) a pH above about pH7.4; or

(ii) a pH of about pH 7.5

5. The method of claim 4, wherein the differentiated stem cells are maintained and/or cultured under the predetermined pH for the duration of an erythrocyte/reticulocyte culture protocol or for about 1 to about 1 1 days of an erythrocyte/reticulocyte culture protocol.

6. The method of claim 4 or 5, wherein the differentiated stem cells are maintained and/or cultured under conditions which comprise a pH above about pH 7.4 for the duration of a erythrocyte/reticulocyte culture protocol or in the final 1 -10 days of maturation.

7. The method of any one of claims 1 -3, wherein the maturation conditions comprise a specific level of oxygen.

8. The method of claim 7, wherein the specific level of oxygen is less than about 50% of atmospheric oxygen and/or less than or equal to 1 1 % dissolved oxygen.

9. The method of claims 7 or 8, wherein the differentiated stem cells are exposed to a specific oxygen level at any time during an erythrocyte/reticulocyte culture protocol.

10. The method of any one of claims 7, 8 or 9 wherein the specific oxygen level is applied continuously or continually throughout an erythrocyte/reticulocyte culture protocol or at one or more specific time points.

1 1 . The method of any one of claims 1 -3, wherein the maturation conditions comprise the use of mechanical stress.

12. The method of claim 1 1 , wherein the mechanical stress is applied to the

differentiating stem cells.

13. The method of claims 1 1 or 12, wherein the mechanical stress is created within a bioreactor.

14. The method of any one of claims 1 1 , 12 or 13, wherein the level of mechanical stress is controlled by modulation of the speed of a bioreactor impeller.

15. The method of any one of claims 1 1 -14, wherein the mechanical stress may be applied continuously or continually throughout an erythrocyte/reticulocyte culture protocol.

16. The method of any one of claims 1 1 -15, wherein the mechanical stress is applied at the beginning and/or end of an erythrocyte/reticulocyte culture protocol.

17. A method of providing mature enucleated erythrocytes and/or reticulocytes, said method comprising maintaining and/or culturing differentiated stem cells under a predetermined pH and at a predetermined or specific level of oxygen.

18. The method of claim 17, wherein the method further comprises subjecting the differentiated stem cells to mechanical stress.

19. The method of any preceding claim, wherein the method is conducted in a bioreactor system.

20. The method of any one of claims 1 to 3, wherein the maturation conditions are applied or used concurrently or separately and at different or overlapping times.

21 . A cell culture medium supplemented with one or more compounds selected from the group consisting of:

(i) Stem cell factor (SCF);

(ii) Flt3-Ligand;

(iii) Bone morphogenic protein 4 (BMP4);

(iv) Interleukin 3 (IL3);

(v) Interleukin 1 1 (IL1 1 );

(vi) Erythropoeitin (EPO);

(νϋ) Hydrocortisone; and

(viii) Phosphodiesterase inhibitor;

wherein the medium is formulated or provided at a pH above about pH 7.4 or at pH

7.5.

22. A cell culture medium supplemented with one or more compounds selected from the group consisting of:

(i) Stem cell factor (SCF);

(ii) Insulin Growth Factor I (IGF1

(iii) IL3;

(iv) IL1 1 ; and

(v) EPO;

7.5.

23. A method of generating or providing mature enucleated erythrocytes and/or reticulocytes, said method comprising culturing or maintaining in a bioreactor system, differentiated stem cells in the culture medium of claim 21 and/or claim 22.

24. The method of claim 23, wherein the method further exploits one or more maturation conditions selected from the group consisting of:

(i) a predetermined or specific level of (dissolved) oxygen; and

(ii) mechanical stress.