Method of producing self-renewing erythroid cells from human progenitor cells

Abstract

Novel methods for producing self-renewing erythroblasts (SREs) from induced pluripotent stem cells (iPSCs) or CD34+ cells are provided. Also provided are genetically modified iPSCs or CD34+ cells having a constitutively active stem cell factor (SCF) receptor and a constitutively active Janus kinase 2 (JAK2) that can differentiate into enucleated red blood cells, self-renew, and expand in culture media for an extended period of time. In particular, the disclosed methods completely eliminate the need for SCF and Epo.

Description

METHOD OF PRODUCING SELF-RENEWING ERYTHROID CELLS FROM HUMAN PROGENITOR CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent No. 63 / 602,742, filed November 27, 2023, which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01HL130764 awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (182219.00244SeqList.xml; Size: 15,602 bytes; and Date of Creation: October 28, 2024) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0004] The present invention relates generally to methods for producing self-renewing erythroblasts (SREs) by differentiating culturing genetically modified pluripotent stem cells or CD34+ cells.BACKGROUND OF THE INVENTION

[0005] Cultured Red Blood Cells (cRBCs) have the potential to supplement blood transfusion needs, and offer promise for drug delivery and immune tolerization. While cRBCs derived from Cord Blood (CB) or Peripheral Blood (PB) Hematopoietic Progenitor Cells (HPCs) can address local blood shortages, they are not optimal for large-scale RBC production due to the continual need for primary cell collection from volunteers.

[0006] Erythroid cell lines that can be cultured for months, and in some cases indefinitely, have been developed using over-expression methods involving HPV E6 / E7, hTERT, SV40T antigen, c-myc, Bcl-XL, Spil or Bmi-1. Some of these lines can be differentiated intoenucleated cRBCs and could potentially serve as an unlimited source of cells for transfusion and other cRBCs applications. However, concerns about their karyotypic stability and high maintenance costs hinder large-scale cRBC production. Notably, all cell lines produced so far are karyotypically unstable, exhibit a low growth rate, and can only be cultured at low density.

[0007] In particular, the high costs of using cytokines such as stem cell factor (SCF) and erythropoietin (Epo) to differentiate induced pluripotent stem cells (iPSCs) into cRBCs as well as the duration of existing differentiation protocols renders large-scale production of cRBCs difficult. Consequently, there remains a need to develop self-renewing erythroblasts (SREs) that proliferate without cytokines. Such SREs could be used to improve large-scale production of cRBCs.SUMMARY OF THE INVENTION

[0008] Provided herein are methods of producing self-renewing erythroblasts (SREs). In one aspect, provided is a method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified CD34+ cell comprising a constitutively active stem cell factor (SCF) receptor and a constitutively active Janus kinase 2 (JAK2); and culturing the genetically modified CD34+ cell in one or more culture media free of a stem cell factor (SCF) and erythropoietin (Epo).

[0009] In one aspect, provided is a method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified CD34+ cell comprising a constitutively active stem cell factor (SCF) receptor; and culturing the genetically modified CD34+ cell in one or more culture media free of a stem cell factor (SCF).

[0010] In one aspect, provided is a method of producing a self-renewing erythroblast (SRE), comprising:providing a genetically modified induced pluripotent stem cell (iPSC) comprising a constitutively active stem cell factor (SCF) receptor and a constitutively active Janus kinase 2 (JAK2); and culturing the genetically modified iPSC in one or more culture media free of a stem cell factor (SCF) and erythropoietin (Epo).

[0011] In one aspect, provided is a method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified induced pluripotent stem cell (iPSC) comprising a constitutively active stem cell factor (SCF) receptor and culturing the genetically modified iPSC in one or more culture media free of a stem cell factor (SCF).

[0012] In one embodiment, the SRE is cultured in the presence of Epo.

[0013] In one embodiment, the SCF receptor comprises a mutation at position D816. In one embodiment, the SCF receptor comprises a D816V mutation. In one embodiment, the SCF receptor comprises the amino acid sequence of SEQ ID NO: 2.

[0014] In one embodiment, the JAK2 comprises a deletion at C616 and V617 or comprises a mutation at position V617. In one embodiment, the JAK2 comprises a V617F mutation. In some embodiments, the JAK2 comprises the amino acid sequence of SEQ ID NO: 4 or 5.

[0015] In some embodiments, the SRE is consistently passaged at ratios from 1 : 10 to 1 : 15 every 3 to 4 days.

[0016] In one embodiment, the SRE is capable of self-renewing in a medium for at least 15 passages. In one embodiment, the CD34+ cell is a human cell. In one embodiment, the iPSC is a human cell.

[0017] In one embodiment, the CD34+ cell is collected from the blood of a subject aged 0 to 100 years old. In one embodiment, the CD34+ cell is collected from embryonic or fetal tissues.

[0018] In one embodiment, the CD34+ cell is produced from a pluripotent stem cell or an iPSC.

[0019] In one embodiment, the one or more culture media comprise dexamethasone (Dex) and isobutylmethylxanthine, l-Methyl-3-Iso-butyl-xanthine (IBMX). In some embodiments, the one or more culture media comprise from about 0.01 pM to about 100 pM of Dex and from about 1 pM to 500 pM of IBMX. In one embodiment, the one or more culture media comprise about 1 unit of Epo, about 1 pM Dex, and about 30 pM of IBMX.

[0020] In some embodiments, the one or more culture media comprises Dex and IBMX from day 17 to at least day 38.

[0021] In some embodiments, the one or more culture media comprise one or more of transferrin, activin A, Wnt, GM-CSF, vascular endothelial growth factor (VEGF), bone morphogenic proteins (BMP), inhibitor VIII, P-Estradiol, heparin, UM171 or UM729, thrombopoietin (TPO), insulin-like growth factor-2 (IGF-2), RU-486, and basic fibroblast growth factor (bFGF).

[0022] In some embodiments, the method comprises supplementing the one or more culture media with thrombopoietin (TPO), insulin-like growth factor-2 (IGF-2), and basic fibroblast growth factor (bFGF) only from day 10 to day 17.

[0023] In some embodiments, the one or more culture media comprises between 1 to 1000 pg of recombinant transferrin.

[0024] In some embodiments, the one or more culture media comprises a serum-free medium or a defined differentiation medium.

[0025] In some embodiments, the one or more culture media comprise a supplement selected from inositol, folic acid, monothioglycerol, insulin, ferrous nitrate, ferrous sulfate, BSA, L- glutamine, penicillin-streptomycin, animal plasma or serum, Fe(III)-EDTA or an equivalent chelator, lipids, and combinations thereof.

[0026] In some embodiments, the method comprises removing Dex and IBMX from the medium to induce differentiation of the SRE into a red blood cell.

[0027] In some embodiments, the red blood cell expresses ADAMTS13, asparaginase, Factor VIII, Factor IX, or phenylalanine hydroxylase.

[0028] In one embodiment, the method further comprises expanding the CD34+ cell prior to being differentiated into a SRE. In some embodiments, the method further comprises differentiating the CD34+ cell into a SRE and sorting a population of SREs derived from the CD34+ cell using magnetic-activated cell sorting (MACS), flow cytometry, fluorescence-activated cell sorting (FACS), or physical means. In some embodiments, the method further comprises sorting the population of SREs based on the expression of one or more of CD31, CD34, CD43, and CD45.

[0029] In some embodiments, provided is a method of obtaining a red blood cell, comprising producing a SRE according to the methods as described herein to obtain a red blood cell.

[0030] In one aspect, provided is a self-renewing erythroblast (SRE), comprising a constitutively active stem cell factor (SCF) receptor and a constitutively active Janus kinase 2 (JAK2).

[0031] In some embodiments, provided is a pharmaceutical composition comprising the SRE as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIGS. 1A, IB, 1C, and ID show the differentiation of KitD816V iPSCs. FIG. 1A is a diagram and table illustrating the chemically-defined PSC-RED protocol to differentiate iPSCs into erythroid cells. STIF: S =SCF; T = Tpo; I = IGF-2; F = bFGF. SEDI: S = SCF; E = Epo; D = Dex; I = IBMX. SER and SER2: S = SCF; E = Epo; R = RU486. See Table 4 for concentrations. FIG. IB shows a growth curve of iPSCs hemizygous (line A4) or homozygous (line B34) for the D816V mutation. The control 01 cells were differentiated according to the PSC-RED protocol. The A4 and B34 lines were differentiated according to the same protocol but SCF was omitted at all steps. FIG. 1C shows the results of a 15-color flow cytometry assay that was used to examine the antigen expression profiles of differentiating cells that were collected weekly between day 10 and 45. The chart summarizes the relative expression of the 10 markers used to define the 8 populations. Populations expressing CD34 were labeled HPC1 to 4 while populations negative for this marker but positive for erythroid markers were labeled Eryl to 4. FIG. ID is a chart summarizing the evolution of the 8 populations during erythroid differentiation. At day 10, the phenotype of the A4 and B34 cells is similar to the control cells with populations of 43+;34+;45- HPC1 and HPC2 (which differed by expression of CD235a)dominating the culture. At days 17 and 24, the control cells differentiated progressively into populations of 45+ HPC4 and 45+ Ery 1 cells. At day 31, Ery2 cells (341ow; 45+; 361ow; 71+; 235a+) resembling pro-Erythroblasts and Ery3 cells (34-; 45- ; 36+; 71+; 235a+) resembling basophilic erythroblasts became prominent and matured into Ery4 late erythrocytes (34-; 45-; 36-; 711ow; 235a+) by day 38.

[0033] FIGS. 2A, 2B, and 2C show differentiation in the absence of SCF and Epo. FIG. 2A is a diagram illustrating the differentiation conditions. The * indicates the absence of SCF in supplement S2 and S3. See FIG. 1A for details. FIG. 2B is a growth curve illustrating the proliferation of the A4 and B34 in the absence of SCF and Epo. FIG. 2C is a diagram illustrating the 15-color FACS analysis of differentiation in the absence of SCF and Epo.

[0034] FIGS. 3A, 3B, 3C, 3D, and 3E illustrate terminal differentiation of KitD816V iPSCs. FIG. 3A is a dot plot illustrating the enucleation of the A4 cells differentiated in the absence of SCF, with or without 200nM dasatininb. FIG. 3B is a micrograph illustrating the morphology of the cells obtained at day 45 of the PSC-RED protocol. FIG. 3C is a graph illustrating the evolution of the cultures between day 31 and day 45 in the presence or absence of dasatinib. Cells were classified using morphological criteria after Romanowski staining and light microscopy examination. Baso = basophilic; Erythroblasts; Poly = poly-chromatophilic erythroblasts; Ortho = ortho-chromatophilic erythroblasts; retie = reticulocytes. FIG. 3D shows HPLC profiles of erythroblasts obtained from clone A4 (in the absence of SCF). FIG. 3E is a bar graph illustrating globin chain expression. RBCs produced by clone A4 express mostly fetal hemoglobins.

[0035] FIGS. 4A, 4B, and 4C depict D816V heterozygous clones. FIG. 4A illustrates iPSCs 01 and 02 that were transfected with Cas9 mRNA, a sgRNA and two oligo donors DNA: a mutated and an unmutated oligo. Mbol was used to identify 96 clones having at least one V816 allele. Top line: wild type genomic sequence; middle: donor oligonucleotide coding for a D at position 816. Bottom line: mutated donor oligonucleotide coding for a V at position 816. The bottom chromatogram illustrates the sequence of one of the heterozygous clones selected for further studies. FIG. 4B is a representative growth curve illustrating the proliferation of clones heterozygous for the D816V mutation isolated from donors 01 (clone D14) and 02 (clone CP)during the PSC-RED protocol (n=2) as compared to clone A4. FIG. 4C is a bar graph illustrating enucleation rate of the same clones as evaluated by staining with DRAQ5™ (n=2). FIG. 4D depicts dot plots illustrating morphology (FSC=H and SS-H) and expression of 11 markers during PSC-RED erythroid differentiation of iPSCs clone A4 (hemizygous D816V), D14 (heterozygous D816V, donor 01) and P ((heterozygous D816V, donor 02).

[0036] FIGS. 5A, 5B, 5C, 5D, 5E, and 5F show the production of SREs. FIG. 5A is a diagram illustrating the differentiation conditions. The * indicates the absence of SCF in supplement S2 and S3 (also see FIG. 1A for details). FIG. 5B shows growth curves illustrating that day- 17 HPCs derived from the A4 and B34 cells can differentiate into SREs for about 45 to 55 days in the presence of Dex and IB MX but in the absence of any cytokines, yield more than 1,000- fold amplification. FIG. 5C is a growth curve illustrating that day-17 HPCs derived from the A4 cells can differentiate into SREs for > 140 days in the presence of Dex and ZBMX and lu / ml of erythropoietin, yielding more than a IO20fold amplification. FIG. 5D shows growth curves illustrating the differentiation of 4 lines of iPSCs hemizygous for the KitD816V mutation and either hemizygous (H5 and Hl 1) or homozygous for the Jak2V617F mutations. All four iPSC lines proliferate and differentiate in the absence of SCF and Epo at a rate greater than that of control cells (01) differentiated in the presence of SCF and Epo. FIG. 5E shows growth curves illustrating that day-17 HPCs derived from the H5 / H11 and G18 / H12 iPSC lines can differentiate into erythroblasts that can respectively self-renew for about 120 and 100 days in the presence of Dex and IBMX but in the absence of any cytokines, yielding > 1018-fold amplification in the case of the H5 and Hl l lines and > 101?-fold amplification for the G19 and H12 lines. FIG. 5F is a bar graph illustrating the expression of 11 markers in the A4 kitD816V line and in the kitJak2 line H5 and Hl 1 (n=2).

[0037] FIGS. 6A, 6B, 6C, 6D, and 6E illustrate the differentiation of the SREs. FIG. 6A shows differentiation conditions. FIG. 6B depicts bar graphs illustrating the fold-amplification, rate of enucleation and yield of RBCs / SREs calculated as the fold-amplification * rate of enucleation). (n=2). FIG. 6C shows various graphs. Top: dot plots and bar graphs illustrating a flow cytometry analysis of H5 cell enucleation at day 10 after having optimized the feeding schedule to keep the concentration of cells below lE6 / mL at all times (n=2). Bottom:micrograph illustrating the morphology of the cells after elimination of the nuclei using a PALL Acrodisc® filter. FIG. 6D shows graphs illustrating a reverse phase HPLC analysis of globin chain expression of RBCs obtained by differentiation of A4 and H5 SREs. FIG. 6E illustrates the morphology of A4 and H5 red blood cells obtained using an AD VIA® blood count analyzer (n=3) and by microscopy (right).

[0038] FIG. 7 shows the copy-number analysis by low-pass sequencing. Genomic DNA was sequenced at a depth of 1 ,5Gb and analyzed using the CNVkit software. Graphs illustrate that PB MNC 01, passage 40 iPSCs 01 (derived from PB MNC 01), passage 38 iPSCs A4 (derived from iPSC 01) and passage 38 iPSCs H5 (derived from iPSCs A4) exhibit no detectable copy number variants greater than Imb (the limit of detection for this read depth). By contrast, transformed cells, HUDEP-2 and K562 cells, cultured for a long period of time exhibit a high level of aneuploidy.

[0039] FIG. 8 illustrates the copy-number analysis by low-pass sequencing. Genomic DNA was sequenced at a depth of 1 to 2 x and analyzed using the CNVkit software. Graphs illustrate that PB MNCs 01 and 02, passage 40 iPSCs G19 and H12 (derived from iPSC 01), passage 30 iPSCs 02 (derived from iPSC 01) and passage 20 iPSCs CE (derived from iPSCs 02) exhibit no detectable copy number variants greater than 1Mb (the limit of detection for this read depth).

[0040] FIG. 9 illustrates the use of cRBC derived from SREs as reagent RBCs in a solid-phase red cell adherence assay. Treated-capture strips were coated as recommended by the manufacturer (Immucor) with about 5 million SRE-derived cRBCs or with control adult RBCs of known (New York Blood Center (NYBC) samples) RhCcE phenotypes. The phenotypes of the coated cells were then determined by solid phase red cell adherence assay using test antibodies and IgG-coated indicator RBCs per manufacturer’s recommendations. In this system, a visible red pellet at the bottom of the well indicates absence of the antigen on the test-ed RBCs, while the absence of a pellet indicates presence of the antigen. The phenotypes determined with this capture assay agreed in all cases with the expected (known) phenotypes, demonstrating that cRBCs can be used as reagent RBCs in this system.

[0041] FIGS. 10A and 10B illustrate the medium requirement. FIG. 10A is a graph showing that during the PSC-RED protocol the cell number doubles in average every 48 hours, leading to >200,000 fold amplification. FIG. 10B is graph illustrating medium consumption during the 38 days of the PSR-RED protocol to produce about 5.109RBCs (the number of RBCs present in 1 mL of blood), in low density cell culture (< . 106 / mL). More than 99 % of the cell culture medium is consumed after day 37 during the PSC-RED protocol. Increasing the cell culture density helps decrease the volume of the culture medium, but not the amount of cytokines, because cytokines are generally internalized and are not recycled.

[0042] FIGS. 11A, 11B, and 11C illustrate adult CD34-derived kitD816V cells. FIG. 11A shows Genomic and HDR donor sequences around position 816 of the kit gene, and a chromatogram of the sequence of the CD34-derived D816V cells 5 weeks after transfection. All of the cells are homozygous for the D816V allele. FIG 11B (Left) is a growth curve of the CD34-derived D816V cells cultured in EDI conditions. Cells amplified 8 million-fold before senescing. FIG. 11B (Right) shows FACS analysis of Draq5-stained CD34-derived D816V cells induced to differentiate 5 weeks after transfection. Cells retained the capacity to enucleate at a relatively high rate. FIG. 11C is HPLC analysis showing that RBCs produced from CD34-derived D816V cells express >99% adult hemoglobin.DETAILED DESCRIPTION OF THE INVENTION

[0043] The present disclosure provides novel methods for producing SREs from iPSCs or CD4+ cells. The disclosed methods completely eliminate the need for SCF and Epo. As demonstrated, all iPSC lines, regardless of genotype, were able to proliferate and differentiate into erythroblasts in the absence of these cytokines at about the same rate as control cells in the presence of cytokines.Methods of Producing SREs

[0044] In one aspect, this disclosure provides a novel, improved method of producing SREs. In some embodiments, the methods disclosed herein allow the large-scale production of SREs from the CFU-E (cultured erythroid colonies) / pro-erythroblasts (proE) stage. Accordingly, obtaining a large number of cells from the CFU-E / proE stage facilitates the generation of RBCs by reducing the time it takes to obtain RBCs without having to start the process with cells such as iPSCs.

[0045] In one aspect, provided is a method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified CD34+ cell comprising a constitutively active stem cell factor (SCF) receptor and a constitutively active Janus kinase 2 (JAK2); and culturing the genetically modified CD34+ cell in one or more culture media free of a stem cell factor (SCF) and erythropoietin (Epo).

[0046] In one aspect, provided is a method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified CD34+ cell comprising a constitutively active stem cell factor (SCF) receptor; and culturing the genetically modified CD34+ cell in one or more culture media free of a stem cell factor (SCF).

[0047] In one aspect, provided is a method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified induced pluripotent stem cell (iPSC) comprising a constitutively active stem cell factor (SCF) receptor and a constitutively active Janus kinase 2 (JAK2); and culturing the genetically modified iPSC in one or more culture media free of a stem cell factor (SCF) and erythropoietin (Epo).

[0048] In one aspect, provided is a method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified induced pluripotent stem cell (iPSC) comprising a constitutively active stem cell factor (SCF) receptor and culturing the genetically modified iPSC in one or more culture media free of a stem cell factor (SCF). In one embodiment, the SRE is cultured in the presence of Epo of this method as described herein.

[0049] In some embodiments, the donor of the IPSCs or the CD34+ cells used in the methods of producing SREs as described herein, exhibit phenotypes that are useful for identifying clinically significant antibodies in the plasma of the transfusion recipients. These phenotypesinclude being blood group O, O negative, and / or null or homozygous for any of the RHCE, KELL, DUFFY, KIDD, LEWIS, MNS, Lutheran, and Dumbrock blood group antigens.

[0050] In some embodiments, the method comprises obtaining IPSCs or the CD34+ cells from a subject exhibiting a characteristic selected from blood group O, O negative, and / or null or homozygous for any of the RHCE, KELL, DUFFY, KIDD, LEWIS, MNS, Lutheran, and Dumbrock blood group antigens.

[0051] As used herein, the term “differentiate” refers to the production of a cell type that is more differentiated than the cell type from which it is derived. In some embodiments, the term “differentiate” means to produce a cell that has fewer fate choices than the cell from which it was derived. The term, therefore, encompasses cell types that are partially and terminally differentiated. Differentiated cells derived from pluripotent stem cells are generally referred to as pluripotent stem cell-derived cells or pluripotent stem cell-derived cell aggregate cultures, pluripotent stem cell-derived single cell suspensions, pluripotent stem cell-derived cell adherent cultures, or the like.

[0052] In some embodiments, the method comprises culturing the genetically modified pluripotent stem cell or a derivative thereof in a medium comprising erythropoietin, dexamethasone, and / or isobutylmethylxanthine, l-Methyl-3-Iso-butyl-xanthine (IB MX).

[0053] In some embodiments, the method comprises culturing the genetically modified pluripotent stem cell or a derivative thereof in a medium comprising erythropoietin, dexamethasone, and isobutylmethylxanthine, l-Methyl-3-Iso-butyl-xanthine (IB MX). In some embodiments, a derivative of a genetically modified pluripotent stem cell can be obtained from culturing or expansion in a medium different from the medium comprising erythropoietin, dexamethasone, and isobutylmethylxanthine, l-Methyl-3-Iso-butyl-xanthine (IBMX). In some embodiments, a derivative of a genetically modified pluripotent stem cell can be a product of division or differentiation of the genetically modified pluripotent stem cell.

[0054] In some embodiments, the medium comprises from about 0.1 to about 10 units (e.g., 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7, 1.9, 2.1 , 2.3, 2.5, 2.7, 2.9, 3.1, 3.3, 3.5, 3.7, 3.9, 4.1, 4.3, 4.5, 4.7, 4.9, 5.1, 5.3, 5.5, 5.7, 5.9, 6.1, 6.3, 6.5, 6.7, 6.9, 7.1, 7.3, 7.5, 7.7, 7.9, 8.1, 8.3, 8.5,8.7, 8.9, 9.1, 9.3, 9.5, 9.7, 9.9, 10 units) of erythropoietin. In some embodiments, the medium comprises about 1 unit of erythropoietin.

[0055] In some embodiments, the medium comprises from about 0.01 pM to about 100 pM (e.g., 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 pM) of dexamethasone. In some embodiments, the medium comprises about 1 pM of dexamethasone.

[0056] In some embodiments, the medium comprises from about 1 pM to 500 pM e.g., 1, 5, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 pM) of IBMX. In some embodiments, the medium comprises about 33 pM of IBMX. In some embodiments, the medium comprises about 1 unit of erythropoietin, about 1 pM dexamethasone, and about 33 pM of IBMX.

[0057] In some embodiments, the method comprises removing the dexamethasone and the IBMX from the medium to induce differentiation of the genetically modified pluripotent stem cell into an enucleated cell.

[0058] In some embodiments, the method comprises refreshing the medium at least once every first interval by adding a concentrated medium comprising erythropoietin, dexamethasone, and IBMX, and passaging cells by dilution at least once every second interval, while maintaining the concentration of the cells between 200,000 and 2,000,000 cells.

[0059] In some embodiments, the first interval is from about 1 day to about 7 days (e.g., 1, 2, 3, 4, 5, 6, 7 days). In some embodiments, the first interval is about 2 days.

[0060] In some embodiments, the second interval is from about 1 day to about 30 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days). In some embodiments, the second interval is about 7 days.

[0061] In some embodiments, one or more culture media free of or substantially free of a stem cell factor (SCF) and / or erythropoietin (Epo).

[0062] As used herein, the term “substantially free,” such as “substantially free of Epo,” “substantially free of SCF,” or “substantially free of serum,” is meant that the solution, media, supplement, excipient, and the like, is at least 98%, or at least 98.5%, or at least 99%, or atleast 99.5%, or at least 100% free of Epo, SCF, or serum. In some embodiments, a defined culture media contains no SCF or is 100% SCF-free, or is substantially free of SCF. In some embodiments, a defined culture media contains no Epo or is 100% Epo-free, or is substantially free of Epo. In some embodiments, a defined culture media contains no Epo and SCF or is 100% Epo-free and SCF-free, or is substantially free of Epo and SCF.

[0063] In some embodiments, the method comprises differentiating the pluripotent stem cell into enucleated erythroid cells (such as reticulocyte and red blood cells). “Erythroid cells,” as used herein, include nucleated red blood cells, red blood cell precursors, and enucleated red blood cells. As used herein, the term “enucleated” refers to a cell, e.g., a reticulocyte or mature red blood cell (erythrocyte), that lacks a nucleus. In some embodiments, the method comprises differentiating the pluripotent stem cell into nucleated red erythroid precursor and progenitor cells.

[0064] As used herein, the term “self-renewing erythroblast (SRE)” refers to a cell that (i) expresses at least three of the following antigens: CD45, CD43, CD36, CD71, CD235a, CD117; (ii) is capable of self-renewing in a medium for at least 15 passages without losing expression of said antigens; and (iii) can differentiate into cells that produce a pellet with a distinct red color upon centrifugation due to their expression of hemoglobin. Differentiated SREs exhibit the typical morphology of mature erythroid cells such as orthochromatic erythroblasts, and reticulocytes (enucleated cells).

[0065] In some embodiments, the SREs are karyotypically stable.

[0066] As used herein the term “CD34+ cell” refers to cells that express CD34 antigens. CD34+ cells can be collected from a subject of any age or from fetal tissues. In some embodiments, CD34+ cells can be produced from pluripotent cells or by culturing CD34- cells as CD34- cells are precursors of CD34+ cells (Sonoda et al. Exp Hematol. 2021. 96: 13-26).

[0067] CD34+ cells can be manipulated to become SREs after purification using methods known to the art (flow cytometry, magnetic beads etc ), or without purification, for instance by transfecting on infecting white blood cells fractions collected from individual of any ages that are composed in part of CD34+ cells.

[0068] As used herein, the term “constitutively active SCF receptor” refers to a SCF receptor capable of performing all or some of the functions of the unmodified SCF receptor in a ligandindependent manner, for example, without contacting the SCF or Epo molecule or any other natural ligand. In some embodiments, the unmodified SCF receptor comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, a constitutively active SCF receptor comprises the amino acid sequence of SEQ ID NO: 2.

[0069] In some embodiments, the SCF receptor (also referred to as kit) comprises one or more mutations that render the SCF receptor constitutively active. In some embodiments, the receptor comprises a mutation at position D816. In some embodiments, the SCF receptor comprises a D816V mutation, rendering the SCF receptor constitutively active. Because the SCF receptor interacts with the Epo receptor, the D816V mutation eliminates the need for both Epo and SCF. Examples of mutations that render the SCF receptor constitutively active include, but are not limited to mutations in the juxtamembrane (JM) domain at position 560 or in the D5 domain (one of the five Ig-like modules) at position 417, 418, 419, 502, 503, or 505 have been shown to have abnormal activity (Shi et al. PNAS. 2016.113(33): E4784-93).

[0070] In some embodiments, a constitutively active SCF receptor is transiently or stably introduced into cells by ectopic expression of a kit gene using nucleic acid vectors and other methods known in the art (e.g., plasmid, cosmid, bacterial artificial chromosome transfection, viral vector infection (retroviral, lentiviral, adenoviral, AAV), micro-injection, mRNA transfection, etc.).

[0071] In some embodiments, the SCF receptor is constitutively active without having one or more mutations. For example, the endogenous kit gene can be modulated by modifying its promoter or enhancer, or inserting or deleting additional coding sequences to produce higher level expression or modified proteins that confer constitutive SCF receptor signaling activity.

[0072] SCF receptor (SEQ ID NO: 1)RGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLL CTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAK LFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVK RAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIK DVSSSVYSTWKRENSQQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMN RTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNT KPEILT YDRLVNGMLQC VAAGFPEPTIDWYFCPGTEQRC S AS VLPVDVQTLNS SGPPFG KLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFV IVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNR LSFGKTLGAGAFGKVVEATAYGL1KSDAAMTVAVKMLKPSAHLTEREALMSELKVLSY LGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKN LLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELAL DLEDLL SFSYQ VAKGMAFL ASKNCIHRDL A ARNILLTHGRITKICDFGL ARDIKND SNYV VKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMI KEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCS PNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

[0073] SCF receptor with D816V mutation (SEQ ID NO: 2)RGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLL CTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAK LFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVK RAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIK DVSSSVYSTWKRENSQQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYA NNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMN RTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNT KPEILT YDRLVNGMLQC VAAGFPEPTIDWYFCPGTEQRC SAS VLPVDVQTLNS SGPPFG KLVVQ S SID S S AFKHNGTVECKAYND VGKT S AYFNF AFKGNNKEQIHPHTLFTPLLIGF V 1VAGMMC11VM1LTYKYLQKPMYEVQWKVVEE1NGNNYVY1DPTQLPYDHKWEFPRNR LSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSY LGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKN LLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELAL DLEDLL SF SYQ VAKGMAFL ASKNCIHRDL A ARNILLTHGRITKICDFGL ARVIKND SNYV VKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMI KEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCS PNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

[0074] In some embodiments, the SCF receptor comprises the amino acid sequence of SEQ ID NO: 2.

[0075] In some embodiments, the SCF receptor comprises a substitution of D816V or a conservative substitution of Vai at residue D816 of the SCF receptor. Examples of conservative substitutions of Vai may include He, Leu, Met, Phe, and Ala.

[0076] In some embodiments, the SCF receptor comprises at least one nucleotide substitution that encodes for a constitutively active protein.

[0077] Also within the scope of this disclosure are the variants and homologs with significant identity to the SCF receptor. For example, such variants and homologs may have sequences with at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the sequences of the SCF receptor described herein.

[0078] As used herein, “constitutively Janus kinase 2 (JAK2)” refers to JAK2 capable of performing all or some of the functions of unmodified JAK2, for example, without undergoing tyrosine phosphorylation with the receptor cytoplasmic domain. In some embodiments, the unmodified JAK2 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, a constitutively active JAK2 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, a constitutively active JAK2 comprises the amino acid sequence of SEQ ID NO: 5.

[0079] In some embodiments, JAK2 comprises at least one nucleotide substitution that encodes for a constitutively active protein. In some embodiments, JAK2 comprises at least one mutation that renders it constitutively active. Examples of mutations that render JAK2 constitutively active include, but are not limited to mutations in or near the FERM (JH4 to JH7), JH2 (pseudokinase domain), and JHl (kinase domain) domain at positions 204, 535, 547, 683, 815, 875, or 1007 (Skoda et al. Exp. Hematol. 2015. 43(8): 599-608).

[0080] In some embodiments, JAK2 comprises a V617F mutation, rendering JAK2 constitutively active. Because JAK2 transduces the signal from several cytokine receptors including the Epo receptor and is involved in SCF-induced proliferation, the V617F mutation eliminates the need for both Epo and SCF.

[0081] In some embodiments, constitutively active JAK2 is transiently or stably introduced into cells by ectopic expression of a kit gene using nucleic acid vectors and other methods known in the art (e.g., plasmid, cosmid, bacterial artificial chromosome transfection, viral vector infection (retroviral, lentiviral, adenoviral, AAV), micro-injection, mRNA transfection, etc.).

[0082] In some embodiments, JAK2 is constitutively active without having one or more mutations.For example, JAK2 can be modulated by modifying its promoter or enhancer, or inserting or deleting additional coding sequences to produce higher level expression or modified proteins that confer constitutive JAK2 signaling activity.

[0083] JAK2 (SEQ ID NO: 3)MGMACLTMTEMEGTSTSSIYQNGDISGNANSIDPVLQVYLYHSLGKSEADYLT FPSGEYVAEEICIAASKACGITPVYHNMFALMSETERIWYPPNHVFHIDESTRHNVLYRIR FYFPRWYCSGSNRAYRHGISRGAEAPLLDDFVMSYLFAQWRHDFVHGWIKVPVTHETQ EECLGMAVLDMMRIAKENDQTPLAIYNSISYKTFLPKCIRAKIQDYHILTRKRIRYRFRRF IQQFSQCKATARNLKLKYLINLETLQSAFYTEKFEVKEPGSGPSGEEIFATIIITGNGGIQW SRGKHKESETLTEQDLQLYCDFPNIIDVSIKQANQEGSNESRVVTIHKQDGKNLEIELSSL REALSFVSLIDGYYRLTADAHHYLCKEVAPPAVLENIQSNCHGPISMDFAISKLKKAGNQ TGLYVLRCSPKDFNKYFLTFAVERENVIEYKHCLITKNENEEYNLSGTKKNFSSLKDLLN CYQMETVRSDNIIFQFTKCCPPKPKDKSNLLVFRTNGVSDVPTSPTLQRPTHMNQMVFH KIRNEDLIFNESLGQGTFTKIFKGVRREVGDYGQLHETEVLLKVLDKAHRNYSESFFEAA SMMSKLSHKHLVLNYGVCVCGDENILVQEFVKFGSLDTYLKKNKNCINIL WKLEV AKQ LAWAMHFLEENTLIHGNVCAKNILLIREEDRKTGNPPFIKLSDPGISITVLPKDILQERIPW VPPECIENPKNLNLATDKWSFGTTLWEICSGGDKPLSALDSQRKLQFYEDRHQLPAPKW AELANLINNCMDYEPDFRPSFRAIIRDLNSLFTPDYELLTENDMLPNMRIGALGFSGAFE DRDPTQFEERHLKFLQQLGKGNFGSVEMCRYDPLQDNTGEVVAVKKLQHSTEEHLRDF EREIEILKSLQHDNIVKYKGVCYSAGRRNLKLIMEYLPYGSLRDYLQKHKERIDHIKLLQ YTSQICKGMEYLGTKRYIHRDLATRNILVENENRVKIGDFGLTKVLPQDKEYYKVKEPG ESPIFWYAPESLTESKFSVASDVWSFGVVLYELFTYIEKSKSPPAEFMRMIGNDKQGQMI VFHLIELLKNNGRLPRPDGCPDEIYMIMTECWNNNVNQRPSFRDLALRV

[0084] JAK2 V617F mutation (SEQ ID NO: 4)

[0085] Note: Bold and underline indicates mutation.MGMACLTMTEMEGTSTSSIYQNGDISGNANSIDPVLQVYLYHSLGKSEADYLTFPS GF.YVAF.F.ICIAASKACGITPVYHNMFALMSF.TERIWYPPNHVFHIDF.STRHNVLYRIRFY FPRWYCSGSNRAYRHGISRGAEAPLLDDFVMSYLFAQWRHDFVHGWIKVPVTHETQEE CLGMAVLDMMRIAKENDQTPLAIYNSISYI<TFLPI<CIRAI<IQDYHILTRKRIRYRFRRFIQ QFSQCKATARNLKLKYLINLETLQSAFYTEKFEVKEPGSGPSGEEIFATIIITGNGGIQWSR GKHKESETLTEQDLQLYCDFPNIIDVSIKQANQEGSNESRVVTIHKQDGKNLEIELSSLRE ALSFVSLIDGYYRLTADAHHYLCKEVAPPAVLENIQSNCHGPISMDFAISKLKKAGNQT GLYVLRCSPKDFNKYFLTFAVERENVIEYKHCLITKNENEEYNLSGTKKNFSSLKDLLNC YQMETVRSDNIIFQFTKCCPPKPKDKSNLLVFRTNGVSDVPTSPTLQRPTHMNQMVFHKI RNEDLIFNESLGQGTFTKIFKGVRREVGDYGQLHETEVLLKVLDKAHRNYSESFFEAAS MMSKLSHKHLVLNYGVCVCGDENILVQEFVKFGSLDTYLKKNKNCINILWKLEVAKQL AWAMHFLEENTLIHGNVCAKNILLIREEDRKTGNPPFIKLSDPGISITVLPKDILQERIPWVPPECIENPKNLNLATDKWSFGTTLWEICSGGDKPLSALDSQRKLQFYEDRHQLPAPKWA ELANLINNCMDYEPDFRPSFRAIIRDLNSLFTPDYELLTENDMLPNMRIGALGFSGAFED RDPTQFEERHLKFLQQLGKGNFGSVEMCRYDPLQDNTGEVVAVKKLQHSTEEHLRDFE REIEILKSLQHDNIVKYKGVCYSAGRRNLKLIMEYLPYGSLRDYLQKHKERIDHIKLLQY TSQICKGMEYLGTKRYIHRDLATRNILVENENRVKIGDFGLTKVLPQDKEYYKVKEPGE SP1FWYAPESLTESKFSVASDVWSFGVVLYELFTYIEKSKSPPAEFMRM1GNDKQGQM1V FHLIELLKNNGRLPRPDGCPDEIYMIMTECWNNNVNQRPSFRDLALRV

[0086] JAK2 deletion at C816 and F817 (SEQ ID NO: 5)MGMACLTMTEMEGTSTSSIYQNGDISGNANSMKQIDPVLQVYLYHSLGKSEAD YLTFPSGEYVAEEICIAASKACGITPVYHNMFALMSETERIWYPPNHVFHIDESTRHNVL YRIRFYFPRWYCSGSNRAYRHGISRGAEAPLLDDFVMSYLFAQWRHDFVHGWIKVPVT HETQEECLGMAVLDMMRIAKENDQTPLAIYNSISYKTFLPKCIRAKIQDYHILTRKRIRY RFRRFIQQFSQCKATARNLKLKYLINLETLQSAFYTEKFEVKEPGSGPSGEEIFATIIITGN GGIQWSRGKHKESETLTEQDLQLYCDFPNIIDVSIKQANQEGSNESRVVTIHKQDGKNLE IELSSLREALSFVSLIDGYYRLTADAHHYLCKEVAPPAVLENIQSNCHGPISMDFAISKLK KAGNQTGLYVLRCSPKDFNKYFLTFAVERENVIEYKHCLITKNENEEYNLSGTKKNFSS LKDLLNCYQMETVRSDNIIFQFTKCCPPKPKDKSNLLVFRTNGVSDVPTSPTLQRPTHMN QMVFHK1RNEDL1FNESLGQGTFTK1FKGVRREVGDYGQLHETEVLLKVLDKAHRNYSE SFFEAASMMSKLSHKHLVLNYGVCGDENILVQEFVKFGSLDTYLKKNKNCINIL WKLEV AKQLAWAMHFLEENTLIHGNVCAKNILLIREEDRKTGNPPFIKLSDPGISITVLPKDILQE RIPWVPPECIENPKNLNLATDKWSFGTTLWEICSGGDKPLSALDSQRKLQFYEDRHQLP APKWAELANLINNCMDYEPDFRPSFRAIIRDLNSLFTPDYELLTENDMLPNMRIGALGFS GAFEDRDPTQFEERHLKFLQQLGKGNFGSVEMCRYDPLQDNTGEVVAVKKLQHSTEEH LRDFEREIEILKSLQHDNIVKYKGVCYSAGRRNLKLIMEYLPYGSLRDYLQKHKERIDHI KLLQYTSQICKGMEYLGTKRYIHRDLATRNILVENENRVKIGDFGLTKVLPQDKEYYKV KEPGESPIFWYAPESLTESKFSVASDVWSFGVVLYELFTYIEKSKSPPAEFMRMIGNDKQ GQMIVFHLIELLKNNGRLPRPDGCPDEIYMIMTECWNNNVNQRPSFRDLALRVDQIRDN MA

[0087] In some embodiments, JAK2 comprises a substitution of V617F or a conservative substitution of Phe at residue V617 of JAK2. Examples of conservative substitutions of Phe may include Tyr and Trp.

[0088] In some embodiments, JAK2 comprises amino acid deletions to confer constitutive activity. In some embodiments, JAK2 comprises a deletion at positions C616 and V617.

[0089] Also within the scope of this disclosure are the variants and homologs with significant identity to JAK2. For example, such variants and homologs may have sequences with at least about 70%, about 71%, about 72%>, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the sequences of JAK2 described herein.

[0090] As used herein, the term “variant” refers to a first composition (e.g, a first molecule) that is related to a second composition (e.g., a second molecule, also termed a “parent” molecule). The variant molecule can be derived from, isolated from, based on or homologous to the parent molecule. The term variant can be used to describe either polynucleotides or polypeptides.

[0091] As applied to polynucleotides, a variant molecule can have an entire nucleotide sequence identity with the original parent molecule, or alternatively, can have less than 100% nucleotide sequence identity with the parent molecule. For example, a variant of a gene nucleotide sequence can be a second nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in nucleotide sequence compared to the original nucleotide sequence. Polynucleotide variants also include polynucleotides comprising the entire parent polynucleotide, and further comprising additional fused nucleotide sequences. Polynucleotide variants also include polynucleotides that are portions or subsequences of the parent polynucleotide; for example, unique subsequences (c.g, as determined by standard sequence comparison and alignment techniques) of the polynucleotides disclosed herein are also encompassed by this disclosure.

[0092] In another aspect, polynucleotide variants include nucleotide sequences that contain minor, trivial or inconsequential changes to the parent nucleotide sequence. For example, minor, trivial or inconsequential changes include changes to nucleotide sequence that (i) do not change the amino acid sequence of the corresponding polypeptide, (ii) occur outside the protein-coding open reading frame of a polynucleotide, (iii) result in deletions or insertions that may impact the corresponding amino acid sequence, but have little or no impact on the biological activity of the polypeptide, or (iv) result in the substitution of an amino acid with a chemically similar amino acid. In the case where a polynucleotide does not encode for a protein (for example, a tRNA or a crRNA or a tracrRNA), variants of that polynucleotide can include nucleotide changes that do not result in loss of function of the polynucleotide. In another aspect, conservative variants of the disclosed nucleotide sequences that yield functionally identicalnucleotide sequences are encompassed by the invention. One of skill in the art will appreciate that many variants of the disclosed nucleotide sequences are encompassed by this disclosure.

[0093] As applied to proteins, a variant polypeptide can have an entire amino acid sequence identity with the original parent polypeptide, or alternatively, can have less than 100% amino acid identity with the parent protein. For example, a variant of an amino acid sequence can be a second amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in amino acid sequence compared to the original amino acid sequence.

[0094] Polypeptide variants include polypeptides comprising the entire parent polypeptide, and further comprising additional fused amino acid sequences. Polypeptide variants also include polypeptides that are portions or subsequences of the parent polypeptide; for example, unique subsequences (e.g, as determined by standard sequence comparison and alignment techniques) of the polypeptides disclosed herein are also encompassed by the invention.

[0095] A “functional variant” of a protein as used herein refers to a variant of such protein that retains at least partially the activity of that protein. Functional variants may include mutants (which may be insertion, deletion, or replacement mutants), including polymorphs, etc. Also included within functional variants are fusion products of such protein with another, usually unrelated, nucleic acid, protein, polypeptide or peptide. Functional variants may be naturally occurring or may be man-made.

[0096] In some embodiments, a variant of the SCF receptor or JAK2 may include one or more conservative modifications. The variant of the SCF receptor or variant of JAK2 with one or more conservative modifications may retain the desired functional properties, which can be tested using the functional assays known in the art.

[0097] As used herein, the term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families include: amino acids with basic side chains e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); nonpolar side chains (e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); beta-branched side chains (e.g, threonine, valine, isoleucine); and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine). The SCF receptor or JAK2 with one or more conservative modifications may retain the desired functional properties, which can be tested using the functional assays known in the art.

[0098] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0099] Additionally or alternatively, the protein sequences of this disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. (ncbi.nlm.nih.gov).

[0100] In some embodiments, the method comprises genetically modifying the pluripotent stem cell prior to differentiation. In some embodiments, the genetic modification comprisesintroducing a D816V substitution into the SCF receptor. In some embodiments, the genetic modification comprises introducing a V617F substitution into JAK2. In some embodiments, the genetic modification comprises introducing a deletion in JAK2 at C616 and V617.

[0101] In some embodiments, genome-editing techniques, such as CRISPR / Cas9 systems, designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases, are available to carry out the genetic modification. In general, “CRISPR / Cas9 system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. One or more elements of a CRISPR system may be derived from a type I, type II, or type III CRISPR system. Alternatively, one or more elements of a CRISPR system may be derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).

[0102] In addition, a wide variety of vectors can be used for the genetic modification as described. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells. Accordingly, in some embodiments, a viral vector is used to introduce a nucleotide sequence encoding a SCF receptor and / or JAK2 or fragment thereof. The viral vector will comprise a nucleotide sequence encoding a SCF receptor and / or JAK2 or fragment thereof operably linked to one or more control sequences, for example, a promoter. Alternatively, the viral vector may not contain a control sequence and will instead rely on a control sequence within the host cell to drive expression of the SCF receptor and / or JAK2 or fragment thereof.Non-limiting examples of viral vectors that may be used to deliver a nucleic acid molecule include adenoviral vectors, AAV vectors, and retroviral vectors.

[0103] In some embodiments, an adeno-associated virus (AAV) can be used to introduce a nucleotide sequence encoding an ADAMTS13 protein or fragment thereof into a host cell for expression. AAV systems have been described previously and are generally well known in the art (Kelleher and Vos, Biotechniques, 17(6): 1110-7, 1994; Cotten et al., Proc Natl Acad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3): 141-64, 1994; Muzyczka, Cun- Top Microbiol Immunol, 158:97-129, 1992). Details concerning the generation and use of rAAV vectors are described, for example, in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein by reference in its entirety for all purposes.

[0104] In some embodiments, a retroviral expression vector can be used to introduce a nucleotide sequence encoding a SCF receptor or fragment thereof into a host cell for expression. These systems have been described previously and are generally well known in the art (Nicolas and Rubinstein, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986).

[0105] Examples of vectors for eukaryotic expression in mammalian cells include AD5, pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and P-actin.

[0106] Combinations of retroviruses and an appropriate packaging line may also find use, where the capsid proteins will be functional for infecting the target cells. Usually, the cells and virus will be incubated for at least about 24 hours in the culture medium. The cells are then allowed to grow in the culture medium for short intervals in some applications, e.g., 24-73 hours, or for at least two weeks, and may be allowed to grow for five weeks or more, before analysis. Commonly used retroviral vectors are “defective,” i.e., unable to produce viral proteins required for productive infection. Replication of the vector requires growth in the packaging cell line. The host cell specificity of the retrovirus is determined by the envelope protein env (pl20). The envelope protein is provided by the packaging cell line. Envelopeproteins are of at least three types, ecotropic, amphotropic, and xenotropic. Retroviruses packaged with ecotropic envelope protein, e.g., MMLV, are capable of infecting most murine and rat cell types. Ecotropic packaging cell lines include BOSC23. Retroviruses bearing amphotropic envelope protein, e.g., 4070A, are capable of infecting most mammalian cell types, including human, dog, and mouse. Amphotropic packaging cell lines include PA12 and PA317. Retroviruses packaged with xenotropic envelope protein, e.g., AKR env, are capable of infecting most mammalian cell types, except murine cells. The vectors may include genes that must later be removed, e.g., using a recombinase system such as Cre / Lox, or the cells that express them destroyed, e.g., by including genes that allow selective toxicity such as herpesvirus TK, bcl-xs, etc. Suitable inducible promoters are activated in a desired target cell type, either the transfected cell or progeny thereof.

[0107] In some embodiments, the method may include more than one differentiation step. Accordingly, more than one differentiation medium may be employed. For example, a first differentiation medium may be used to initiate the differentiation of pluripotent stem cells into progenitor cells, followed by a step in which a second differentiation medium is used to expand and maintain the progenitor cells or to further differentiate the progenitor cells.

[0108] In some embodiments, the pluripotent stem cell comprises an embryonic stem cell or an embryo-derived cell. In some embodiments, the pluripotent stem cell comprises an induced pluripotent stem cell (iPSC). In some embodiments, the pluripotent stem cell is a human cell.

[0109] As used herein, the term “pluripotent stem cells” includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are derived. Pluripotent stem cells are defined functionally as stem cells that are: (a) capable of inducing teratomas when transplanted in immunodeficient (SCTD) mice; (b) capable of differentiating to cell types of all three germ layers (i.e., can differentiate to ectodermal, mesodermal, and endodermal cell types); and (c) express one or more markers of embryonic stem cells (e.g., express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REXI, etc.). Exemplary pluripotent stem cells can be generated using, for example, methods known in theart. Exemplary pluripotent stem cells include embryonic stem cells derived from the ICM of blastocyst stage embryos, as well as embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo (optionally without destroying the remainder of the embryo). Such embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells or iPSCs) generated by reprogramming a somatic cell by expressing a combination of factors (herein referred to as reprogramming factors). Induced pluripotent stem cells can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. In some embodiments, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, a combination of Oct 4, Sox2, Nanog, and Lin28. In other embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or at least four reprogramming factors. In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram a somatic cell to a pluripotent stem cell. Induced pluripotent stem cells are defined functionally and include cells that are reprogrammed using any of a variety of methods (integrative vectors, non-integrative vectors, chemical means, etc ).

[0110] The pluripotent stem cells can be from any species. Embryonic stem cells have been successfully derived in, for example, mice, multiple species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one of skill in the art can generate embryonic stem cells and embryo-derived stem cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like. In some embodiments, the species is an endangered species. In some embodiments, the species is a currently extinct species.

[0111] Similarly, induced pluripotent stem cells can be from any species. Induced pluripotent stem cells have been successfully generated using mouse and human cells. Induced pluripotent stem cells have been successfully generated using embryonic, fetal, newborn, and adult tissue. Accordingly, one can readily generate induced pluripotent stem cells using a donor cell from any species. Thus, one can generate induced pluripotent stem cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like. In some embodiments, the species is an endangered or currently extinct species.

[0112] Induced pluripotent stem cells can be generated using, as a starting point, virtually any somatic cell of any developmental stage. For example, the cell can be from an embryo, fetus, neonate, juvenile, or adult donor. Exemplary somatic cells that can be used include fibroblasts, such as dermal fibroblasts obtained by a skin sample or biopsy, synoviocytes from synovial tissue, foreskin cells, cheek cells, or lung fibroblasts. Although skin and cheek provide a readily available and easily attainable source of appropriate cells, virtually any cell can be used. In some embodiments, the somatic cell is not a fibroblast.

[0113] The pluripotent stem cells can be, for example, embryonic stem cells or induced pluripotent stem cells. Induced pluripotent stem cells can be produced by expressing a combination of reprogramming factors in a somatic cell. In some embodiments, at least two reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In other embodiments, at least three reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In other embodiments, at least four reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell.

[0114] In some embodiments, the method may include steps of dispersing a pluripotent stem cell colony or clonal cell grouping to form dispersed essentially individual cells and seeding the dispersed cells into a culture that may contain a survival factor. For example, the cells may be seeded at a density of from about 10,000 stem cells per square centimeter ofculturing surface to about 70,000 stem cells per square centimeter of culturing surface. In some embodiments, the cells may be seeded at a density of from about 10,000 stem cells per square centimeter of culturing surface to about 50,000 stem cells per square centimeter of culturing surface, or at a density of from about 20,000 stem cells per square centimeter of culturing surface to about 70,000 stem cells per square centimeter of culturing surface.

[0115] In some embodiments, the cells may be dispersed by mechanical or enzymatic means. For example, the cells may be dispersed by treatment with an effective amount of one or more enzymes, such as trypsin or trypLE, or a mixture of enzymes such as Accutase®.

[0116] In some embodiments, the method may include steps of seeding the pluripotent stem cells in a culturing medium, which may contain a matrix component and / or a survival factor, to form a culture; introducing a differentiation medium into the culture, wherein the differentiation medium is free or essentially free of feeder cells and includes at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF; and differentiating the cells under a hypoxic atmosphere having less than about 5.5% oxygen for a period of time sufficient to generate progenitor cells. In some embodiments, one or more of these steps may be employed to produce CD34+ progenitor cells, CD31+ progenitor cells, CD43+ progenitor cells, or CD34+ CD43+ progenitor cells. The progenitor cells may then be harvested, and they may further be sorted. At this point, the progenitor cells may be maintained, expanded, or further differentiated.

[0117] In some embodiments, one or more culture media may include a growth factor. Examples of growth factors may include, but are not limited to, BMP -4, VEGF, bFGF, stem cell factor (SCF), Flt-3 ligand, interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 9 (IL-9), interleukin 11 (IL-11), insulin-related growth factor 1 (IFG1), insulin-related growth factor 2 (IGFII), thrombopoietin (TPO), granulocyte-macrophage-colony-stimulating factor (GM- CSF), and granulocyte colony-stimulating factor (G-CSF). One or more culture media may include one, two, three, or more of these growth factors; for example, other growth factors may be included in a defined medium in order to increase proliferation or modulate the differentiation state of the cells. Various amounts of these factors may be used to stimulatecellular responses (e.g., in the amounts described in Yamamura et al., 2007; Fadilah et al., 2007; Bashey et al., 2007).

[0118] In some embodiments, one or more culture media may include a supplement selected from inositol, folic acid, monothioglycerol, insulin, ferrous nitrate, ferrous sulfate, BSA, L-glutamine, penicillin-streptomycin, transferrin, animal or human plasma or serum, Fe(III)-EDTA or an equivalent chelator, lipids, and combinations thereof. In one embodiment, the one or more culture media comprises between 1 to 1000 pg of recombinant transferrin.

[0119] In some embodiments, one or more culture media comprise a serum-free culture medium or a culture medium substantially free of serum. In some embodiments, one or more culture media comprise a defined differentiation medium.

[0120] As used herein, the term “serum-free culture medium” refers to a culture medium that has not been supplemented with animal serum. The serum-free medium is a known composition medium, but the serum-free medium can be supplemented with individual animal or plant proteins, or protein fractions. The term “serum,” as used herein, refers to a non-human animal product that may be added to a culture to provide nutrients to growing cells.

[0121] As used herein, the terms “defined conditions,” “defined medium,” and “defined differentiation” refer to culture conditions, wherein the culture has known quantities of all ingredients and does not utilize undefined ingredients, serum, or feeder cells (e.g., mouse embryonic fibroblasts). An “undefined ingredient” is an ingredient that contains unknown components, or contains known components in unknown amounts. Defined conditions may be particularly useful, e.g., in applications where differentiated cells may be therapeutically administered to a subject, such as a human patient.

[0122] In some embodiments, the differentiation medium may include a survival factor. The survival factor may be, for example, an inhibitor of a Rho-associated kinase (ROCK), such as HA100 or Hl 152, or an inhibitor of myosin II, such as blebbistatin.

[0123] In some embodiments, the method comprises supplementing one or more culture media with a cytokine only at the early stage of culturing, e.g., from day 0 to day 17 (e.g., from day 0 to day 1, from day 0 to day 2, from day 0 to day 3, from day 0 to day 4, from day 0 today 5, from day 0 to day 6, from day 0 to day 7, from day 0 to day 8, from day 0 to day 9, from day 0 to day 10, from day 0 to day 11, from day 0 to day 12, from day 0 to day 13, from day 0 to day 14, from day 0 to day 15, from day 0 to day 16, from day 0 to day 18). The advantages of only supplementing one or more culture media with a cytokine at the early stage of culturing include significantly reduced overall costs to produce red blood cells, thus enabling large-scale production of red blood cells for therapeutic use.[00124J In some embodiments, the method further comprises expanding the CD34+ cell prior to being differentiated into the red blood cell.

[0125] The term “culturing” or “expanding” refers to maintaining or cultivating cells under conditions in which they can proliferate and avoid senescence. For example, cells may be cultured in media optionally containing one or more growth factors, i.e., a growth factor cocktail. In some embodiments, the cell culture medium is a defined cell culture medium. Stable cell lines may be established to allow for the continued propagation of cells.

[0126] In some embodiments, a robot may be employed to automate at least a portion of the disclosed method. For example, a plurality of human embryonic stem cells may be cultured using a bioreactor (e.g., a hollow fiber bioreactor). For example, one or more steps for the culture of stem cells and / or differentiation of progenitor cells from pluripotent stem cells may be automated. Automating a process using robotic or other automation can allow for more efficient and economical methods for the production, culture, and differentiation of cells. For example, robotic automation may be utilized as described in US patent application 20090029462, incorporated herein by reference in its entirety.

[0127] A bioreactor may also be used to culture, maintain, and / or differentiate cells (e.g., human embryonic stem cells, CD34+ cells, CD31+ cells, hematopoietic cells, etc.) according to the present disclosure. Bioreactors provide the advantage of allowing for the “scaling up” of a process in order to produce an increased amount of cells. Various bioreactors may be used with this disclosure, including batch bioreactors, fed batch bioreactors, continuous bioreactors (e.g., a continuous stirred-tank reactor model), and / or a chemostat. Pluripotent stem cells may be cultured on the robot, using flat plates in order to induce differentiation into CD34 / 43+ cells. Once separation of the cells has occurred, spinner flasks or a bioreactor may be used to generatelarge numbers of cells. Robotics may include liquid handling tools such as cap-piercing probes and disposable tips to minimize carry-over between samples. In some embodiments, robotics may be utilized in conjunction with one or more bioreactors for culturing cells (e.g., during the maintenance or growth of pluripotent stem cells, the differentiation of pluripotent stem cells into red blood cells, etc.).

[0128] In some embodiments, the method further comprises differentiating the CD34+ cell into a SRE and sorting a population of SREs derived from the CD34+ cell using magnetic- activated cell sorting (MACS), flow cytometry, fluorescence-activated cell sorting (FACS), or physical means such as centrifugation or fdtration. In some embodiments, the method further comprises sorting the population of SREs based on the expression of one or more of CD31 , CD34, CD43, and CD45.

[0129] In some embodiments, provided is a method of obtaining a red blood cell, comprising producing a SRE according to the methods as described herein to obtain a red blood cell.

[0130] A “progenitor cell,” as used herein, refers to a lineage-committed cell derived from a pluripotent stem cell. Thus, progenitor cells are more differentiated than pluripotent stem cells, but still have the capacity to differentiate into more than one type of cell. For example, a hematopoietic progenitor cell is more differentiated than a pluripotent stem cell, but the hematopoietic progenitor cell still has the capacity to differentiate into, for example, an erythrocyte, a macrophage, a granulocyte, a megakaryocyte, a dendritic cell, or a mast cell. In some embodiments, the progenitor cell is a hematopoietic progenitor cell. In yet other embodiments, the progenitor cell is a hematoendothelial (or hemangioblast) progenitor cell, which is capable of differentiating into hematopoietic cells or endothelial cells.Methods of Use

[0131] This disclosure further provides methods of treating a disease, disorder, or injury by administering to a subject a pharmaceutically effective amount of red blood cells obtained by the SREs as described herein. In some embodiments, provided is a method of using a pharmaceutical composition comprising the SREs as described herein. Administration of these compositions will be via any common route so long as the target tissue is available via thatroute. This includes administration by systemic or parenteral methods, including intravenous injection.

[0132] Diseases or disorders that may be treated by methods disclosed here include, but are not limited to, a vascular disease or disorder, an immunological disease or disorder, a neuronal disease or disorder, a blood disease or disorder, or an injury. For example, hematopoietic progenitor cells may be differentiated into red blood cells to be used in blood transfusions. One important application is production of red blood cells for transfusion purpose, particularly of cells carrying a rare blood group that are difficult to procure (for instance, to treat allo-immunized sickle cell patients). Similarly, the cells can be used to generate reagent red blood cells that are used by blood banks to cross match the patient and the cells to be transfused and to help identify antibodies against some blood groups that are present in some patients (so-called allo-immunized patients).

[0133] The red blood cells can be administered by infusion. In some embodiments, the method may include producing the red blood cells in vitro by the disclosed methods before administrating them to the subject. In some embodiments, the red blood cells can be produced in a bioreactor, e.g., a hollow fiber culturing system. The red blood cells can be administered to individuals through infusion or injection (for example, intravenous) or other methods known in the art. Administration may be once every two weeks, once a week, or more often, but the frequency may be decreased during a maintenance phase of the disease or disorder.

[0134] The red blood cells may be administered in a pharmaceutical formulation as described above. The dose of the red blood cells for an optimal therapeutic benefit can be determined clinically. A certain length of time is allowed to pass for the circulating or locally delivered modified red blood cells. The waiting period will be determined clinically and may vary depending on the composition of the composition. For example, the dose and the administration frequency will depend on the clinical signs, which confirm maintenance of the remission phase, with the reduction or absence of at least one or more clinical signs of the acute phase known to the person skilled in the art. More generally, dose and frequency will depend in part on the recession of pathological signs and clinical and subclinical symptoms of a disease condition or disorder contemplated for treatment with the above-describedcomposition. Dosages and administration regimens can be adjusted depending on the age, sex, and / or physical condition of administered, as well as the benefit of the treatment and side effects in the patient or mammalian subject to be treated and the judgment of the physician, as is appreciated by those skilled in the art. In all of the above-described methods, the cells can be administered to a subject at 1 * 104to 1 x 1010 / time.Additional Definitions

[0135] To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0136] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term “amino” acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0137] The term “amino acid sequence” refers to an amino acid sequence of a protein molecule, “amino acid sequence” and like terms, such as “polypeptide” or “protein,” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. Furthermore, an “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein.

[0138] As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “geneproducts.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0139] As used herein, the term “subject” refers to a vertebrate, and in some exemplary aspects, a mammal. Such mammals include, but are not limited to, mammals of the order Rodentia, such as mice and rats, and mammals of the order Lagomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and canines (dogs), mammals from the order Artiodactyla, including bovines (cows) and swines (pigs) or of the order Perissodactyla, including Equines (horses), mammals from the order Primates, Ceboids, or Simoids (monkeys) and of the order Anthropoids (humans and apes). In exemplary aspects, the mammal is a mouse. In more exemplary aspects, the mammal is a human.

[0140] The term “disease” as used herein is intended to be generally synonymous and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.

[0141] As used herein, “treatment,” “treating,” “palliating,” and “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit and / or a prophylactic benefit. As used herein, the term “therapeutic benefit” refers to any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

[0142] As used herein, the term “administering” refers to the delivery of cells by any route, including intravenous administration.

[0143] As used herein, the term “effective amount” or “therapeutically effective amount” refers to an amount which results in measurable amelioration of at least one symptom or parameter of a specific disorder. A therapeutically effective amount of the above-described cells can be determined by methods known in the art. An effective amount for treating adisorder can be determined by empirical methods known to those of ordinary skill in the art. The exact amount to be administered to a patient will vary depending on the state and severity of the disorder and the physical condition of the patient. A measurable amelioration of any symptom or parameter can be determined by a person skilled in the art or reported by the patient to the physician. It will be understood that any clinically or statistically significant attenuation or amelioration of any symptom or parameter of the above-described disorders is within the scope of this disclosure. Clinically significant attenuation or amelioration means perceptible to the patient and / or to the physician.

[0144] Doses are often expressed in relation to bodyweight. Thus, a dose which is expressed as [g, mg, or other unit] / kg (or g, mg etc.) usually refers to [g, mg, or other unit] “per kg (or g, mg etc.) bodyweight”, even if the term “bodyweight” is not explicitly mentioned.

[0145] The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a “therapeutic agent,” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

[0146] The terms “therapeutic agent,” “therapeutic capable agent,” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.

[0147] Combination” therapy, as used herein, unless otherwise clear from the context, is meant to encompass administration of two or more therapeutic agents in a coordinated fashion and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration,provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time (Kohrt et al. Blood. 2011. 117:2423).

[0148] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism.

[0149] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0150] The term “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of this disclosure within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxideand aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

[0151] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof. As used herein, the term “bz vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0152] As used herein, the term “in vivo" refers to events that occur within a multi-cellular organism, such as a non-human animal.

[0153] It is noted here that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0154] The terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0155] The phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0156] The terms “and / or” or means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0157] The word “substantially” does not exclude “completely,” e.g., a composition that is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

[0158] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

[0159] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of this disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

[0160] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0161] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. When used in this document, the term “exemplary” is intended to mean “by way of example” and is not intended to indicate that a particular exemplary item is preferred or required.

[0162] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise.

[0163] In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0164] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of this disclosure. Nothing herein is to be construed as an admission that this disclosure is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.EXAMPLES

[0165] To further decrease costs, this disclosure provides a method for producing SREs from iPSCs or CD34+ cells by eliminating the need for SCF and Epo, the only cytokines necessary after day 17. The following Examples illustrate, inter alia, that iPSCs or CD34+ cells carrying a constitutively active SCF receptor (such as a kitD816V allele associated with mastocytosis; Worobec, A. S. et al. Cancer. 1998. 83, 2120-2129 and Shi, X. et al. PNAS. 2016. 113, E4784-E4793) and a constitutively active jak2 adaptor protein (such as a V617F allele associated with polycythemia vera (James, C. et al. Nature. 2005. 434, 1144-1148)) can produce SRE cell lines that proliferate without any cytokines.Example 1. Materials and Methods

[0166] This Example details the materials and methods used in Examples 2-6.Example 1A, PSC-RED protocol

[0167] On day -1 : Three-day-old iPSC colonies were dissociated with 5mM EDTA to generate small clumps that yielded colonies of about 50 cells on day 0. Clumps were plated at1-2 x KF cells / well in 2mL / well of E8 medium on vitronectin-coated six-well plates and allowed to attach overnight (Olivier, E. N. et al. Experimental Hematology. 2019. 75, 31-52 and International Application No. PCT / US23 / 66608).

[0168] On day 0 : Differentiation was induced by replacing the E8 medium with an fMIT medium containing supplement 1 (SI).

[0169] On day 2, 6x concentrated supplement 2 (S2) was added.

[0170] On day 3, the cells were dissociated with Tryple-Select, centrifuged, and plated in an fMIT medium containing supplement 3 (S3) at IxlO3cells / mL in a 6-well plate (3mL / well).

[0171] On day 6, the cells were centrifuged and plated at 5.105 / mL in an fMIT medium containing supplement 3, excluding SB431542 but including 30nM of UM171 (or with UM729 at 500 nM). An additional dose of supplement 3 (provided from a 6x concentrated stock) was added on day 8.

[0172] On day 10, the cells were centrifuged and re-suspended at 0.66 x io5cells / mL in an fMIT medium containing the STIF supplement.

[0173] On day 17, the cells were centrifuged and plated at 2 x 105cells / m in an IMIT medium plus GM-CSF and the SED supplement.

[0174] Note: cells obtained on day 17 (z.e., day-17 HPCs) from kit or kitjak2 mutant lines give rise to SREs when cultured as described in the SRE section.

[0175] On day 24, the cells were centrifuged and plated at 2 x 10?cells / mL in an IMIT medium containing the SER supplement. RU486, a Dex antagonist, is included in the SER supplement to block residual traces of Dex.

[0176] On day 31, the cells were centrifuged and plated in a R5 medium containing the SER2 supplement.

[0177] On day 38, the cells were centrifuged and diluted to 0.5xl06 / mL and maintained in pure R6 medium for up to 8 days.

[0178] Cells cultured between days 6 to 38 were diluted to 0.5xl06 / mL in the appropriate media and supplement whenever their concentrations reached more than about1.5xl 06cells / mL. Cytokine concentrations between days 6 and 38 were refreshed by addition of 6x concentrate of the appropriate supplement every other day. 6x concentrates of each supplement were made by multiplying by 6 the concentrations of cytokines and small molecules indicated in Table 1. Concentrated supplements were used to keep the concentration of cytokine and small molecules high enough at all times without having to spin the cells and without overly increasing the cell culture volume, lx and 6x concentrates of supplements 1, 2, 3, 4, STIF, SED, and SER were prepared in IMIT. SER2 supplement was prepared in R6 medium. All centrifugations were performed at 250g for 5 to 10 minutes (depending on volume). All culture vessels used were tissue culture-treated. Experiments were generally performed in 6-well plates, but flasks were used to obtain larger volumes.

[0179] Table 1. Media composition.Example IB, SREs

[0180] Long-term culture of SREs: kitD816V day 17 HPCs (obtained as described earlier) can be cultured for a duration of 45 to 55 days in IMIT combined with 1 pM Dex and 30 pM IBMX. They can also be cultured for up to 140 days in the same conditions when supplemented in lU / mL Epo.

[0181] Kitjak2 double mutants (kitD816V; Jak2V617F) day 17 HPCs can be cultured for about 120 days in IMIT combined with luM Dex and 30uM IBMX for about 120 days.

[0182] All SRE lines were passaged every 3 to 5 days by dilution to 1.25 to 2.5x10’ cells / mL once the culture concentration exceeded 1.5xl06 / mL. The passage frequency depended on the passage number because the rate of proliferation of the SREs diminishes gradually over time.

[0183] Terminal differentiation of SREs:

[0184] Expanded SREs can be differentiated up to the point of senescence as follows.

[0185] Day 0 : Cells were centrifuged, rinsed once in PBS to eliminate all traces of Dex and IBMX and plated at about 1.5xl05cells / mL in R6 media containing 4U / ml of Epo, and 5% human AB plasma and IpM RU 486.

[0186] Day 3 : Cells were diluted 1 to 2 to about 3.5xl05cells / mL in the same media without Epo.

[0187] Days 5, 7, and 9: Cells were diluted in pure RPMI to about 3 to 5xl05cells / mL.

[0188] Total cell amplification at day 10 was about 40-fold and enucleation rates were consistently >50%.Example 1C. Analysis and characterization

[0189] Cell enumeration: Cells were counted with a Luna-FL dual channel Automated Cell Counter (Logos) using acridine orange to visualize the live cells and propidium iodide to exclude the dead cells. Alternatively, cells were counted using a Cytek Aurora flow cytometer. Apoptosis was detected by staining with AnnexinV-FITC and propidium iodide.

[0190] Flow cytometry: iPSCs undergoing differentiation were evaluated by FACS using a Cytek aurora spectral cytometer and a 15-color antibodies panel described in Table 2. Data were analyzed with FlowJo™ software using the FlowSOM and UMAP plugins, essentially as suggested by the manufacturer.

[0191] Table 2. Antibody panel.

[0192] Enucleation: The enucleation rate was measured using the DRAQ5™ DNA nuclear stain (ThermoFisher) after exclusion of dead cells with Propidium Iodide or DAPI. The cells were analyzed with a Cytek Aurora flow cytometer and FlowJo™ software.

[0193] Cell morphology: Erythroid differentiation and enucleation were also assessed microscopically by Rapid Romanovsky staining of cytospin preparations using the HEMA-3 kit from Fisher Scientific according to the manufacturer’s instructions. Cell sizes were estimated on a Nikon TE-2000S microscope using software provided by the manufacturer.

[0194] RBC filtration: 99.5% pure populations of enucleated RBCs were generated by filtration of the cells obtained after 10 days of differentiation of SREs through PAL Acrodisc 25mm WBC filters as recommended by the manufacturer. Filtered cRBCs were stored for up to one month with little signs of hemolysis in Alsever’s solution (Sigma).

[0195] HPLC analysis: Cells were washed twice with PBS and lysed in water by 3 rapid freeze-thaw cycles in dry ice and in a 37 °C water bath. Debris was eliminated by centrifugation at 16,000g, and the lysates were stored at - 80 °C. HPLC was performed as described by Fabry et al. (Fabry, M. E. et al. in Hemoglobin Disorders Vol. 82 Methods in Molecular Biology (ed R. Nagel) 213-241 (Humana Press, 2003)). Briefly, a few pL of lysate containing about 50 pg of protein in about 100 pL of 40% acetonitrile and 0.18% TFA was filtered and loaded on a VYDAC C4 column. The globins were then eluted with increasing concentration of acetonitrile during a period of about 80 minutes. The starting elution buffer was programmed to be 80% buffer A and 20% buffer B and to rise to 50% buffer B in 50 minutes. Buffer A = 36% acetonitrile and 0.18% TFA and buffer B = 56% acetonitrile and 0.18% TFA. Globin chain elution was monitored by measuring O.D. at 220 nm.

[0196] CRISPR editing: A nucleic acid solution containing 1 pg of capped, poly adenylated Cas9 mRNA substituted with modified Uridine (CLEAN-Cap Cas9 mRNA, cat # L-7206 from Trilink Biotechnologies) was mixed with 4 pg of synthetic sgRNA (Sigma technologies) and 4ug of a 200-mer oligonucleotides HDR donor in 15 pl of H2O. About 150,000 iPSCs dissociated with Accutase were resuspended in 15 pl of buffer P (lOOmM phosphate buffer pH 7.4; 15mM NaHCO3; 2 mM glucose, 12mM MgCu) and mixed with 15 pl of the nucleic acid solution in a 1 cm 1 / 16 of an inch inner diameter electroporation chambermade of platinum-cured silicone tubing. The tube was then subjected to two 10 msec pulses of 115V using a NEPA21 (Nepagene) electroporator hooked up to Cell-Porator Voltage Booster (Life technology) resulting in a 600V pulse. Subclones were then isolated and screened for the presence of the desired mutations by amplifying and sequencing PCR fragments representing the regions of interest. In some experiments, the PCR fragments were pre-screened by digestion with an appropriate restriction enzyme. To obtain heterozygous clones, donor HDR oligonucleotides coding for the wt sequences were mixed-in with the mutated donor HDR oligonucleotide. Sequences of the sgRNA and HDR donor DNA are provided in Table 3.

[0197] Table 3. Sequences of the sgRNA and HDR donor oligonucleotides used to generate the kitD816V and Jak2V617F lines.

[0198] Low-pass sequencing: Genomic DNA was extracted, and 1.5Gb of sequence was obtained on an Illumina sequencer (2xl50bp configuration). Reads were aligned to the hg38 genome using the bwa aligner47, and copy-number variants were detected using the CNVKit software package (Talevich, E. et al. PLOS Computational Biology. 2016. 12, el004873). A library of 10 normal genomes sequenced to the same depth was used as a baseline control. iPSCs genomic DNA was also compared with the DNA obtained from peripheral blood cells from the same donor.Example 2. Generation of induced pluripotent stem cells (iPSCs) and cultured red blood cells (cRBCs) carrying the D816V mutation. iPSCs

[0199] To generate iPSCs with the kitD816V mutation, iPSC clone 019 was transfected with Cas9 mRNA, an sgRNA targeting exon 17 of the kit gene, and a 200 bp homology- directed recombination donor oligonucleotide (HDR) (Olivier, E. N. et al. Experimental Hematology. 2019. 75, 31-52). Screening 24 iPSC clones showed a targeting frequency of about 50%. Cell lines homozygous (B34) and hemizygous (A4) for the D816V were selected for further characterization. cRBCs

[0200] The above-described iPSCs were differentiated into cRBCs using the Pluripotent Stem Cell Robust Erythroid Differentiation (PSC-RED) protocol (FIG. 1A) (Olivier, E. N. et al. Experimental Hematology. 2019. 75, 31-52 and International Patent Application No. PCT / US2023 / 066608). During the PSC-RED protocol, iPSCs underwent sequential culture over 17 days with four supplements (SI to S4; See Table 4), which produced a mixture of hematopoietic progenitor cells (HPCs). These HPCs were differentiated into early erythroidcells over an additional 7 days in the presence of SCF, Epo, Dexamethasone (Dex), and 3- Isobutyl-1 -methylxanthine (IBMX). After another 14 days in the same media, minus the IBMX and Dex, the cells progressed to late erythroid cells. Finally, the removal of SCF and Epo triggered the terminal differentiation into enucleated cRBCs.

[0201] Table 4. Supplement composition as used in the protocol.*: UM171 can be replaced with 500nM UM729Example 3. Characterization of kitD816V iPSCs

[0202] To evaluate the phenotype of the kitD816V iPSCs, the A4, B34 and the unedited 01 (control) iPSC cell lines were differentiated using the PSC-RED protocol with the omission of SCF (FIG. 1A). As anticipated, the unedited 01 iPSCs could not survive beyond day 17 in the absence of SCF. In contrast, the A4 and B34 lines generated over 200,000 cells / iPSC by day 38. This yield was comparable to the cell production from control 01 cells differentiated in the presence of SCF (FIG. IB).A, Differentiation in the absence of both Epo and SCF

[0203] Analysis of the cells generated in these cultures through a 15-color flow cytometry assay demonstrated that the progression of cells during the PSC-RED protocol could be succinctly represented using the flowSOM (Quintelier, K. et al. Nature Protocols. 2021. 16, 3775-3801) and U-MAP algorithms (Mcinnes, L., et al. Journal of Open Source Software. 2018. 3, 861). These algorithms categorized the cells into four distinct populations of HPCs (HPC1 to HPC4) and four populations of erythroid cells (Eryl to Ery4), based on the expression patterns of ten surface antigens (FIGS. 1C and ID). Dimensionality reduction analysis using flowSOM and U-MAP revealed that dividing the cells into 8 major populations according to the expression of 10 markers provided a useful summary of the evolution of the cells undergoing the PSC-RED protocol (FIG. 1C). In the kit mutated clones, the HPC2 and Eryl cells did not amplify to the same degree as in the control cells, and the Ery2 cells were barely detectable (FIG. ID). Instead, an HPC3 population with a phenotype intermediate between HPC1 and 2 briefly expanded and, most notably, the Ery3 population became prominent much earlier, particularly in the homozygous B4 cells. The Ery3 cells from the A4 cells eventually differentiated into Ery4 cells, but those from the B34 clone did not, resulting,at days 31 and 38, in cultures composed almost exclusively of Ery3 cells (FIG. ID). Due to massive cell death of the B34 cells during the last week of differentiation, a FACS analysis for these cells was not performed at day 45.

[0204] The data indicated that the rate of differentiation of the HPC populations was accelerated in the presence of the kitD816V mutation, while the overall pattern remained consistent. Upon completion of the differentiation process, the hemizygous kitD816V A4 clone yielded a combination of basophilic erythroblasts (referred to as Ery3), orthochromatic erythroblasts, and enucleated cells (referred to as Ery4), a pattern similar to that of the control cells. The homozygous (B34) clone, which differentiated at an even quicker rate than the A4 clone, generated Ery3 cells but not Ery4 cells. Thus, cRBCs can be produced without SCF.

[0205] It has previously been reported that SCF interacts with the Epo receptor (EpoR) by phosphorylating its cytoplasmic domain, and that this interaction is essential for erythropoiesis (Wu, H. et al. Nature. 1995. 377, 242-246 and Wu, H. et al. PNAS. 1997. 94, 1806-1810). A more recent study has shown that HUDEP (human umbilical cord blood-derived erythroid progenitor)-2 transformed cord blood erythroblast cells (Kurita, R. et al. PLoS One. 2013. 8, e59890), which are dependent on SCF and Epo can be rendered both Epo and SCF independent by introducing a mutation in the kit gene similar to the D816V mutation (Couch, T. et al. Experimental Hematology. 2019. 74, 19-24).

[0206] To investigate the potential of iPSC-derived kitD816V cells to differentiate in the absence of both Epo and SCF, differentiation was induced in the A4 and B34 lines omitting both cytokines (FIG. 2A). This demonstrated that both cell lines could proliferate without Epo and SCF, yielding a substantial number of cells (>200,000 cells / iPSC). Interestingly, the block of differentiation at the Ery3 stage that was observed in the B4 lines was partly alleviated in the absence of Epo, since Ery4 cells were produced in these conditions (FIG. 2B).

[0207] Both hemizygous and homozygous D816V lines can differentiate in the absence of both SCF and Epo. In the absence of SCF and Epo, B34 Ery3 cells did not die as quickly upon withdrawal of Dex when Epo was absent from the culture media, and some of the cells were even able to complete their terminal differentiation and enucleate at a rate of about 5%. This indicates that the block of differentiation of the Ery3 cells in the B34 cells is likely due tooverstimulation by the homozygous kitD816V, and that this overstimulation can be partly relieved by omitting Epo from the media.B, Enucleation rate

[0208] To measure the enucleation rate, day 45 cells cultured without SCF and induced to terminally differentiate by discontinuing Epo on day 38 were analyzed by flow cytometry after staining with DRAQ5™, a cell permeant DNA dye, and by light-microscopy following Romanowsky staining. In certain experiments, the potential of dasatinib (an inhibitor of the constitutive kinase activity of the kitD817V SCF receptor; also termed SPRYCEL®) was also investigated, to enhance terminal differentiation (Akin, C. et al. J Allergy Clin Immunol. 2022. 149, 1912-1918).

[0209] In the absence of dasatinib, the enucleation rate of A4 cells was about 18%, similar to the control cells. This rate rose to around 24% in the presence of 200nM of dasatinib (FIG. 3A). Further analysis revealed that dasatinib also accelerated the differentiation process (FIGS. 3B and 3C). Subsequently, the globin chain composition of cells obtained on day 45 was analyzed through reverse-phase HPLC (high-performance liquid chromatography). As previously reported, reticulocytes and orthochromatic erythroblasts produced from the control 01 cells expressed mostly fetal y-globin chains (FIG. 3D) (Olivier, E. N. et al. Experimental Hematology. 2109. 75, 31-52). Cultured RBCs obtained from the A4 line also expressed predominantly fetal y-globin, alongside detectable embryonic globin chains. Further experiments indicated that A4 cells grown without SCF and Epo enucleated at similar rates.

[0210] To replicate these findings and assess the phenotype of heterozygous kitD816V iPSCs (which were not obtained in the experiments described above), CRISPR editing was repeated on cells from two distinct individuals, mixing in an additional HDR donor oligonucleotide encoding the wild-type sequence in equal proportions with the kitD816VHDR oligonucleotide. This approach yielded multiple clones heterozygous for the D816V mutation for both the 01 and 02 donors (FIG. 4A). For both 01 and 02 about 20% of the clones were positive for the Mbol restriction sites and of those about half were heterozygous for the D816 V mutation. Differentiation experiments using the PSC-RED protocol revealed that these clones could produce enucleated cells, although they proliferated at a rate slightly lower than the A4hemizygous clones (FIGS. 4B and 4C). These results illustrate that the kitD816V mutation represents a reliable broadly applicable approach to generate iPSC lines capable of differentiating into RBCs without the need for SCF.C. Self-renewal capabilities

[0211] Both human and mouse CFU-E / pro-Erythroblasts can self-renew for a limited time when cultured in the presence of SCF, Epo and Dex (Wessely, O. et al. EMBO Journal. 1997. 16, 267-280; Migliaccio, G. et al. Blood Cells, Molecules, and Diseases. 2002. 28, 169-180; England, S. J. et al. Blood. 2011. 117, 2708-2717; and Leberbauer, C. et al. Blood. 2005. 105, 85-94). To investigate whether kitD816V erythroid progenitors also exhibit self-renewal capabilities, the PSC-RED protocol was used to generate day 17 HPCs from the A4 and B34 iPSCs lines. These cells were expanded without cytokines, but in the presence of Dex and IB MX (referred to as DI conditions). Both lines demonstrated the ability to expand under these conditions for approximately 45 to 55 days, resulting in a greater than 1,000-fold expansion before reaching a plateau and ceasing to proliferate (FIG. 5B).

[0212] Crucially, further experiments indicated that adding small amounts of Epo to Dex and IBMX (referred to as EDI conditions) enabled the A4 erythroblasts to proliferate without differentiation for over 25 passages (approximately 120 days), resulting in a 1020-fold amplification (FIG. 5C). These findings indicated that semi-permanent cell lines capable of sustained growth without the need for SCF can be generated from kitD816V iPSCs.

[0213] To investigate the possibility of obtaining cells capable of such long-term selfrenewal without requiring Epo, the jak2V617F mutation was introduced through CRISPR- mediated mutagenesis in the A4 line using the method described earlier. Once again, over 50% of the screened clones had acquired the jak2V617F mutation. The double-mutant was termed the kitjak2 line. When subjected to differentiation using the PSC-RED protocol, iPSC lines hemizygous for the kitD816V mutation and either homozygous (lines G19 and H12) or hemizygous (lines H5 and Hl l) for the jak2V617F mutation demonstrated the ability to undergo erythroid differentiation in the absence of both SCF and Epo (FIG. 5D) and exhibited antigen profiles similar to the A4 cells.

[0214] Significantly, additional experiments demonstrated that day 17 HPCs from the G19 and H12 kitjak2 cell lines proliferated exponentially in the absence of SCF and Epo for about 3 months before entering a state of senescence. Even more remarkable, the H5 and H12 cells proliferated for more than 4 months, resulting in an amplification > 10ls-fold (FIG. 5E). Flow cytometry analysis indicated that the self-renewing A4 cells and the kitjak2 lines resembled iPSC-derived late CFU-Es and pro-erythroblasts, and expressed erythroid antigen profiles that were strikingly similar across all lines and that varied minimally over a span of seventy days (FIG. 5F)

[0215] To investigate the potential of the A4 (expanded in EDI conditions) and of the kitjak2 (expanded in DI conditions) lines, their differentiation was initiated by withdrawing Epo, Dex and IBMX from the culture media. This resulted in a rapid 7 day differentiation, which produced a few enucleated cells, but also resulted in a yield of RBCs per SREs of only about 0.01% due to high cell mortality.

[0216] Epo serves as the primary cytokine shielding erythroblasts from apoptosis (Lacombe, C. et al. Haematologica. 1998. 83, 724-732). To enhance the differentiation of these cells, a high-concentration pulse of Epo (4u / ml) was administered to cushion the cells from the abrupt withdrawal of Dex and IBMX and supplemented with human plasma during the initial five days of differentiation (FIG. 6A). These adjustments lengthened the differentiation to ten days, dramatically decreased cell death, and significantly elevated the rate of enucleation to 25% to 30%. As a result, the yield of RBCs per SREs escalated nearly a thousand-fold, from less than 0.01 to a range of 4% to 8% (FIG. 6B). Both the A4 and all kitjak2 cell lines could differentiate into RBCs until senescence. The feeding routine was modulated to alternate days, ensuring that the cell concentration remained under 1 million / ml throughout the terminal differentiation. As a result, the enucleation rate was further boosted to beyond 50%, without compromising cell yield (FIG. 6C).D. Quality of the RBCs originating from the A4 and the kitjak2 SREs

[0217] To evaluate the quality of the RBC originating from the A4 and the kitj ak2 SREs, enucleated cells were purified using PALL Acrodisc filters. Then HPLC globin profiles were generated, and morphological data was obtained via light microscopy, and with the AD VIA®2120i blood count analyzer. The purified RBCs expressed 78-82% fetal (Ay and Gy), 12-17% embryonic (e) and 5 to 7 % adult ( ) 0-like globins. They also expressed small amounts of embryonic (< ) a-like globins (FIG. 6D). Morphologically the cells were larger than standard adult RBCs (MCV of about 125fL vs 85 fL, and diameter of about 10.5um vs 8.3um) and were well hemoglobinized since they exhibited hemoglobin concentrations similar to control cells (about 28g / dL) (FIG. 5E).E, Karyotypic stability

[0218] To examine if the kitD816V and jak2V617F mutations induce karyotypic instability, chromosome copy-numbers in iPSCs before and after CRISPR / Cas9 editing through low-pass whole-genome sequencing were compared, using 01 and 02 donor-derived peripheral blood (PB) mononuclear cells (MNC) as controls. CNVKit24 software analysis showed no detectable copy-number variations in any cell line studied while confirming the marked aneuploidy of control transformed cells sequenced at an equivalent depth (FIG. 7 and FIG. 8). In particular, FIG. 7 illustrates thatPB MNC 01, passage 40 iPSCs 01 (derived from PB MNC 01), passage 30 iPSCs A4 (derived from iPSC 01), and passage 30 iPSCs H5 (derived from iPSCs A4) exhibit no detectable copy number variants greater than Imb, (the limit of detection for this read depth). In contrast, transformed cells, HUDEP-2 and K562 cells, cultured for a long period of time exhibit a high level of aneuploidy. As shown in FIG. 9, homozygous clones grew poorly, even in the presence of Epo. Heterozygous clones grew as well as control cells in the presence of Epo, but did not grow well in the complete absence of Epo. Thus, cells carrying both the kitD816V and jak2V617F can be propagated in culture for long periods of time without acquiring karyotypic abnormalities.

[0219] The cell lines that we generated appear karyotypically stable, likely because mutations in cytokine receptors and signaling adaptors don't directly drive chromosomal instability. While the kitD816V and jak2V617F mutations can be leukemogenic when associated with other mutations, this should not preclude the use of these cells for transfusion, as RBCs lack nuclei, and as any residual nucleated cells can be killed by irradiation.Example 4. Generation of iPSCs with deletions at C616 and V817

[0220] Multiple mutations can constitutively activate the JAK2 gene (Saharinen, P.et al. Molecular and Cellular Biology. 2000. 20, 3387-3395). To determine if the production of SREs was specific for the V617F mutation, or if other mutations also led to the generation of SREs, homozygous or heterozygous iPSC clones were generated that contained a two amino acid deletion (Cys and Vai position 616 and 617, respectively) in the JAK2 gene. These mutations, which are located in the JH2 pseudokinase domain were expected to render the JAK2 adaptor constitutively active.

[0221] Differentiation of these clones according to the PSC-RED protocol yielded as many erythroid cells as clones with the V817F mutations. Likewise, culture of day 17 HPC carrying these mutations in medium containing only Dex and IBMX yielded selection of SREs with similar growth and differentiation characteristics as SREs with the V817F mutation. Therefore, kitJak2 SREs can be generated with constitutive mutations other than the V617F mutation.Conclusions:

[0222] The results from the above-presented experiments demonstrate that SREs can reproducibly be generated from iPSCs carrying the kitD816V and jak2V671F mutations (as well as other constitutively active JAK2 mutations such as deletion of C616 and V617). While the single-mutant kitD816V cells exhibited modest self-renewal — lasting approximately 50 days without cytokines — their proliferation potential surged to 140 days in the presence of Epo. Most remarkably, double-mutant kitjak2 cells thrived for about 120 days sans cytokines. All of the cell lines that were developed retained the ability to enucleate at a high rate up until senescence, and could be consistently passaged at ratios from 1 : 10 to 1 : 15 every 3-4 days, utilizing a cost-effective, chemically-defined, albumin-free medium supplemented with a minimal amount of recombinant transferrin.

[0223] The self-renewal of kitjak2 cells hinges on the inclusion of two specific small molecules, Dex and IBMX. Dex has long been known for its role in erythroblast self-renewal (Zingariello, M. et al. Frontiers in Physiology. 2019. 10, 1-17). Notably, Dex is essential for the self-renewal of the A4 and kitjak2 SREs since withdrawal of this molecule leads to rapid differentiation.

[0224] IBMX, a non-specific inhibitor of cAMP and cGMP phosphodiesterases, elevates intracellular cAMP levels. See Ahmad, F. et al. Oral Diseases. 2015. 21, e25-e50. IBMX was incorporated in the PSC-RED protocol because high cAMP concentrations facilitate early HPC specification (Olivier, E. N. et al. Stem Cells Transl Med. 2016. 5, 1394-1405 and Diaz, M. F. et al. J Exp Med. 2015. 212, 665-680). IBMX also regulates erythropoiesis. The EpoR doesn't directly control cAMP, but agents such as forskolin and prostaglandins, which modify cAMP concentration, influence Epo-mediated erythropoiesis (Boer, A. K. et al. Leuk Lymphoma. 2003. 44, 1893-1901). Notably, cAMP -inducing agents have been shown to amplify the proliferation of colony-forming erythroid progenitors (Belegu, M. et al. Am J Physiol. 1983. 245, C322-327 and Fisher, J. W. et al. Exp Hematol. 1980. 8 Suppl 8, 65-89). It was determined that IBMX is essential for the self-renewal of A4 and kitjak2 erythroblasts, as its omission markedly reduced their proliferation and viability. Consequently, kitD816V, Iak2V617F, Dex and IBMX jointly extend the self-renewal of kitjak2 cells.

[0225] Additionally, the generation of kitjak2 cells was reproducible because these cells successfully produced multiple lines from two distinct donors. SREs differentiate spontaneously from iPSC-derived HPCs on a slightly quicker timeline than controls. This contrasts with immortalization using the HPV6 / 7 proteins, which demands several months of transduced cell cultivation before cell lines emerge. See Kurita, R. et al. Experimental Hematology. 2019. 69, 11-16. This reproducibility, paired with the rapid differentiation, indicates that no additional genetic or epigenetic alterations, beyond the engineered modifications, are necessary for the erythroblasts to achieve self-renewal without cytokines.Example 5. Generation of adult SREs.

[0226] The cRBCs produced from iPSCs are larger than adult cells and express mostly fetal hemoglobin. To determine if SREs could be generated from adult cells, CD34+ cells were electroporated with the kitD816V sgRNA and an HDR oligonucleotide donor. After three days of culture in a medium rich in cytokines, the cells were transferred in a medium containing only Epo, Dex and IBMX (EDI conditions). Control CD34+ cells died within 10 days, likely due to the absence of SCF. By contrast, transfected cells thrived in the EDI conditions for more than 7 weeks and yielded over 8.106-fold amplification, before starting to senesce (FIG. 11B).Remarkably, sequencing after 5 weeks of culture revealed that all of the cells carried a GTG (valine) at position 816 of the kit gene, suggesting that all of the non-edited and partially edited had been select-ed out during the culture under EDI conditions.

[0227] Similarly, co-transfection of sgRNAs and HGR donor targeting to introduce the D816V mutation and the V817F mutation into adult CD34+ resulted in the production of SREs that could proliferate in culture for at least 2 months in the presence of DEX and IB MX and in the absence of any cytokine. Withdrawal of Dex and BMX demonstrated that the cells had retained their ability to differentiate and enucleated at a high rate. HPLC analysis demonstrated that the cells produced almost exclusively adult hemoglobin (Hb A) and had a diameter smaller than iPSC-derived cRBCs. Consequently, these experiments demonstrate a method to produce KitJak2 cells from adult primary CD34+ progenitor cells.Example 6. Generation of reagent RBCs.

[0228] One application of cRBCs is the production of reagent RBCs for identifying allo- antibodies in chronic transfusion recipients, minimizing transfusion reactions. Reagent cells typically come from volunteer blood, but there are shortages of cells to detect allo-antibodies in sickle cell and myelodysplastic patients who constitute over 80% of all allo-immunized individuals at most blood banks. To investigate whether cRBCs could fill this role, a solid phase red cell adherence assay was used to determine whether the cRBCs could detect Rhesus C, c, E, and e antigens on the surface of A4 cRBCs. See Sinor, L. et al. Transfusion. 1985. 25, 21-23 and Ching, E. et al. Transfus Apher Sci. 2012, 46, 287-291. The results indicated that 5 million RBCs per test could be used to identify all four antibodies (FIG. 9), demonstrating that cRBCs produced without cytokines can be used as reagent RBCs.

[0229] Differentiating a single iPSC using the PSC-RED protocol generates about 103day 17 HPCs. Each of these HPCs can produce about 1018kitjak2 SREs, which each yield up to 8 RBCs. Thus, theoretically, a single iPSCs is sufficient to produce the number of RBCs present in >109liters of blood (1021cells), surpassing the global annual transfusion volume. The cytokine-independent SREs from 01 and 02 donors, who have blood group 0 and test negative for RhD and other clinically significant RBCs antigens, therefore have ample proliferation capacity to meet global RBCs needs for the foreseeable future.

[0230] The above-discussed findings highlight the profound influence of constitutive alleles of the kit and jak2 genes on the erythroid differentiation of iPSCs. The kitD816V mutation completely alleviated the need for SCF and Epo, as all iPSC lines that were generated, regardless of genotype, were able to proliferate and differentiate into erythroblasts in the absence of these cytokines at about the same rate as control cells in the presence of cytokines. Homozygosity for the kitD816V hindered terminal erythroid differentiation, but hemizygous and heterozygous underwent terminal differentiation and showed high enucleation rates. iPSCs with both the kitD816V and jak2V671F mutations also thrived and differentiated without any cytokines, matching or even surpassing control cells supplemented with SCF and Epo.

[0231] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified CD34+ cell comprising a constitutively active stem cell factor (SCF) receptor and a constitutively active Janus kinase 2 (JAK2); and culturing the genetically modified CD34+ cell in one or more culture media free of a stem cell factor (SCF) and erythropoietin (Epo).

2. A method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified CD34+ cell comprising a constitutively active stem cell factor (SCF) receptor; and culturing the genetically modified CD34+ cell in one or more culture media free of a stem cell factor (SCF).

3. A method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified induced pluripotent stem cell (iPSC) comprising a constitutively active stem cell factor (SCF) receptor and a constitutively active Janus kinase 2 (JAK2); and culturing the genetically modified iPSC in one or more culture media free of a stem cell factor (SCF) and erythropoietin (Epo).

4. A method of producing a self-renewing erythroblast (SRE), comprising: providing a genetically modified induced pluripotent stem cell (iPSC) comprising a constitutively active stem cell factor (SCF) receptor and culturing the genetically modified iPSC in one or more culture media free of a stem cell factor (SCF).

5. The method according to claim 4, wherein the SRE is cultured in the presence of Epo.

6. The method of any one of claims 1-5, wherein the SCF receptor comprises a mutation at position D816.

7. The method of claim 6, wherein the SCF receptor comprises a D816V mutation.

8. The method of claim 6 or 7, wherein the SCF receptor comprises the amino acid sequence of SEQ ID NO: 2.

9. The method of claim 1 or 3, wherein the JAK2 comprises a deletion at C616 and V617 or comprises a mutation at position V617.

10. The method of claim 9, wherein the JAK2 comprises a V617F mutation.

11. The method of claim 9, wherein the JAK2 comprises the amino acid sequence of SEQ ID NO: 4 or 5.

12. The method of any one of claims 1-11, wherein the SRE is consistently passaged at ratios from 1 : 10 to 1 :15 every 3 to 4 days.

13. The method of claim 12, wherein the SRE is capable of self-renewing in a medium for at least 15 passages.

14. The method of claim 1 or 2, wherein the CD34+ cell is a human cell.

15. The method of claim 3 or 4, wherein the iPSC is a human cell.

16. The method of claim 14, wherein the CD34+ cell is collected from the blood of a subject aged 0 to 100 years old.

17. The method of claim 14, wherein the CD34+ cell is collected from embryonic or fetal tissues.

18. The method of claim 14, wherein the CD34+ cell is produced from a pluripotent stem cell or an iPSC.

19. The method of any one of claims 1-18, wherein the one or more culture media comprise dexamethasone (Dex) and isobutylmethylxanthine, l-Methyl-3-Iso-butyl-xanthine (IBMX).

20. The method of claim 19, wherein the one or more culture media comprise from about 0.01 pM to about 100 pM of Dex and from about 1 pM to 500 pM of IBMX.

21. The method of claim 19 or 20, wherein the one or more culture media comprise about 1 unit of Epo, about 1 pM Dex, and about 30 pM of IBMX.

22. The method of any one of claims 19-21, wherein the one or more culture media comprises Dex and IBMX from day 17 to at least day 38.

23. The method of any one of claims 1-22, wherein the one or more culture media comprise one or more of transferrin, activin A, Wnt, GM-CSF, vascular endothelial growth factor (VEGF), bone morphogenic proteins (BMP), inhibitor VIII, -Estradiol, heparin, UM171 or UM729, thrombopoietin (TPO), insulin-like growth factor-2 (IGF-2), RU-486, and basic fibroblast growth factor (bFGF).

24. The method of any one of claims 1-23, comprising supplementing the one or more culture media with thrombopoietin (TPO), insulin-like growth factor-2 (IGF-2), and basic fibroblast growth factor (bFGF) only from day 10 to day 17.

25. The method of any one of claims 1-24, wherein the one or more culture media comprises between 1 to 1000 pg of recombinant transferrin.

26. The method of any one of claims 1-25, wherein the one or more culture media comprises a serum-free medium or a defined differentiation medium.

27. The method of any one of claims 1-25, wherein the one or more culture media comprise a supplement selected from inositol, folic acid, monothioglycerol, insulin, ferrous nitrate, ferrous sulfate, BSA, L-glutamine, penicillin-streptomycin, animal plasma or serum, Fe(III)-EDTA or an equivalent chelator, lipids, and combinations thereof.

28. The method of claim 27, comprising removing Dex and IBMX from the medium to induce differentiation of the SRE into a red blood cell.

29. The method of claim 28, wherein the red blood cell expresses ADAMTS13, asparaginase, Factor VIII, Factor IX, or phenylalanine hydroxylase.

30. The method of claim 27, further comprising expanding the CD34+ cell prior to being differentiated into a SRE.

31. The method of claim 30, further comprising differentiating the CD34+ cell into a SRE and sorting a population of SREs derived from the CD34+ cell using magnetic-activated cell sorting (MACS), flow cytometry, fluorescence-activated cell sorting (FACS), or physical means.

32. The method of claim 31, further comprising sorting the population of SREs based on the expression of one or more of CD31, CD34, CD43, and CD45.

33. A method of obtaining a red blood cell, comprising producing a SRE according to the method of any one of claims 1-32 to obtain a red blood cell.

34. A self-renewing erythroblast (SRE), comprising a constitutively active stem cell factor (SCF) receptor and a constitutively active Janus kinase 2 (JAK2).

35. The SRE of claim 34, wherein the SCF receptor comprises a mutation at position D816.

36. The SRE of claim 35, wherein the SCF receptor comprises a D816V mutation.

37. The SRE of any one of claims 34-36, wherein the SCF receptor comprises the amino acid sequence of SEQ ID NO: 2.

38. The SRE of claim 34, wherein the JAK2 comprises a deletion at C616 and V617 or comprises a mutation at position V617.

39. The SRE of claim 38, wherein the JAK2 comprises a V617F mutation.

40. The SRE of claim 38, wherein the JAK2 comprises the amino acid sequence of SEQ ID NO: 4 or 5.

41. A pharmaceutical composition comprising the SRE of any one of claims 34-40.

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