Erythroid lineage derived from pluripotent cells

Abstract

The present disclosure, in various aspects and embodiments, provides methods for generating hematopoietic lineages for cell therapy, including erythroid progenitors, progenitor erythroblasts, granulocyte-macrophage progenitors (GMPs), and megakaryocytic erythroid progenitors (MEPs), as well as erythroid cells. In various embodiments, the present invention provides an efficient ex vivo process for developing such hematopoietic lineages, including, but not limited to, progenitor erythroid and erythroid cell lineages, from human induced pluripotent stem cells (iPSCs). In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or bone marrow. The present invention also provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 413,439, filed October 5, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in XML format and is incorporated herein by reference in its entirety. The XML copy, created on September 26, 2023, is named GRU-012PC_Sequence_Listing.xml and is 30,040 bytes in size. [Background technology]

[0003] According to the American Red Cross, we are facing a nationwide blood crisis, the worst blood shortage in a decade, posing concerning risks to patient care. Amid this crisis, physicians are being forced to make difficult decisions about who receives transfusions and who must wait until more product becomes available. Therefore, there is an urgent need to develop commercially available red blood cells (RBCs) or erythrocytes or their progenitors. Hematopoietic stem cells derived from induced pluripotent stem cells offer such an opportunity. However, current methodologies for generating RBCs from iPSCs have proven inadequate due to limitations in erythroid lineage cell proliferation or insufficient enucleation. Therefore, successful generation of red blood cell products from iPSCs ex vivo would fill a significant need. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure, in various aspects and embodiments, provides methods for generating hematopoietic lineages for cell therapy, including gene-edited hematopoietic stem cells (HSCs), erythroid progenitors, progenitor erythroblasts, granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), and erythroid cells. In various embodiments, the present invention provides an efficient ex vivo process for developing such hematopoietic lineages, including, but not limited to, progenitor erythroid and erythroid cell lineages, from human induced pluripotent stem cells (iPSCs). In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or bone marrow. The present invention also provides isolated cells and cell compositions generated by the methods disclosed herein, as well as methods for cell therapy.

[0005] In other aspects and embodiments, the present disclosure provides HSCs or erythroid progenitor cells derived from iPSCs that have been gene-edited to encode a hyperresponsive EPO receptor. These populations of HSCs or erythroid progenitor cells can be used for more efficient ex vivo red blood cell production, or in other aspects, can be used to deliver HSCs or erythroid progenitor cells to patients in need of reducing or eliminating the need for regular blood transfusions.

[0006] In one aspect, the present disclosure provides a method for preparing a cell population of hematopoietic lineage. The method includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population, differentiated into embryoid bodies (EBs) and enriching for CD34+ cells, thereby preparing a CD34+-enriched population. Endothelial-hematopoietic transition (EHT) is induced in the CD34+-enriched population, thereby preparing a hematopoietic stem cell (HSC) population, optionally followed by further enrichment of CD34+ cells. In some embodiments, the resulting HSC population (or a fraction thereof) can be differentiated into an erythroid hematopoietic lineage (e.g., erythroid progenitor cells). In various embodiments, the hematopoietic lineage is selected from erythrocytes (i.e., red blood cells), progenitor erythroblasts, granulocyte-macrophage progenitors (GMPs), and megakaryocyte-erythroid progenitors (MEPs).

[0007] In various embodiments, the HSCs and erythroid lineage cells derived therefrom are derived from iPSCs that have been gene-edited to be one of: (i) HLA-A-B+C+DP-DR+DQ+, (ii) HLA-A-B+C+DP+DR+DQ-, (iii) HLA-A-B+C+DP-DR+DQ-, (iv) HLA-AB-C+DP-DR+DQ+, (v) HLA-AB-C+DP+DR+DQ-, or (vi) HLA-AB-C+DP-DR+DQ-. In some embodiments, the HSCs and erythroid lineage cells are derived from iPSCs that have been gene-edited to be one of: HLA-A-B+C+DP-DR+DQ+, (ii) HLA-A-B+C+DP+DR+DQ-, (iii) HLA-A-B+C+DP-DR+DQ-, (iv) HLA-AB-C+DP-DR+DQ+, (v) HLA-AB-C+DP+DR+DQ-, or (vi) HLA-AB-C+DP-DR+DQ-. neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0008] In some embodiments, iPSCs of the present disclosure are gene-edited to encode a hyperresponsive EPO receptor. Erythropoiesis is the process for the production of red blood cells. Erythropoietin (EPO) is a key hormone involved in efficient red blood cell production. Erythropoietin receptor (EPOR) is a protein encoded by the EPOR gene in humans. The most well-established function of EPOR is to promote the proliferation and rescue of erythroid (red blood cell) progenitor cells from apoptosis. Beneficial mutations in EPOR increase red blood cell numbers and enable improved oxygen delivery. For example, a truncation mutation that removes only the C-terminal portion of the intracellular EPOR that binds to negative regulatory factors can make EPOR hyperresponsive to EPO.

[0009] These HSC populations or erythroid progenitor cells can be used for more efficient ex vivo red blood cell production, or in other embodiments, to deliver HSCs or erythroid progenitor cells to patients in need of reduced or eliminated regular transfusions. Furthermore, such cells can be genetically edited (as described) to delete certain HLA genes, so that the HSC populations and erythroid progenitor cells can be readily HLA-matched for the recipient. HSCs according to the present disclosure (like bone marrow CD34+ cells) can differentiate into various hematopoietic lineages and restore hematopoietic function in the recipient.

[0010] In some embodiments, iPSC differentiation proceeds until the cells are at least about 10% CD34+, or at least about 20% CD34+, or at least about 25% CD34+, or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced on days 7-14 of iPSC differentiation, such as on days 8, 9, 10, 11, 12, 13, or 14. Induction of EHT in CD34+ cells harvested from differentiated iPSCs can be by any known process. In some embodiments, EHT generates HSCs through endothelial or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification).

[0011] In some embodiments, the method comprises increasing the expression or activity of DNA (cytosine-5-)-methyltransferase 3 beta (Dnmt3b) in PSCs, embryoid bodies, CD34+ enriched cells, ECs, HECs, or HSCs, which may be by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the induction of EHT comprises increasing the expression or activity of dnmt3b in hemogenic endothelial cells. In some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol.

[0012] In various embodiments, CD34+ cells (e.g., floating and / or adherent cells) are harvested from the culture to undergo endothelial-to-hematopoietic transformation and optionally expanded in culture. Hematopoietic stem cells (HSCs), which give rise to erythroid, myeloid, and lymphoid lineages, can be identified and optionally sorted or enriched based on the expression of markers of CD34+ cells and the absence of lineage-specific markers (termed Lin-).

[0013] In some embodiments, the HSC population or a fraction thereof is differentiated into erythrocytes or their progenitors or derivatives. For example, the HSC population (or cells isolated therefrom) is cultured with EPO, IL-3, and SCF, e.g., (and / or other extracellular matrix component(s)), and / or combinations thereof, to produce a population comprising an erythroid progenitor or derivative cell population (e.g., erythrocytes). The HSC population of cells gives rise to a high percentage of burst-forming unit-erythroid (BFU-E) cells and colony-forming unit-erythroid (CFU-E) cells, indicative of induction of erythropoiesis.

[0014] In another aspect, the present invention provides an erythroid lineage population produced by the methods described herein, or a pharmaceutically acceptable composition thereof. In various embodiments, the composition comprises a desired cell population (e.g., erythrocytes) and a pharmaceutically acceptable vehicle. The pharmaceutical composition may comprise at least about 10 erythroid lineage cells per mL. 7 The pharmaceutical composition may be provided in units of about 50 mL to about 500 mL, or about 100 mL to about 500 mL, or about 250 to about 500 mL.

[0015] In some aspects, HSC compositions or erythroid progenitor cells are provided that have a hyperreactive EPOR and can be produced by the methods described herein. In some embodiments, the HSC or erythroid progenitor cells are HLA-A neg , homozygous for both HLA-B and HLA-C, HLA-DPB1 neg , and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1. The cell compositions of the present disclosure may further comprise a pharmaceutically acceptable carrier or vehicle suitable for intravenous infusion or other administration routes, and the compositions may include a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). The cell compositions may be provided in unit vials or bags and stored frozen until use.

[0016] Cells produced according to the present disclosure can be administered or used in therapy for, for example, inherited or acquired red blood cell disorders, bone marrow failure disorders, high altitude-associated physiological and pathological conditions, anemia (e.g., sickle cell anemia), red blood cell enzyme deficiencies (e.g., G6PD), red blood cell membrane disorders (e.g., hereditary spherocytosis), hemoglobinopathies (e.g., sickle cell disease and thalassemia), hemolytic anemia, nutritional anemia (e.g., iron deficiency anemia and folate deficiency), disorders of heme production (e.g., sideroblastic anemia), hemochromatosis, conditions associated with chemical or radiation exposure, and / or for the treatment of subjects undergoing HSC transplantation. In further embodiments, red blood cells prepared according to the present disclosure are provided as pharmaceutically acceptable compositions that deliver or encapsulate drugs (including, but not limited to, enzymes), oxygen carriers, or other suitable substances for treating human diseases or physiological or pathological conditions.

[0017] In certain embodiments, the present disclosure provides compositions and methods for treating anemia. In embodiments, the present disclosure provides hematopoietic stem cell (HSC) or erythroid progenitor cell compositions that provide durable and potent cellular therapy for anemia. In embodiments, the HSCs are overloaded with a shortened and / or mutated EPOR, resulting in a hyperresponsive EPOR. In embodiments, the compositions and methods of the present disclosure can provide a patient with sufficient red blood cells to supply healthy oxygenation levels.

[0018] In various embodiments, the present disclosure provides a method of treating a subject in need of red blood cell production, comprising administering to the subject an HSC composition or erythroid progenitor cell composition of the present disclosure. Thus, HSCs produced according to the present disclosure can be used to produce red blood cells in vivo in a durable and potent manner. In some embodiments, the recipient subject has anemia. In some embodiments, the recipient subject has sickle cell anemia, aplastic anemia, anemia associated with bone marrow disease or failure, anemia from blood loss, or hemolytic anemia. In some embodiments, the recipient subject has Fanconi anemia. In some embodiments, the subject has thalassemia. In some embodiments, the HSCs or erythroid progenitor cells are used to prepare a blood product for treating a co-morbidity associated with blood, bone marrow, immune, metabolic, or mitochondrial disorders.

[0019] Other aspects and embodiments of the present disclosure will be apparent from the following detailed disclosure and examples. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows that ETV2 overexpression (OE) does not affect pluripotency. Figure 1 shows a FACS plot showing the transduction efficiency of iPSCs using an adenoviral vector to overexpress ETV2 and GFP sequences. ETV2 overexpression does not affect iPSC stemness, as indicated by the expression of the TRA-1-60 stemness marker. [Figure 2] ETV2 overexpression (OE) increases the yield of hemogenic endothelial cells. Representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (described as CD235a-CD34+CD31+) demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells. [Figure 3] Figure 1 shows that ETV2 overexpression (OE) enhances CD34+ cell formation during iPSC differentiation. Representative flow cytometry analysis and relative quantification of CD34+ cells demonstrates that ETV2-OE enhances CD34+ cell formation. [Figure 4A] Figure 1 shows that iPSC-derived HSCs derived by EHT of CD34+ cells (using Piezo1 activation in this example) undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Figure 2 shows FACS plots of the differentiation efficiency of bone marrow (BM)-HSCs to CD34+CD7+ pre-T cells and iPSC-HSCs derived by EHT of 34+ cells (using Piezo1 activation in this example). [Figure 4B] Figure 1 shows that iPSC-derived HSCs derived from EHT of CD34+ cells (in this example, using Piezo1 activation) undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Quantification (%) of CD34+CD7+ cells derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation). Averages of three experiments are shown. [Figure 5A] Figure 1 shows that iPSC-derived HSCs generated in EHT (using Piezo1 activation in this example) can undergo T cell differentiation and be activated with CD3 / CD28 beads, similar to BM-HSCs. Figure 2 shows FACS plots of the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs (generated using Piezo1 activation in this example). [Figure 5B] We show that iPSC-derived HSCs generated in EHT (using Piezo1 activation in this example) can undergo T cell differentiation and be activated with CD3 / CD28 beads, similar to BM-HSCs. Quantification (%) of CD3+CD69+ cells derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation). Averages of three experiments are shown. [Figure 6]We show that iPSC-derived HSCs generated in EHT of CD34+ cells (using Piezo1 activation in this example) can differentiate into functional T cells. IFNγ expression is a consequence of T cell activation after T cell receptor (TCR) stimulation via CD3 / CD28 beads. IFNγ expression in T cells differentiated from iPSC-derived HSCs (including EHT of D8 34+ cells using Piezo1 activation) enhances the HSC capacity to further differentiate into functional hematopoietic lineages (T cells in this example). Averages of three experiments are shown. [Figure 7A]

[0023] Figure 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence. Global expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface is shown, with HLA-edited cells being positive for global HLA class I expression to a similar extent as wild-type cells (gHSCs). [Figure 7B] Figure 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence. Cellular expression of HLA-A via immunofluorescence is shown, where HLA-A is not expressed in HLA-edited clones. [Figure 8] We show that the HLA-edited clones retain their pluripotency (maintain tri-lineage differentiation) as illustrated by immunofluorescence, where ectodermal differentiation is indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation is indicated by GATA-488 staining, and endodermal differentiation is indicated by CXCR4-488 and FOX2A-594 staining. [Figure 9] Immunocompatibility of HLA-edited HSCs: HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were cocultured with peripheral blood mononuclear cells (PBMCs) bearing HLA-B and HLA-C matched but mismatched HLA-A, and PBMC-mediated cytotoxicity was measured by Annexin V staining assay. [Figure 10]Figure 1 shows the in vivo engraftment potential of HLA-edited HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed for competitive transplantation into mice, and bone marrow (BM) and peripheral blood samples were assessed by FACS to compare the relative amounts of each cell type present in the samples. [Figure 11A] Figure 1 shows that HLA-A deletion does not affect class I peptide presentation. Schematic diagram of immunopeptidome analysis. [Figure 11B] Figure 1 shows that HLA-A deletion does not affect class I peptide presentation. Figure 1 shows the results of immunopeptidome analysis, revealing little difference in the number of peptides and representative proteins presented by class I molecules in WT and HLA-edited cells. [Figure 12A] Figure 1 shows that deletion of HLA-DP and DQ does not affect class II peptide presentation. Figure 2 shows the immunopeptidome analysis scheme. [Figure 12B] Figure 1 shows that deletion of HLA-DP and DQ does not affect class II peptide presentation. Figure 2 shows that despite deletion of HLA-DP and DQ, cells retain the ability to present a wide range of peptides through HLA class II. [Figure 13] 1A-1D are schematic diagrams of in vivo tests of antigen-mediated immune responses: delayed-type hypersensitivity assay (DTH), sensitization phase, and elimination phase, respectively. [Figure 14A] This shows that HLA-edited HSCs reconstitute a functional immune system, as evidenced by DTH responses in immunodeficient mice. A delayed-type hypersensitivity assay, an assay involving crosstalk between different types of immune cells, was performed on transplanted mice. Mice were sensitized by subcutaneous injection of sheep red blood cells (antigen). A functional immune system results in swelling of the left paw, as measured with a microcaliper. As can be seen in Figure 14A, non-transplanted mice, due to their immunodeficiency, did not exhibit swelling of the left paw. Conversely, mice transplanted with umbilical cord blood CD34+ cells exhibited tissue swelling, doubling the diameter of their left paw. [Figure 14B]14A-B are graphical evaluations of the results shown in Figure 14A, showing that HLA-edited HSCs reconstitute a functional immune system as demonstrated by DTH responses in immunodeficient mice. [Figure 15] Figure 15 shows the differentiation potential of HSCs into T cell subtypes. After a 35-day differentiation period, pre-T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. Figure 15 compares the differentiation potential of bone marrow-derived CD34+ cells, embryoid body CD34+ cells, and HSCs prepared according to the present disclosure (e.g., using Piezo1 activation) ("gHSCs"). [Figure 16]

[0023] Figure 1 shows the degree of T cell-mediated cytotoxicity measured from co-culture of HSC-derived T cells with CD19+ lymphoma cells in the presence of an anti-CD3 / CD-19 bispecific antibody. T cells prepared from HSCs according to the present disclosure ("gHSCs") exhibit high levels of cytotoxicity against target cells. [Figure 17] The ability of HSCs to evolve into pre-T cells as measured by their CD34-CD7+ markers is shown. [Figure 18A] 10 demonstrates increased expression of T cell-specific transcription factors and thymic engraftment molecules using pre-T cells derived from HSCs according to the present disclosure. TCF7 mRNA expression is shown. [Figure 18B] 10 demonstrates increased expression of T cell-specific transcription factors and thymic engraftment molecules using pre-T cells derived from HSCs according to the present disclosure. CCR7 mRNA expression is shown. [Figure 19A] 1 shows that HSC-derived pre-T cells engraft and differentiate in the thymus. Engraftment and analysis procedures are illustrated. [Figure 19B] Figure 1 shows that HSC-derived pre-T cells engraft and differentiate in the thymus. Figure 2 shows FACS analysis of the CD3 cell population of cells gated on the CD45+ cell population, demonstrating the superior engraftment and differentiation potential of HSC-derived pre-T cells in the thymus. [Figure 20]Figure 1 shows that HSC-derived T cells can be activated in vitro. The top panel shows FACS analysis of activated T cells from different sources, including from HSCs prepared according to the present disclosure. T cells of the present disclosure show comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, where activated T cells express inflammatory cytokines. HSC-derived T cells express higher levels of inflammatory cytokines, as exemplified by TNF-α and interferon gamma expression levels. [Figure 21A] We show that WT and HLA-edited HSCs can differentiate into the monocyte / macrophage lineage, which also preserves the overall expression of both class I and class II molecules, as identified by the CD11b+CD14+ marker. [Figure 21B] Analysis of HLA-I and HLA-II on cells gated on CD11b+CD14+ is shown. [Figure 22A] 1 shows that deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules. 2 is a schematic diagram showing differentiation of HLA-edited iPSCs into macrophages. [Figure 22B] 1 shows that deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules. Immunofluorescence experiments confirm the specific deletion of DPB1 and DQB1 molecules. [Figure 22C] Figure 1 shows that deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules, and that the same cells preserve class II DRB1 expression. [Figure 23] This shows that HLA-edited HSCs can differentiate into megakaryocytes (MKs), which can further differentiate into platelets. The image on the left shows the increased percentage of platelets among HSCs by light microscopy at 1000x magnification. The graph on the right shows a statistically significant increase in the percentage of platelets differentiated from HLA-edited HSCs compared to BM CD34+ and iPSC-34+ cell populations. DETAILED DESCRIPTION OF THE INVENTION

[0021] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0022] The terms "wild-type" (WT), "unedited," and "non-HLA edited" are used interchangeably herein to refer to non-gene-edited cells of the present disclosure.

[0023] EB34+ cells refer to embryonic body-derived CD34+ cells, which contain hemogenic endothelial cells.

[0024] The present disclosure, in various aspects and embodiments, provides methods for generating hematopoietic lineages for cell therapy, including gene-edited hematopoietic stem cells (HSCs), erythroid progenitors, progenitor erythroblasts, granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), and erythroid cells. In various embodiments, the present invention provides an efficient ex vivo process for developing such hematopoietic lineages, including, but not limited to, progenitor erythroid and erythroid cell lineages, from human induced pluripotent stem cells (iPSCs). In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or bone marrow. The present invention also provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

[0025] In other aspects and embodiments, the present disclosure provides HSCs derived from iPSCs that have been gene-edited to encode a hyperresponsive EPO receptor. These HSC populations can be used for more efficient ex vivo red blood cell production, or in other aspects, to deliver HSCs or erythroid progenitor cells to patients in need of reducing or eliminating the need for regular blood transfusions.

[0026] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to produce essentially unlimited pluripotent stem cells (PSCs) is exploited to generate an unlimited supply of hematopoietic cells, including, but not limited to, therapeutic human erythrocytes or red blood cells ("RBCs") or their erythroid progenitors. The use of RBCs for therapeutic purposes is severely limited by their limited availability, cell number, and limited proliferation capacity. Furthermore, compared to primary cells, hiPSCs are more easily subject to genetic modification in vitro, thereby offering opportunities for improved cell targeting specificity, cell number, and avoiding, for example, HLA matching issues. In addition, fully engineered hiPSC clones can serve as a stable and safe source compared to primary cells (Nianias and Themeli, 2019). Furthermore, unlike human embryonic stem cells (hESCs), hiPSCs are of non-embryonic origin, eliminating ethical concerns. Thus, the use of hiPSCs according to the present disclosure offers several advantages over primary cells for generating therapeutic hematopoietic lineages, such as erythroid lineages.

[0027] In one aspect, the present disclosure provides a method for preparing a cell population of hematopoietic lineage. The method includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population differentiated into embryoid bodies, and enriching for CD34+ cells, thereby preparing a CD34+-enriched population. Endothelial-hematopoietic transition (EHT) is induced in the CD34+-enriched population, thereby preparing a hematopoietic stem cell (HSC) population, optionally followed by further enrichment of CD34+ cells. In some embodiments, the resulting HSC population (or a fraction thereof) can be differentiated into an erythroid hematopoietic lineage. In various embodiments, the hematopoietic lineage is selected from erythrocytes (i.e., red blood cells), progenitor erythroblasts, granulocyte-macrophage progenitors (GMPs), and megakaryocytic erythroid progenitors (MEPs).

[0028] Traditionally, hematopoietic lineages are prepared by differentiating iPSCs into embryoid bodies up to day 8 and harvesting CD34+ cells. CD34 is commonly used as a marker for hemogenic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been discovered that inducing endothelial-hematopoietic transition (EHT) of CD34+ cell populations, which may be derived from iPSC embryoid bodies, can be used for the ex vivo generation of hematopoietic lineages, including potent hematopoietic stem cells and erythroid lineages.

[0029] In some embodiments, the present disclosure provides methods for generating an erythroid population (e.g., early erythroid progenitors, late erythroid progenitors, and morphologically recognizable erythroid precursors), or derivatives of this population. For example, the methods include generating a hematopoietic stem cell (HSC) population comprising human long-term hematopoietic stem cells (LT-HSCs) from iPSCs (e.g., hiPSCs). The HSC population is derived by inducing endothelial-to-hematopoietic transformation of CD34+ cells (e.g., CD34+ cells derived from embryoid bodies). The HSC population (or cells isolated therefrom) is cultured with, for example, EPO, IL-3, and SCF (and / or other extracellular matrix component(s)), and / or combinations thereof, to produce a population comprising erythroid progenitor or derivative cell populations (e.g., red blood cells). The HSC population of cells gives rise to a high percentage of burst-forming unit-erythroid (BFU-E) cells and colony-forming unit-erythroid (CFU-E) cells, indicative of induction of erythropoiesis.

[0030] Aspects and embodiments of the present disclosure can be utilized to generate reticulocytes and / or enucleated mature red blood cells from cells differentiated from or derived from hematopoietic stem cell (HSC) populations, including human long-term hematopoietic stem cells (LT-HSCs) derived from iPSCs (e.g., hiPSCs). For example, iPSC-derived HSCs can be cultured under erythroid differentiation conditions in a bioreactor (e.g., a stirred bioreactor) without gas sparging, using mechanical agitation (impeller speed of approximately 300-450 rpm) under optimal physical conditions: a pH range of approximately 7.0 to approximately 7.5, and approximately 25% to approximately 75% oxygen (e.g., approximately 50% oxygen), resulting in enucleated cells of approximately 80% purity. Supplementary compounds, such as cytokines and growth factors, can be added to the erythroid differentiation conditions to enhance the yield of enucleated cells.

[0031] In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cells" or "iPSCs" refers to cells derived from somatic cells, such as skin or blood cells, that have been reprogrammed back to an embryonic-like pluripotent state. In some embodiments, iPSCs are generated from somatic cells, such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from lymphocytes (e.g., T cells, B cells, NK cells, etc.), umbilical cord blood cells, PBMCs, CD34+ cells, or other primary human tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood, bone marrow, or umbilical cord blood. In various embodiments, iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to the recipient (the subject in need of the treatment described herein). In various embodiments, iPSCs can be genetically edited to support HLA matching (such as deletion of one or more HLA class I and / or HLA class II alleles or their master regulators, including, but not limited to, beta-2-microglobulin (B2M), CIITA, etc.), or to delete or express other functions. For example, iPSCs can be genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C, and one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, iPSCs are derived from cord blood CD34+ cells or CD36+ erythroblasts. iPSCs can be derived from CD34+ cells that form universal donor red blood cells (i.e., type O). Other blood types can also be used.

[0032] In various embodiments, the HSCs and erythroid lineage cells derived therefrom are derived from iPSCs that have been gene-edited to be one of: (i) HLA-A-B+C+DP-DR+DQ+, (ii) HLA-A-B+C+DP+DR+DQ-, (iii) HLA-A-B+C+DP-DR+DQ-, (iv) HLA-AB-C+DP-DR+DQ+, (v) HLA-AB-C+DP+DR+DQ-, or (vi) HLA-AB-C+DP-DR+DQ-. For retained HLA (e.g., HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or can retain only a single copy of the gene. For example, the modified cells are identified as at least (a) HLA-C+ and HLA-DR+, and optionally as one or more of (b) HLA-B-, (c) HLA-DP-, and (d) HLA-DQ-. In an exemplary embodiment, the modified cells are HLA-B+, HLA-DP-, and HLA-DQ-.

[0033] In some embodiments, HSCs and erythroid lineage cells derived from iPSCs are HLA-A neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0034] As used herein, the term "neg," (-), or "negative" with respect to a particular HLA class I or HLA class II molecule indicates that both copies of the gene have been disrupted in a cell line or population, and therefore the cell line or population does not exhibit significant functional expression of the gene. Such cells can be generated by complete or partial gene deletion or disruption, or alternatively by other techniques such as siRNA. As used herein, the term "deletion" in the context of genetic modification (i.e., gene editing) of a targeted gene refers to the elimination of functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletion or disruption of a coding sequence or deletion of a critical cis-acting expression control sequence.

[0035] In some embodiments, iPSCs are gene-edited using gRNAs that are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more nucleotides in length. In some embodiments, the gRNAs comprise modifications at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and / or at or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end). In some embodiments, the modified gRNAs exhibit increased resistance to nucleases. In some embodiments, the gRNAs comprise two separate RNA molecules (i.e., "duplex gRNAs"). Duplex gRNAs comprise two separate RNA molecules, "crispr RNA" (or "crRNA") and "tracr RNA," and are well known to those of skill in the art.

[0036] Generally, various gene editing technologies are known that can be applied in accordance with various embodiments of the present disclosure, including, but not limited to, zinc finger (ZF), transcription activator-like effector (TALE), etc. Fusion proteins comprising one or more of these DNA binding domains and the cleavage domain of a Fokl endonuclease can be used to generate double-stranded breaks in desired regions of DNA within a cell (see, e.g., U.S. Patent Application Publication No. 2012 / 0064620, U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. 2011 / 0301073, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the entire contents of which are incorporated herein by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system (e.g., CRISPR-Cas9) known in the art. See, for example, US8,697,359, US8,906,616, and US8,999,641, each of which is incorporated herein by reference in its entirety. In various embodiments, gene editing employs a type II Cas endonuclease (such as Cas9) or a type V Cas endonuclease (such as Cas12a). Type II and type V Cas endonucleases are guide RNA dependent. Design of gRNAs to guide desired gene editing (while limiting or avoiding off-target editing) is known in the art. See, for example, Mohr SE, et al., CRISPR guide RNA design for research applications, FEBSJ. 2016 Sep; 283(17):3232-3238.In still other embodiments, non-canonical Type II or Type V Cas endonucleases with homology (albeit low primary sequence homology) to S. pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) can be employed. Many such non-canonical Cas endonucleases are known in the art. Nidhi S, et al. Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives, Int J Mol Sci. 2021 Apr;22(7):3327. In still other embodiments, gene editing employs base editing or prime editing to incorporate mutations without creating double-strand breaks. See, for example, Antoniou P, et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA, Front. Genet., 9 June 2020. Various other gene editing processes are known, including the use of dead Cas (dCas) systems (e.g., Cas fusion proteins) to target DNA-modifying enzymes to desired targets using dCas as a guide RNA-dependent system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications, Int J Mol Sci. 2019 Dec;20(23):6041.

[0037] Base editors that can introduce precise genome modifications without generating double-stranded DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., designing gene therapy vectors). Base editors inherently cannot create DSBs and contain a catalytically inactive nuclease, such as Cas9 nickase (nCas9), fused to a nucleic acid base deaminase enzyme, possibly a DNA glycosylase inhibitor. Currently, there are two major categories of base editors: cytidine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G transversions. Base editors can be delivered, for example, via HDAd5 / 35++ vectors, to efficiently edit promoters and enhancers to activate or inactivate genes. Exemplary methods are described in U.S. Patent Nos. 9,840,699, 10,167,457, 10,113,163, 11,306,324, 11,268,082, 11,319,532, and 11,155,803. Also contemplated are prime editors comprising a reverse transcriptase conjugated to (e.g., fused with) a Cas endonuclease and a polynucleotide useful as a DNA synthesis template conjugated to (e.g., fused with) a guide RNA, as described in WO2020 / 191153.

[0038] Exemplary vectors that can be used for genome editing applications include plasmids, retroviral vectors, lentiviral vectors, adenoviral vectors (e.g., Ad5 / 35, Ad5, Ad26, Ad34, Ad35, Ad48), parvoviruses (e.g., adeno-associated virus (AAV) vectors), herpes simplex virus vectors, baculovirus vectors, negative-stranded RNA viruses such as coronaviruses, orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses. Examples of vectors that can be used include, but are not limited to, double-stranded DNA viruses, including canarypox, vaccinia, or modified vaccinia viruses. Vectors containing a nucleic acid molecule of interest can be delivered to cells (e.g., iPS cells, endothelial cells, hemogenic endothelial cells, HSCs (ST-HSCs or LT-HSCs)) via any method known in the art, including, but not limited to, transduction, transfection, infection, and electroporation. Any of these vectors may contain a transposable element (such as a piggyBac transposon or a Sleeping Beauty transposon). Transposons insert specific sequences of DNA into the vertebrate genome. A gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar excision site present in the cell's nuclear genome.

[0039] For increased efficiency, in some embodiments, Cas and gRNA can be combined before delivery into cells. The Cas-gRNA complex is known as a ribonucleoprotein (RNP). Several methods have been developed for direct delivery of RNP into cells. For example, RNP can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nucleofection protocol can be employed, which allows RNP to rapidly enter the cell's nucleus and immediately begin cleaving the genome. See, for example, Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics. 2021 Jan 1;11(2):614-648, incorporated herein by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNP.

[0040] Generally, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage and is typically found 3-4 nucleotides downstream of the cleavage site. The PAM is a short DNA sequence (usually 2-6 base pairs in length) following the DNA region targeted for cleavage by a CRISPR system such as CRISPR-Cas9. In some embodiments, the PAM sequence, sgRNA, or base editing tool targeting a haplotype or polymorphism at an HLA locus does not contain four Gs, four Cs, a GC repeat, or a combination thereof.

[0041] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed by using the gRNAs described herein to design a single gRNA that targets each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example). To perform gene knockout, the gRNA targets the Cas9 protein to the appropriate site for editing. The Cas9 protein can then perform a double-strand break (DSB), which repairs the DNA through a non-homologous end joining (NHEJ) mechanism, generating an indel that results in a frameshift mutation and terminates the function of the resulting protein. However, off-target gene recombination can occur, altering the function of an otherwise intact gene. For example, the Cas9 endonuclease can generate DSBs at undesired off-target locations, even in the presence of some degree of mismatch. This off-target activity can result in genomic instability events such as point mutations and genomic structural perturbations. In various embodiments, an sgRNA targeting HLA-A can target a region of chromosome 6 defined as 29942532-29942626. In various embodiments, an sgRNA targeting HLA-DQB1 can target a region of chromosome 6 defined as 32665067-32664798. In various embodiments, an sgRNA targeting HLA-DPB1 can target a region of chromosome 6 defined as 33080672-33080935.

[0042] gRNAs can be used to develop clonal iPSCs. Such iPSC lines can be assessed for (i) on-target editing, (ii) off-target editing, and (iii) translocation editing, for example, using sequencing as described herein. Specifically, such assays can be performed by multiplex PCR using primers designed to target and enrich for the region of interest, followed by next-generation sequencing (e.g., Amplicon sequencing, AMP-seq). On-target and translocation panels can amplify the intended edited region and allow for the selection of iPSC clones with the expected edits that do not contain chromosomal translocations resulting from unintended DSB cleavage site fusions. Off-target panels can enrich for any potential off-target regions identified through sequencing, allowing for the selection of iPSC clones with negligible off-target mutations. Together, these assays enable the screening of iPSC clones to select clones with the desired edits while ruling out potential CRISPR / Cas9-related genome integrity issues.

[0043] In some embodiments, to further ensure the genomic stability and integrity of the reprogrammed and edited iPSCs, genetic and genomic assays can be performed to select clones that have not undergone translocation and mutation events and have not integrated the episomal vector. For example, whole genome sequencing (WGS) can be performed on CD34+ cells and iPSC clones after reprogramming, and the genomes can be compared for differences resulting from editing. These analyses provide an assessment of which iPSC clone genomes differ from the CD34+ starting material, allowing for the informed selection of iPSC clones that have not undergone mutations during reprogramming.

[0044] In some embodiments, karyotyping using a system such as the KARYOSTAT assay is used to select iPSC clones that did not develop indels or translocations during reprogramming, as described, for example, in Ramme AP, et al., "Supporting dataset of two integration-free induced pluripotent stem cell lines from related human donors," Data Brief. 2021 May 15;37:107140, incorporated herein by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal abnormalities with a resolution similar to G-banding karyotyping. The size of structural abnormalities that can be detected is >2 Mb for chromosome gains and >1 Mb for chromosome losses. The KARYOSTAT array is functionalized for balanced whole-genome coverage with low-resolution DNA copy number analysis, and the array covers all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay allows for the detection of aneuploidy, submicroscopic abnormalities, and mosaic events.

[0045] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that have not developed copy number aberrations (CNAs) during reprogramming, as described, for example, in Wiesner et al. "Molecular Techniques," Editor(s): Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, "Pathology of Melanocytic Tumors," Elsevier, 2019, pp. 364-373, ISBN 9780323374576, and Hussein SM, et al. "Copy number variation and selection during reprogramming to pluripotency," Nature. 2011 Mar 3;471(7336):58-62, which are incorporated herein by reference in their entireties. aCGH is a technique that analyzes the entire genome for CNAs by comparing sample DNA with reference DNA.

[0046] In some embodiments, a targeted hematologic malignancy NGS panel analysis is used to select iPSC clones that did not develop hematologic malignancy mutations during reprogramming. For example, a targeted hematologic malignancy NGS panel focuses on genes associated with myeloid leukemia, lymphoma, and / or other hematologic malignancies, generating smaller, more manageable datasets than more broad methods. A targeted hematologic malignancy NGS panel analysis involves the use of highly multiplexed PCR to amplify regions associated with hematologic malignancies, followed by next-generation sequencing.

[0047] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that have not integrated the episomal vector and have been passaged sufficiently for episomal vector clearance. As discussed herein, iPSC reprogramming of CD34+ cells can be achieved by delivering episomal vectors encoding reprogramming factors. However, episomal vectors can randomly integrate into the cellular genome, although this is rare, which can disrupt developmental processes, homeostasis, and the like. Therefore, ddPCR can be used to detect residual episomal vectors in iPSC cultures, allowing for the selection of iPSC clones that have not integrated the episomal vector.

[0048] In some embodiments, after assessing that selected clones do not contain editing-related genomic abnormalities, the clones can be further tested for spontaneous mutations that may arise during expansion, such as mutations affecting hematologic malignancies, indels, translocations, and numerical abnormalities, as described for pre-edited reprogrammed clones. Analysis of spontaneous mutations can include whole genome sequencing (WGS), KARYOSTAT analysis, array comparative genomic hybridization (aCGH) analysis, targeted hematologic malignancies NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0049] Somatic cells can be reprogrammed by expression of reprogramming factors selected from Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, and klf4. In some embodiments, the reprogramming factors include Oct-4, Sox-2, Klf-4, 1-Myc, Lin-28, SV40 large T antigen ("SV40LT"), and short hairpin RNA targeting p53 ("shRNA-p53"). Methods for preparing iPSCs are described, for example, in U.S. Pat. Nos. 10,676,165, 9,580,689, 10,221,395, and 9,376,664, which are incorporated herein by reference in their entireties. In various embodiments, reprogramming factors are expressed using well-known viral vector systems, such as lentivirus, Sendai, or measles virus systems. Alternatively, reprogramming factors can be expressed by introducing mRNA(s) encoding the reprogramming factors into somatic cells. Furthermore, iPSCs can be generated by introducing non-integrating episomal plasmids expressing the reprogramming factors, i.e., for the generation of transgene-free and virus-free iPSCs. Known episomal plasmids, which have limited replication capacity and are therefore lost over several cell generations, can be employed. In some embodiments, the method comprises reprogramming CD36+ basophilic erythroblasts to pluripotency using a vector (e.g., viral or episomal) expressing POU5F1 / OCT4, SOX2, KLF4, and c-MYC as a polycistronic unit. In some embodiments, the method comprises reprogramming PBMCs with Oct4, Sox2, Lin28, Klf4, and L-myc.

[0050] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are gene-edited, which may include, but is not limited to, HLA gene modifications (e.g., deletion of one or more HLA class I and / or HLA class II genes), deletion of β2 microglobulin (β2M), and deletion of CIITA.

[0051] In some embodiments, iPSCs of the present disclosure are gene-edited to encode a hyperresponsive EPO receptor. Erythropoiesis is the process for the production of red blood cells. Erythropoietin (EPO) is a key hormone involved in efficient red blood cell production. The erythropoietin receptor (EPOR) is a protein encoded by the EPOR gene in humans. EPOR is a 52 kDa peptide with a single carbohydrate chain, resulting in a protein of approximately 56-57 kDa found on the surface of EPO-responsive cells. The most well-established function of EPOR is to promote proliferation and rescue from apoptosis of erythroid (red blood cell) progenitor cells.

[0052] Beneficial mutations in EPOR have been reported, where increased red blood cell counts allow for improved oxygen delivery in competitive endurance events without any apparent adverse effects on the athlete's health. de La Chapelle et al., PNAS 1993;90.10:4495-4499. Truncating mutations that remove only the C-terminal portion of the intracellular EPOR that binds to negative regulatory factors have generally been reported to be associated with primary polycythemia with low EPO and high hemoglobin levels. These mutations render EPOR hyperresponsive to EPO, with the secondary effect of increasing hemoglobin levels. See Juvonen, E., Ikkala, E., Fyhrquist, F. & Ruutu, T. Autosomal dominant erythrocytosis caused by increased sensitivity to erythropoietin. Blood 1991;78,3066-3069. SHP-1 is known to play an important role in EPOR signal transduction by associating with the receptor via its SH2 domain and dephosphorylating key substrates. Jiao, H., Berrada, K., Yang, W., Tabrizi, M., Platanias, L.C., & Yi, T. Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1. Molecular and Cellular Biology, 1996;16(12),6985-6992.

[0053] Thus, in various embodiments, the HSC populations of the present disclosure express EPOR with a truncating mutation. In some embodiments, the HSC populations of the present disclosure express EPOR that lacks or has a mutant SHP-1 inhibitory domain. In some embodiments, the HSC populations of the present disclosure express EPOR with one or more missense or frameshift mutations that result in hyperresponsiveness, optionally due to mutation or deletion of the SHP-1 inhibitory domain.

[0054] These HSC populations can be used for more efficient ex vivo red blood cell production, or in other embodiments, to deliver HSCs or erythroid progenitor cells to patients in need of reduced or eliminated regular transfusions. Furthermore, such cells can be genetically edited to delete certain HLA genes (as described), so that the HSC populations and erythroid progenitor cells can be readily HLA-matched for the recipient. HSCs according to the present disclosure (like bone marrow CD34+ cells) can differentiate into various hematopoietic lineages and restore hematopoietic function in the recipient.

[0055] In various embodiments, iPSCs are prepared and expanded using conventional culture systems. The expanded iPSCs can be harvested from the culture to generate embryoid bodies (EBs). EBs generated by differentiation of iPSCs are three-dimensional aggregates of iPSCs and contain three (or alternatively, two or one) embryonic germ cell layers based on the differentiation method(s). Preparation of EBs is described, for example, in US 2019 / 0177695, which is incorporated herein by reference in its entirety. In some embodiments, EBs prepared by differentiation of iPSCs are expanded in a bioreactor, as described, for example, in Abecasis B. et al., "Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches." J. of Biotechnol. 246 (2017) 81-93. EBs can be used to generate any desired cell type. Other methods involving 3D suspension culture for the growth or differentiation of EBs are described in WO2020 / 086889, which is incorporated herein by reference in its entirety.

[0056] In some embodiments, processes according to each aspect can include generating CD34+ enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial-hematopoietic transformation. HSCs, including relatively high frequencies of LT-HSCs, can be generated from cell populations using a variety of stimuli or factors, including mechanical, biochemical, metabolic, and / or local stimuli, as well as factors such as extracellular matrix, niche factors, cell-extrinsic factors, induction of cell-intrinsic properties, and including pharmacological and / or genetic means.

[0057] In some embodiments, the method includes preparing hematopoietic endothelial cells from pluripotent stem cells prior to inducing EHT. In some embodiments, the method includes overexpressing the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSCs. ETV2 can be expressed by introducing an encoding non-integrating episomal plasmid for constitutive or inducible expression of ETV2 and for the production of transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. The mRNA can be introduced using any available method, including electroporation or lipofection. Differentiation of ETV2-expressing cells can include the addition of VEGF-A. See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA. Sci. Adv. Vol. 6 (2020). Cells generated in this manner can be used to produce CD34+ cells and induce EHT according to embodiments of the present disclosure.

[0058] After CD34+ enrichment, HSCs are then generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification.

[0059] In some embodiments, iPSC differentiation proceeds until the cells are at least about 10% CD34+, or at least about 20% CD34+, or at least about 25% CD34+, or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced on days 7-14 of iPSC differentiation, such as on days 8, 9, 10, 11, 12, 13, or 14. In some embodiments, CD34+ cells are harvested on about day 8. Differentiation of iPSCs can be by known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, a combination of bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1. In some embodiments, hPSCs are differentiated using feeder-free, serum-free, and / or GMP-compatible materials, such as pomalidomide or Revlimid. In some embodiments, hPSCs are co-cultured with mouse bone marrow-derived feeder cells, such as the OP9 or MS5 cell line, in serum-containing medium. The culture can contain growth factors and cytokines to support differentiation into embryoid bodies or monolayers. The OP9 co-culture system can be used to generate multipotent HSPCs that can further differentiate into several hematopoietic lineages, including T lymphocytes, B lymphocytes, megakaryocytes, monocytes or macrophages, and erythrocytes. See Netsrithong R. et al., Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells, Stem Cell Research & Therapy Vol. 11 Art. 481 (2020). Alternatively, a stepwise process using defined conditions in conjunction with specific signals can be used. For example, the expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs favors direct differentiation into multipotent CD34+ / CD45+ progenitors.Furthermore, expression of factors such as HOXB4, CDX4, SCL / TAL1, or RUNX1a supports the hematopoietic program in human PSCs. See Doulatov S. et al., Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors, Cell Stem Cell. 2013 Oct 3;13(4).

[0060] Induction of EHT can be by any known process. In some embodiments, induction of EHT generates a hematopoietic stem cell (HSC) population including LT-HSC. In some embodiments, EHT generates HSCs through endothelial or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population including one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), and hematopoietic stem progenitor cells.

[0061] In some embodiments, the method comprises increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34+ enriched cells, ECs, HECs, or HSCs, which may be by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the method comprises increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in the cells. See WO2019 / 236943 and WO2021 / 119061, which are incorporated by reference in their entireties. In some embodiments, inducing EHT comprises increasing the expression or activity of dnmt3b.

[0062] In some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. An exemplary Piezol agonist is Yoda1. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Yodal (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine) is a small molecule agonist developed for the mechanosensitive ion channel Piezol. Syeda R, Chemical activation of the mechanotransduction channel Piezol. eLife (2015). Yoda1 has the following structure: [ka]

[0063] Derivatives of Yodal can be employed in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are employed in some embodiments. Exemplary agonists are disclosed in Evans EL, et al., "Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation," British Journal of Pharmacology 175(1744-1759):2018. Still other Piezo1 agonists include Jedi1, Jedi2, single-stranded (ss) RNA (e.g., ssRNA40), and derivatives and analogs thereof. See Wang Y., et al., A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel Nature Communications (2018) 9:1300; Sugisawa, et al., RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis, Cell, Volume 182, Issue 3, 2020, Pages 609-624, which are incorporated herein by reference in their entireties. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of the Piezo1 agonist or derivative is within the range of about 1 μM to about 500 μM, or about 5 μM to about 200 μM, or about 5 μM to about 100 μM, or in some embodiments, about 25 μM to about 150 μM, or about 25 μM to about 100 μM, or about 25 μM to about 50 μM.

[0064] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34-enriched cells). In certain embodiments, pharmacological Piezo1 activation may further be applied to iPSCs, embryoid bodies, ECs, hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineage(s). In certain embodiments, Piezo1 activation is applied to at least one or more of iPSCs, EBs generated from iPSCs, or CD34+ cells isolated from EBs, and / or combinations thereof, which, according to various embodiments, allows for superior generation of erythroid progenitor cells compared to other methods for inducing EHT.

[0065] In some embodiments, Piezo1 activation is not used during the induction of EHT.

[0066] In certain embodiments, expanding hematopoietic cells can also include culturing cells in contact with an immunomodulatory compound, e.g., a TNF-α inhibitory compound, for a time and in an amount sufficient to cause a detectable increase in hematopoietic cell proliferation over a given period of time compared to an equivalent number of hematopoietic cells not contacted with the immunomodulatory compound. See, e.g., U.S. Patent No. 7,498,171 (incorporated herein by reference in its entirety). In embodiments, the immunomodulatory compound is 3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione, 3-(4'aminoisolindoline-1'-one)-1-piperidine-2,6-dione, 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione, 4-amino-2-[(3RS)-2,6-dioxopiperidin-3-yl]-2H-isoindole-1,3-dione, α-(3-aminophthalimido)glutarimide, pomalidomide, lenalidomide, or thalidomide.

[0067] Alternatively, or in addition, Dnmt3b activity or expression can be increased directly in cells, for example, in CD34-enriched cells. For example, Dnmt3b mRNA expression can be increased by transgene-free methods, including but not limited to, delivering a transcript encoding Dnmt3b to cells, or by introducing a transgene encoding Dnmt3b, or by introducing a non-integrating episome into cells. In some embodiments, gene editing is employed to introduce genetic modifications into Dnmt3b-expressing elements in cells, such as, but not limited to, increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0068] In some embodiments, the method includes increasing the activity or expression of Gimap6 in a cell, alone or in combination with Dnmt3b and / or other genes that are up- or down-regulated upon cyclic strain or Piezol activation. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cell; a transgene-free approach can be employed, including, but not limited to, introducing an episome into the cell; or, alternatively, a transgene encoding Gimap6 can be introduced. In some embodiments, gene editing is employed to introduce genetic modifications (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or to affect RNA splicing) into a Gimap6-expressing element in the cell.

[0069] In embodiments of the present disclosure employing mRNA delivery to cells, known chemical modifications can be used to circumvent the innate immune response in cells. For example, synthetic RNAs containing only canonical nucleotides can bind to pattern recognition receptors and induce a strong immune response in cells. This response can result in translation block, secretion of inflammatory cytokines, and cell death. RNAs containing certain non-canonical nucleotides can avoid detection by the innate immune system and can be translated into proteins with high efficiency. See US Pat. No. 9,181,319, incorporated herein by reference, particularly for nucleotide modifications to circumvent the innate immune response.

[0070] In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into cells, which can direct the desired level of overexpression (with varying promoter strength or other selection of expression control elements). Various viral vectors or transfection reagents (including lipid nanoparticles) known in the art can be used to introduce the transgene. In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by transgene-free methods (e.g., episomal delivery). In some embodiments, expression or activity of Dnmt3b and / or Gimap6, or other genes disclosed herein, is increased using gene editing techniques, for example, to introduce one or more modifications to increase promoter strength, ribosome binding, or RNA stability.

[0071] In some embodiments, the method includes applying cyclic 2D, 3D, or 4D stretch to cells. In various embodiments, the cells subjected to cyclic 2D, 3D, or 4D stretch are selected from one or more of CD34-enriched cells, iPSCs, ECs, and HECs. For example, the cell population is introduced into a bioreactor that provides cyclic strain biomechanical stretch, as described in WO 2017 / 096215, which is incorporated herein by reference in its entirety. The cyclic strain biomechanical stretch can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, the mechanical means applies a stretching force to the cells or to a cell culture surface having cells (e.g., ECs or HECs) cultured thereon. For example, cyclic 2D, 3D, or 4D stretching can be applied to cells ex vivo under defined and controlled cyclic strain conditions using a computer-controlled vacuum pump system or other means for providing a stretching force (e.g., the FlexCell™ Tension System, CytoStretcher System) attached to a flexible, biocompatible and / or biomimetic surface. For example, the applied cyclic stretching can be about 1% to about 20% cyclic strain (e.g., about 6% cyclic strain) for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours.

[0072] Alternatively, or in addition, EHT is stimulated by Trpv4 activation, which can be by contacting cells (e.g., CD34-enriched cells, ECs, or HECs) with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0073] When a cell population is described herein as having a particular phenotype, it is understood that the phenotype represents a substantial portion of the cell population, such as at least 25%, at least 40%, or at least about 50%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90% of the cell population. Furthermore, in various steps, the cell population can be enriched for cells of the desired phenotype and / or depleted of cells of an undesired phenotype, such that the cell population contains at least about 75%, or at least about 80%, or at least about 90% of the desired phenotype. Such positive and negative selection methods are known in the art. For example, cells can be sorted based on cell surface antigens (including those described herein) using a fluorescence-activated cell sorter or magnetic beads that bind to certain cell surface antigens. Negative selection columns can be used to remove cells expressing undesirable cell surface markers. In some embodiments, cells are enriched for CD34+ cells (before and / or after undergoing EHT). In some embodiments, the cell population is cultured under conditions that promote the expansion of CD34+ cells, thereby producing an expanded population of stem cells.

[0074] In various embodiments, CD34+ cells (e.g., suspension cells and / or adherent cells) are harvested from the culture to undergo endothelial-to-hematopoietic transformation. In various embodiments, the HSCs or CD34-enriched cells are further expanded. For example, the HSCs or CD34-enriched cells can be expanded according to the methods disclosed in U.S. Pat. Nos. 8,168,428, 9,028,811, 10,272,110, and 10,278,990, which are incorporated by reference in their entireties. In some embodiments, the ex vivo expansion of the HSCs or CD34-enriched cells employs prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSCs comprise at least about 0.01% LT-HSCs, or at least about 0.05% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 0.5% LT-HSCs, or at least about 1% LT-HSCs.

[0075] Hematopoietic stem cells (HSCs), which give rise to erythroid, myeloid, and lymphoid lineages, can be identified based on the expression of CD34+ cell markers and the absence of lineage-specific markers (referred to as Lin-). In some embodiments, a population of stem cells containing HSCs is enriched, for example, as described in US Pat. No. 9,834,754, incorporated herein by reference in its entirety. For example, this process can include sorting the cell population based on the expression of one or more of CD34, CD90, CD38, and CD43 (HSC markers), or CD71 (a marker for early erythroid progenitors), or CD235a (a marker for mature erythroid progenitors). A fraction that is one or more of CD34+, CD90+, CD38-, and CD43- can be selected for further differentiation. In some embodiments, a stem cell population for differentiation into the hematopoietic lineage is at least about 80% CD34+, or at least about 90% CD34+, or at least about 95% CD34+.

[0076] In some embodiments, stem cell populations, or CD34-enriched cell or fraction thereof, or derivative populations are expanded as described in US 2020 / 0308540, which is incorporated herein by reference in its entirety. For example, the cells are expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55, and Boitano A., et al., Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells. Science 2010 Sep 10;329(5997):1345-1348.

[0077] In some embodiments, compounds that promote the proliferation of CD34+ cells include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, incorporated herein by reference).

[0078] In some embodiments, the stem cell population or CD34-enriched cells are further enriched for or modified to express periostin and / or platelet-derived growth factor receptor alpha (pdgfra), as described in WO 2020 / 205969 (incorporated herein by reference in its entirety). Such expression can be by transgene-free methods, including but not limited to, by delivering an encoding transcript to the cells, by introducing an encoding transgene, or by introducing a non-integrating episome into the cells. In some embodiments, gene editing is employed to introduce genetic modifications into expression elements in the cells, such as to modify promoter activity or strength, ribosome binding, RNA stability, or affect RNA splicing.

[0079] In yet another embodiment, the stem cell population or CD34-enriched cells are cultured with an inhibitor of histone methyltransferase EZH1. Alternatively, EZH1 is partially or completely deleted or inactivated, or transiently silenced, in the stem cell population. Inhibition of EZH1 can direct myeloid progenitor cells (e.g., CD34+CD45+) to the erythroid lineage. In yet another embodiment, EZH1 is overexpressed in the stem cell population.

[0080] In some embodiments, the HSC population or a fraction thereof is differentiated into erythrocytes or their progenitors or derivatives. For example, the HSC population (or cells isolated therefrom) is cultured with EPO, IL-3, and SCF, e.g., (and / or other extracellular matrix component(s)), and / or a combination thereof, to produce a population comprising an erythroid progenitor or derivative cell population (e.g., erythrocytes). The HSC population of cells gives rise to a high percentage of burst-forming unit-erythroid (BFU-E) cells and colony-forming unit-erythroid (CFU-E) cells, indicative of induction of erythropoiesis. These cells can be further enriched and / or expanded.

[0081] For example, production of iPS cell lines from peripheral blood samples can include the following steps: erythroblast enrichment and iPSC initiation. In the erythroblast enrichment step, cells are reprogrammed using, for example, POU5F1 / OCT4, SOX2, KLF4, and c-MYC transfection (or as otherwise described herein). In some embodiments, the erythroblast population is greater than 80% after enrichment. Once the pluripotency of iPSC cells is confirmed by the presence of pluripotency markers, such as, but not limited to, POU5F1 / OCT4, SOX2, LIN28, KLF4, and NANOG, the reprogrammed iPSCs are cultured under culture conditions to generate embryoid bodies (EBs). CD34+ cells are isolated / enriched from dissociated EBs on days 8-14 of iPSC differentiation (as previously described). After inducing EHT in CD34+ cells (optionally followed by further enrichment of CD34+ cells and / or enrichment with other erythroid progenitor markers), the cells are cultured under conditions for erythroid differentiation.

[0082] In some embodiments, HSC populations or fractions thereof are differentiated into erythroid cells or their progenitors or derivatives, independent of the use of agonists of mechanosensitive receptors or mechanosensitive channels, such as Yoda1. In some embodiments, the use of agonists of mechanosensitive receptors or mechanosensitive channels, such as Yoda1, is optional. Thus, in some embodiments, CD34+ cells are enriched from a differentiated pluripotent stem cell population to prepare a CD34+ enriched population. Endothelial-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days but not more than 12 days, optionally with the use of agonists of mechanosensitive receptors or mechanosensitive channels, such as Yoda1, jedi1, jedi2, or ssRNA40. HSCs and / or HSPCs are differentiated into pro-erythroid cell populations or erythroid cell populations.

[0083] In some embodiments, endothelial-to-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days, optionally further for at least about 4 hours, or at least about 8 hours, or at least about 12 hours, or at least about 16 hours, or at least about 20 hours, or at least about 24 hours, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days. Generally, EHT is not induced for more than 12 days.

[0084] During differentiation, the proliferative progenitor stage of erythroid-committed cells can be assessed, for example, by assessing their colony-forming ability in semi-solid medium. For example, harvested cells can give rise to hematopoietic colonies with a high percentage of burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells. The Hb content of cells can generally be used to define their developmental stage, for example, by assessing the γ-to-β globin switch in vitro.

[0085] In some embodiments, the process utilizes serum-free and xenogeneic protocols that comply with Good Manufacturing Practice (GMP). In some embodiments, the process utilizes, for example, a feeder layer such as OP9 or co-culture with other cells, such as stromal cells. In some embodiments, the process utilizes small molecules such as StemRegenin (SR1, a dual RasGAP and ERK1 / 2 inhibitor), Yoda1, Jedi1, Jedi2, ssRNA40, or their analogs or derivatives, BIO (a prototypic GSK3b inhibitor), CHIR99021 (a GSK3b inhibitor), IBMX (a nonspecific inhibitor of cAMP and cGMP phosphodiesterase), and A-A014418 (GSK3b inhibitor VIII) as substitutes for growth factors or various cytokines to reduce side effects and media costs.

[0086] The use of bioreactors, modification of the microenvironment using macrophages and small molecules, and the use of genetic modifications to enhance survival of mature RBCs, increase enucleation rates, and promote hemoglobin switching are also contemplated in various embodiments and aspects of the present invention. As an example of the use of bioreactors to generate enucleated cells, HSCs or erythroid progenitor cells can be cultured in a bioreactor under one or more maturation conditions. The maturation conditions can include (i) a predetermined pH, (ii) a predetermined or specific level of (dissolved) oxygen, and (iii) mechanical stress.

[0087] The predetermined pH may be selected from a pH range of about 4.0 to about 7.9. For example, maturation conditions may include a pH range of about 5.0 to about 7.9, or about 6.0 to about 7.9, or about 7.0 to about 7.9 (e.g., about 7.4 to about 7.5). Maturation conditions may include one or more of the pH values ​​listed above; for example, maturation conditions may be modulated between different pH levels, e.g., a first pH and a second pH.

[0088] Maturation conditions may utilize levels of oxygen that are less than about 90% of atmospheric oxygen, or in other embodiments, less than about 80%, or less than about 70%, or less than about 60%, or less than about 50%, or less than about 40%, or less than about 30%. For example, methods of the invention may utilize levels of oxygen that are less than about 50% of atmospheric oxygen (e.g., about 25% to about 50% of atmospheric oxygen). Maturation conditions may utilize levels of dissolved oxygen (i.e., oxygen dissolved in the culture medium) of about 2% to about 29%, such as about 5% to about 20%, or about 5% to about 15% (e.g., about 11%).

[0089] The level of mechanical stress can be generated by controlling the speed at which the bioreactor impeller tip moves through the cell culture. Impeller tip speeds of about 50 to about 500 rpm can be used, for example, about 100 to about 450 rpm, or about 150 to about 300 rpm, or about 200 to about 250 rpm. The level of mechanical stress used can be modulated between two or more predetermined levels of mechanical stress. Under such circumstances, and when a bioreactor is used to impart the necessary mechanical stress, the variable or modulated mechanical stress used can be generated through the selection (and use) of one or more different impeller speeds. One or more levels of mechanical stress can be applied for any suitable period of time. For example, one or more levels of mechanical stress can be applied for several minutes (e.g., 10 to 60 minutes), for one hour or more (e.g., 1 to 10 hours), or for one day or more (e.g., 1 to 10 days). For example, one or more levels of mechanical stress can be applied continuously or intermittently throughout the period.

[0090] In one embodiment, an erythroid growth medium is used, comprising at least EPO, and optionally comprising cytokines and growth factors, which can be supplemented with one or more small molecule compounds selected from (i) a piezo1 agonist, (ii) a Trpv4 agonist, (iii) a phosphodiesterase inhibitor, or (iv) a GSK3 inhibitor, including the compounds and concentrations described herein.

[0091] Another important issue for the clinical use of hESC-derived RBCs is whether they can be enucleated in vitro. Under the conditions disclosed herein, RBCs undergo differentiation events, including a gradual decrease in size and an increase in glycophorin A expression (a mature RBC marker) and chromatin / nuclear condensation, which leads to the extrusion of the condensed nucleus to form enucleated red blood cells with a diameter of 6-8 μm, similar to that of normal RBCs.

[0092] Events associated with enucleation can be assessed by examining several characteristics associated with the process of erythrocyte maturation. For example, there is a gradual decrease in cell size and nucleus-to-cytoplasm (N / C) ratio before enucleation, and the size and N / C ratio of these cells decrease significantly over time, indicating substantial nuclear condensation during the process. Also during this process, cells express high levels of CD71, an early erythrocyte marker, and their expression decreases over time. Initially, cells exhibit low to negligible levels of CD235a (glycophorin A), a mature erythrocyte marker, but their expression increases dramatically with maturation. Benzidine staining can be used to demonstrate the gradual accumulation of intracellular hemoglobin and the decrease in cell size over time.

[0093] In some embodiments, erythrocytes or red blood cells or their precursors (e.g., iPSCs) can be genetically engineered to be capable of carrying a wide variety of useful cargo to specific locations within the body.

[0094] In another aspect, the present invention provides a red cell or erythroid lineage population produced by the methods described herein, or a pharmaceutically acceptable composition thereof. In various embodiments, the composition comprises a desired cell population (e.g., red blood cells) and a pharmaceutically acceptable vehicle. The pharmaceutical composition may be at least about 10 per mL. 5 or at least about 10 6 or at least about 10 7The pharmaceutical composition may be provided in units of about 50 mL to about 500 mL, or about 100 mL to about 500 mL, or about 250 to about 500 mL.

[0095] In some aspects, the HSC composition is provided with hyperreactive EPOR (as described). The HSC composition of the present disclosure comprises at least 0.0001% LT-HSCs. In some embodiments of the present disclosure, the HSCs comprise at least about 0.05% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 0.5% LT-HSCs, or at least about 1% LT-HSCs. In other aspects, the present invention provides cell populations produced by the methods described herein or pharmaceutically acceptable compositions thereof. In various embodiments, compositions for RBC therapy are prepared comprising the cell population and a pharmaceutically acceptable vehicle. The pharmaceutical composition is at least about 10 per kilogram of body weight. 2 cells, or at least about 10 3 or at least about 10 4 or at least about 10 5 or at least about 10 6 or at least about 10 7 or at least about 10 8 cells, or at least about 10 9 cells, or at least about 10 10 cells, or at least about 10 11 cells, or at least about 10 12 cells, or at least about 10 13 cells, or at least about 10 14 For example, in some embodiments, pharmaceutical compositions containing HSCs (e.g., with hyperresponsive EPOR) in the range of about 100,000 to about 400,000 cells per kilogram (e.g., about 200,000 cells / kg) are administered. In other embodiments, RBCs are administered in the range of about 10 per kilogram of recipient body weight. 5 ~Approx. 5×10 5 cells (e.g., approximately 2.5 x 10 5cells / kg), or approximately 10 per kilogram 6 ~Approx. 5×10 6 cells (e.g., approximately 2.55 x 10 6 cells / kg), or approximately 5 x 10 cells per kilogram 6 ~about 10 7 cells (e.g., approximately 5 x 10 6 cells / kg), or approximately 10 per kilogram 7 ~about 10 8 cells (e.g., approximately 5 x 10 7 cells / kg), or approximately 10 per kilogram 8 ~about 10 9 cells (e.g., approximately 5 x 10 8 cells / kg), or approximately 10 per kilogram 9 ~about 10 10 cells, or approximately 10 10 ~about 10 11 cells, or approximately 10 per kilogram 11 ~about 10 12 cells, or approximately 10 per kilogram 12 ~about 10 13 cells, or approximately 10 per kilogram 13 ~about 10 14 It is administered in individual cells.

[0096] In some embodiments (as described), the HSCs are HLA-A neg , homozygous for both HLA-B and HLA-C, HLA-DPB1 neg , and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0097] The cell compositions of the present disclosure may further comprise a pharmaceutically acceptable carrier or vehicle suitable for intravenous infusion or other administration routes, and the compositions may include a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). The cell compositions may be provided in unit vials or bags and stored frozen until use.

[0098] Cells produced according to the present disclosure can be administered or used in therapy for, for example, inherited or acquired red blood cell disorders, bone marrow failure disorders, high altitude-associated physiological and pathological conditions, anemia (e.g., sickle cell anemia), red blood cell enzyme deficiencies (e.g., G6PD), red blood cell membrane disorders (e.g., hereditary spherocytosis), hemoglobinopathies (e.g., sickle cell disease and thalassemia), hemolytic anemia, nutritional anemia (e.g., iron deficiency anemia and folate deficiency), disorders of heme production (e.g., sideroblastic anemia), hemochromatosis, conditions associated with chemical or radiation exposure, and / or for the treatment of subjects undergoing HSC transplantation. In further embodiments, red blood cells prepared according to the present disclosure are provided as pharmaceutically acceptable compositions that deliver or encapsulate drugs (including, but not limited to, enzymes), oxygen carriers, or other suitable substances for treating human diseases or physiological or pathological conditions.

[0099] In certain embodiments, the present disclosure provides compositions and methods for treating anemia. In embodiments, the present disclosure provides hematopoietic stem cell (HSC) or erythroid progenitor cell compositions that provide durable and potent cellular therapy for anemia. In embodiments, the HSCs are overloaded with a shortened and / or mutated erythropoietin receptor (EPOR), resulting in a hyperresponsive EPOR. In embodiments, the compositions and methods of the present disclosure can provide patients with sufficient red blood cells to supply healthy oxygenation levels. In embodiments, the compositions and methods of the present disclosure effectively harness the potential of induced pluripotent stem cells (iPSCs) to generate HSC populations containing significant numbers of long-term (LT)-HSCs for therapy, thereby providing durable production of red blood cells in vivo.

[0100] Anemia is a condition in which there are not enough healthy red blood cells to carry sufficient oxygen to body tissues. Primary symptoms of anemia include fatigue and weakness. There are many forms of anemia, including sickle cell anemia, aplastic anemia, and anemia associated with bone marrow disease or bone marrow failure. For example, sickle cell disease is a group of inherited red blood cell disorders in which patients have a mutation in the beta-globin gene that leads to an abnormal hemoglobin called hemoglobin S. Hemoglobin S transforms flexible red blood cells into rigid, sickle-shaped cells. Sickle cell anemia is the most common and most severe type of sickle cell disease, resulting in premature death of red blood cells and a lack of healthy red blood cells.

[0101] Current treatments for sickle cell disease and other types of anemia include blood transfusions and blood and bone marrow transplants. Patients receiving available treatments continue to face significant challenges and complications. For example, patients treated with chronic transfusion therapy to increase oxygen-carrying capacity and reduce the ratio of sickle cell (HbS) to hemoglobin A (HbA) face significant burdens, including the need for periodic hospitalization, and often require iron chelation therapy. Howard J. Sickle cell disease: when and how to transfuse. Hematology Am Soc Hematol Educ Program. 2016;2016(1):625-631. Patients treated with blood and bone marrow transplants, i.e., replacing the patient's hematopoietic stem cells with those from a donor, continue to face significant risks, including graft-versus-host disease, exposure to infection, and the need for chemotherapy. Rangarajan, HG, Abu-Arja, R., Pai, V., Guilcher, G., & Soni, S. Outcomes of Unrelated Donor Stem Cell Transplantation with Post-Transplant Cyclophosphamide for Graft-versus-Host Disease Prophylaxis in Patients with Severe Sickle Cell Disease.Biology of Blood and Marrow Transplantation.2018;24(2),413-417.

[0102] HSCs or erythroid progenitor cells generated using the methods described herein are administered to a subject (recipient), for example, by intravenous infusion or intrabone marrow transplantation. The methods can be performed following myeloablative, non-myeloablative, or immunotoxin-based (e.g., anti-c-Kit, anti-CD45, etc.) conditioning regimens. In some embodiments, the methods can be performed without myeloablative, non-myeloablative, or immunotoxin-based (e.g., anti-c-Kit, anti-CD45, etc.) conditioning regimens.

[0103] In various embodiments, the present disclosure provides methods of treating a subject in need of red blood cell production, comprising administering to the subject an HSC composition or an erythroid progenitor cell composition of the present disclosure. Thus, HSCs produced in accordance with the present disclosure can be used to produce red blood cells in vivo in a durable and potent manner.

[0104] In some embodiments, the recipient subject has anemia. In some embodiments, the recipient subject has sickle cell anemia, aplastic anemia, anemia associated with bone marrow disease or failure, anemia from blood loss, or hemolytic anemia. In some embodiments, the recipient subject has Fanconi anemia. In some embodiments, the subject has thalassemia. In some embodiments, the HSCs or erythroid progenitor cells are used to prepare blood products for treating co-morbidities related to blood, bone marrow, immune, metabolic, or mitochondrial disorders.

[0105] As used herein, the term "about" means ±10% of the associated numerical value.

[0106] Certain aspects and embodiments of the present disclosure are further illustrated with reference to the following examples. [Example]

[0107] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances CD34+ cell formation during iPSC differentiation, but does not affect pluripotency. method iPSCs were developed from hCD34+ cells by episomal reprogramming, as known in the art and essentially as described in Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells, Science 318, 1917-1920, (2007), and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009). Embryoid bodies and hemogenic endothelial differentiation were performed essentially as described in R. Sugimura, et al., Hematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438, (2017); C. M. Sturgeon, et al., Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554-561, (2014); J. Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920, (2007); and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009).

[0108] Briefly, hiPSCs were dissociated and resuspended in medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holo-transferrin, monothioglycerol, BMP4, and Y-27632. The cells were then seeded onto 10 cm dishes (EZSPHERE or low-attachment plates) for EB formation. On day 1, bFGF and BMP4 were added to the medium. On day 2, the medium was replaced with medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. Cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, EBs were dissociated on day 8, cells were filtered through a 70 μm strainer, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0109] result Induced pluripotent stem cells (iPSCs) were transduced using an adenoviral vector containing both ETV2 and GFP sequences under the control of the EF1A promoter. After transduction, approximately 45% of the iPSC cultures were observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). It was further observed that ETV2-OE in iPSC cells retained the pluripotent properties of iPSCs, as indicated by the stemness marker expression TRA-1-60 (Figure 1). Figure 1 shows a FACS plot demonstrating the transduction efficiency of iPSCs using an adenoviral vector for overexpressing ETV2 and GFP sequences.

[0110] Next, ETV2-OE-iPSCs (along with control iPSCs transduced with a vector carrying the GFP sequence but lacking ETV2) were differentiated into embryoid bodies and then into hemogenic endothelial cells (Strugeon et al., 2014). The results suggest that overexpression of ETV2 enhances the formation of hemogenic endothelial cells, as evidenced by the expression of CD34+ and CD31+ markers within the CD235a- population (Figure 2). Specifically, Figure 2 shows representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (defined here as CD235a-CD34+CD31+), demonstrating that ETV2-OE enhances the formation of hemogenic endothelial cells compared to controls.

[0111] Furthermore, the results suggest that ETV2-OE enhances the formation of CD34+ cells (Figure 3). Figure 3 shows representative flow cytometry analysis and relative quantification of CD34+ cells demonstrating that ETV2-OE enhances CD34+ cell formation.

[0112] Overall, these data indicate that ETV2 overexpression in iPSCs does not affect their pluripotent properties, but promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation.

[0113] Example 2 - iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs. method To analyze EHT, EB-derived CD34+ cells were suspended in medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. After the cells had adhered to the bottom of the well (by visual inspection) for approximately 4-18 hours, Yoda1 was added to the culture. After 4-7 days, cells were collected for analysis.

[0114] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells from the iPSC-derived embryoid bodies were harvested and cultured for an additional 5–7 days to induce endothelial-hematopoietic (EHT) transition. CD34+ cells were then harvested from the EHT cultures on days 5–7 for further hematopoietic lineage differentiation.

[0115] CD34+ cells harvested from EHT cultures on days 5–7 (or days 13–21 total of differentiation from iPSCs) were seeded onto 48-well plates pre-coated with rhDL4 and retronectin. T lineage differentiation was induced in medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1α, and SB203580.

[0116] 80% of the medium was changed every other day from days 2 to 6. On day 7, cells were transferred to new coated plates and analyzed for the presence of pre-T cells (CD34+CD7+CD5+ / -).

[0117] 80% of the medium was changed every other day from days 8 to 13. On day 14, 100,000 cells / well were transferred to new coated plates, and cells were analyzed for the presence of pre-T cells (CD34-CD7+CD5+ / -).

[0118] 80% of the medium was changed every other day from days 15 to 20. Cells were harvested on day 21 and analyzed via FACS for CD3, CD8, CD5, CD7, TCRab expression on behalf of T cells, and / or activated using CD3 / CD28 beads to assess their functional properties.

[0119] After 21 days of differentiation, cells were harvested and approximately 80,000 cells were replated into new 96-well culture plates in RPMI 1640 (without L-glutamine, without phenol red) with FBS, L-glutamine, and IL-2, and then activated with 1:1 CD3 / CD28 beads. After 72 hours of activation with CD3 / CD28 beads, cells were analyzed for CD3, CD69, and CD25 expression by FACS and for IFN-γ expression using RT-qPCR. Supernatants were analyzed by ELISA.

[0120] result Figures 4A and 4B show that iPSC-derived HSCs derived by day 8 34+ cell EHT (using Piezol activation in this example) undergo pre-T cell differentiation, similar to bone marrow (BM)-HSCs. Furthermore, Figures 5A and 5B show that iPSC-derived HSCs generated by D8 34+ cell EHT (using Piezol activation in this example) undergo T cell differentiation, similar to BM-HSCs, and can be activated with CD3 / CD28 beads. Figure 6 shows that iPSC-derived HSCs generated by D8 34+ cell EHT (using Piezol activation in this example) can differentiate into functional T cells, as demonstrated by INFγ expression upon stimulation with CD3 / CD28 beads. Together, these results demonstrate that iPSC-differentiated 34+ cell EHT enhances HSC capacity to further differentiate into hematopoietic lineages, including progenitor and functional T cells, ex vivo.

[0121] Example 3 - Evaluation of off-target editing in HLA knockout HSCs HLA typing of triple knockout (HLA edited) HSC clones was performed to check for unwanted editing and ensure that major editing events, such as deletion(s), did not occur within other regions of chromosome 6. Sequencing methods and analyses were performed to assess the extent of gRNA off-target activity and select gRNAs that represent a low risk of affecting non-target HLA genes.

[0122] Sequencing was performed using in situ cleavage labeling in fixed and permeabilized cells by ligating full-length P5 sequencing adapters to end-prepared DSBs. Genomic DNA was extracted, fragmented, end-prepared, and ligated using chemically modified semi-functional P7 adapters. The resulting DNA library contained a mixture of functional DSB-labeled fragments (P5:P7) and non-functional genomic DNA fragments (P7:P7). Subsequent DNA sequencing of the DNA library enriched for the DNA-labeled fragments, eliminating all irrelevant non-functional DNA. Because the library preparation was PCR-free, each resulting sequencing read was equivalent to a single labeled DSB end from the cell. This generated a readout of DNA cleavage, enabling direct detection and quantification of genomic DSBs by sequencing without the need for error correction, and allowed for the unambiguous mapping of off-target mutations.

[0123] Table 1 below summarizes the results of the editing strategy in two representative clones relative to wild-type cells. [Table 1]

[0124] Table 2 provides non-limiting examples of gRNAs used in experiments that can be used to knock out expression of the indicated HLA genes. [Table 2]

[0125] The results show that the editing strategy was successful in selectively targeting the HLA-A, DPB1, and DQB1 genes without affecting other HLA genes or introducing major deletions elsewhere.

[0126] These results were confirmed by phenotypic analysis of the HLA-edited clones by FACS and immunofluorescence. As shown in Figures 9A and 9B, the HLA-edited cells tested positive for overall expression of HLA class I molecules, comparable to that of wild-type cells. Specific expression of HLA-A via immunofluorescence confirmed that HLA-A was not expressed in the HLA-edited cells, supporting the finding that the gene editing strategy successfully deleted only the HLA-A gene. Specifically, Figure 7A shows that all HLA-edited cells were positive for HLA-like class I molecules to the same extent as wild-type (WT) (i.e., non-HLA-edited) cells. This result indicates that despite the deletion of HLA-A, other class I molecules, such as HLA-B and HLA-C, were expressed and were not affected by the gene editing strategy.

[0127] To confirm that the HLA-A gene had been deleted, the specific expression of HLA-A was analyzed by immunofluorescence. As can be seen in Figure 7B, HLA-A was not expressed in the HLA-edited clones, indicating that the gene editing strategy was effective in specifically deleting only the HLA-A gene. Such preservation of overall class I expression with deletion of HLA-A would facilitate patient matching while avoiding NK cell-mediated rejection.

[0128] Example 4 - Evaluation of pluripotency and immune compatibility of HLA-edited HSCs The ability of HLA-edited cells to preserve pluripotency was assessed. As shown in Figure 8, immunofluorescence evaluation of HLA-edited iPSC clones demonstrated that they maintained tri-lineage differentiation, with ectodermal differentiation indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation indicated by GATA-488 staining, and endodermal differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0129] HLA class I molecules are expressed on the surface of all nucleated cells, and if HLA class I molecules are mismatched between donor and recipient, the cells can be recognized and killed by CD8+ T cells. In addition, HLA mismatches can lead to cytokine release syndrome (CRS) and graft-versus-host disease (GVD). Conversely, complete deletion of HLA-I molecules via B2M KO would render the cells targets for NK cell-mediated cytotoxicity. Preservation of overall class I expression with deletion of HLA-A could facilitate patient matching while avoiding NK cell-mediated rejection. Therefore, the immune compatibility of HLA-edited HSCs was tested by coculture with peripheral blood mononuclear cells (PBMCs) to assess whether immune cells would reject HSC grafts.

[0130] Wild-type (WT) and HLA-edited HSCs were cocultured with PBMCs bearing HLA-B and HLA-C markers matched but mismatched HLA-A. B2M KO HSCs, which lack expression of HLA class I molecules, and CIITA KO HSCs, which lack expression of class II molecules, were used as controls to compare the degree of PBMC-mediated cytotoxicity for HLA-null and HLA-mismatched HSCs, respectively. Figure 9 shows the results of a PBMC-mediated cytotoxicity assay in the coculture, measured by Annexin V staining. The results show that deletion of HLA-A in HLA-edited HSCs protected the cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO HSCs were susceptible to PBMC-mediated cytotoxicity. Coculture of HSCs with sorted CD8+ T cells from the same PBMC donor protected HLA-edited and B2M KO HSCs from CD8+ T cytotoxicity. Conversely, HSCs co-cultured with sorted NK cells protected only WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0131] In summary, the immunocompatibility results indicate that CD8+ T cells present in the PBMC samples were involved in killing cells bearing mismatched HLA molecules (WT and CIITA KO), while NK cells present in the PBMCs were involved in killing HLA-null cells (B2M KO). However, HLA-edited HSCs were protected from CD8+ T cell-mediated cytotoxicity (because mismatched HLA-A had been knocked out) and from NK cell-mediated cytotoxicity (because HLA class I molecule expression was largely preserved).

[0132] Example 5 - Evaluation of in vivo engraftment potential of HLA-edited HSCs To evaluate the engraftment potential of HLA-edited HSCs, the ability of the cells to engraft in vivo was assessed by competitive transplantation against wild-type HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs were mixed and transplanted into mice. Bone marrow (BM) and peripheral blood samples were collected and assessed by FACS to compare the relative amounts of each cell type present in the samples. As shown in Figure 10, both HLA-edited HSCs and wild-type HSCs contributed to approximately equal engraftment in BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to wild-type HSCs in their engraftment and reconstitution potential. Therefore, the properties of wild-type (unedited, parental) HSCs are expected to match those of the HLA-edited HSCs of the present disclosure for generating T cell lineages.

[0133] Example 6 - Differentiation of HLA-edited HSCs into CD4+ / CD8+ T cells Antigen-presenting cells (APCs) present antigens to helper CD4+ T cells through HLA-II molecules. Activation of helper CD4+ T cells promotes the generation of antigen-specific CD8+ T cells, which further evolve into antigen-specific CTLs. Similarly, HLA class I molecules are expressed on the surface of all nucleated cells and present peptide fragments of proteins to CD8+ CTLs from within the cell. CTLs induce cytotoxic killing of target (infected) cells upon recognition of HLA-I-peptide complexes expressed on the cell surface. Therefore, studies were performed to determine whether deletion of HLA-A affects class I peptide presentation of edited HSCs. As shown in Figures 11A and 11B, immunopeptidome analysis indicates that deletion of HLA-A does not affect overall class I peptide presentation. HLA-A-edited cells exhibited comparable peptide and protein presentation compared to wild-type (non-HLA-edited) HSCs. Furthermore, as shown in Figures 12A and 12B, deletion of HLA-DQB1 and HLA-DPB1 does not affect overall class II peptide presentation by macrophages differentiated from HSCs. Together, these data suggest that despite the deletion of HLA-A, HLA-DQ, and HLA-DP molecules, the cells (and their derived lineages) retain the ability to present a wide range of class I and II peptides.

[0134] Example 7 - In vivo testing of antigen-mediated immune responses. Figure 13 is a schematic diagram of the delayed-type hypersensitivity reaction, showing the sensitization and elicitation stages of antigen presentation. Briefly, upon antigen injection, the antigen is processed by antigen-presenting cells (APCs) and presented by MHC class II molecules on the surface of the APCs. CD4+ T cells recognize peptide-MHC on the antigen-presenting cells (APCs). Upon antigen administration, CD4+ helper T cells are activated, and cytokines recruit macrophages and other immune cells that induce tissue swelling.

[0135] A delayed-type hypersensitivity assay was performed on transplanted mice. Specifically, mice were sensitized by subcutaneous injection of sheep red blood cells as an antigen. If the mouse has a functional immune system, APCs process the antigen and present the peptide antigen to CD4+ T cells. The mouse was then stimulated by subcutaneous injection of the same antigen in the left paw. At this point, T cells are activated and secrete cytokines that recruit macrophages and other immune cells to the antigen injection site, resulting in tissue swelling. In this assay, a functional immune system resulted in swelling of the left paw, as measured with a microcaliper.

[0136] As can be seen in Figures 14A and 14B, control (non-transplanted) mice, due to their immunodeficiency, did not show swelling in their left paws. Conversely, mice transplanted with cord blood CD34+ cells showed tissue swelling, doubling the diameter of their left paws. Similar immune system responses were seen in both mice transplanted with WT (non-edited HSCs) and mice transplanted with HLA-edited HSCs.

[0137] Example 8 - Evaluation of differentiation and maturation of HSC-derived T cells (pre-T cells) Next, we tested the ability of HSC-derived T cells (pre-T cells) to differentiate into mature T cells. After a 35-day differentiation period, pre-T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. As shown in Figure 15, pre-T cells differentiated into CD4+, CD8+, and αβ+ T cells more efficiently than bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells.

[0138] Next, to test their functional properties, each T cell population was co-cultured with a CD19+ lymphoma cell line and an anti-CD3 / CD19 bispecific antibody. In this experimental model, the bispecific antibody engaged both the CD3 receptor on T cells and the CD19 cell surface receptor on lymphoma cells, thus inducing T cell activation. The degree of activation was assessed by measuring subsequent T cell-mediated cytotoxicity compared to a pan T cell positive control. As shown in Figure 16, pre-T cells demonstrated statistically significant superiority in cytotoxicity compared to both BM CD34+ T cells and EB CD34+ T cells.

[0139] Example 9 - Evaluation of HSC properties evolving into pre-T cells. The ability of HSCs to evolve into pre-T cells was assessed by measuring the CD34-CD7+ marker on pre-T cells. As shown in Figure 17, FACS analysis demonstrated that HSCs produced according to the present disclosure successfully differentiated into CD34-CD7+ pre-T cells compared to bone marrow-derived CD34+ cells or EB-derived CD34+ cells.

[0140] Next, the expression of T cell-specific transcription factors and thymic engraftment molecules was measured. Figure 18A shows increased TCF7 expression in pre-T cells derived from HSCs of the present disclosure, and Figure 18B shows increased CCR7 expression. Figure 19A shows that pre-T cells derived from HSCs engraft and differentiate in the thymus. Figure 19B shows FACS analysis of the CD3+ cell population of cells gated on the CD45+ cell population, demonstrating the superior engraftment and differentiation potential of pre-T cells derived from HSCs in the thymus. Pre-T cells in this example were prepared from HSCs using Piezo1 activation, as previously described.

[0141] In vitro activation of HSC-derived T cells was also measured, as illustrated in Figure 20. The top panel of Figure 20 shows FACS analysis of activated T cells from different sources, including HSCs of the present disclosure (e.g., prepared using Piezol activation). T cells prepared from HSCs of the present disclosure showed comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, where activated T cells express inflammatory cytokines. T cells derived from HSCs according to the present disclosure (e.g., prepared using Piezol activation) expressed higher levels of inflammatory cytokines, as exemplified by TNF-α and interferon-gamma expression levels.

[0142] Example 10: Differentiation of HLA-edited HSCs into hematopoietic lineage, promonocyte / macrophage cells Experiments were conducted to determine whether HLA deletion affects the ability of HSCs to differentiate into different types of immune cells. HLA-edited HSCs were differentiated into promonocyte / macrophage cells using a process essentially as described in Example 2. It was determined that HLA-edited HSCs were capable of differentiating into the monocyte / macrophage lineage equivalent to wild-type (non-HLA-edited) HSCs, as measured by their CD11b+-CD14+ expression (Figure 21A). Furthermore, the CD11b+-CD14+ gated population showed equivalent HLA-I and HLA-II expression (Figure 21B), also indicating that HLA-edited HSCs preserved their overall expression of both class I and class II molecules.

[0143] The global expression of other class II molecules in HLA-DQB1 and HLA-DPB1 supported by edited HSCs was assessed by evaluating their expression in macrophages differentiated from HSCs. The design of this study is shown schematically in Figure 22A. It was found that deletion of HLA-DQB1 and HLA-DPB1 did not affect the expression of other HLA class II molecules (Figure 22B). For example, HLA-DR was equally expressed in both WT and HLA-edited cells (Figure 22C). In Figures 22B and 22C, CIITA-KO is the positive control.

[0144] Example 11: Differentiation of HLA-edited HSCs into proplatelets It was determined that HLA-edited HSCs can differentiate into megakaryocytes (MKs) and further differentiate into platelets. Differentiation was compared to bone marrow (BM)-derived CD34+ cells and iPSC-CD34+ cells. As shown in Figure 23, HLA-edited HSCs showed a statistically significant increase in platelet content compared to BM CD34+ and iPSC-CD34+ cell populations. Thus, HLA-edited HSCs can differentiate into megakaryocytes (MKs), which can further support differentiation into platelets.

[0145] References 1. Nianias, A. & Themeli, M. Induced Pluripotent Stem Cell (iPSC)-Derived Lymphocytes for Adoptive Cell Immunotherapy: Recent Advances and Challenges. Curr Hematol Malig Rep 14, 261-268 (2019). 2. Brauer, PM, Singh, J., Xhiku, S. & Zuniga-Pfluecker, JCT Cell Genesis: In Vitro Veritas Est?Trends Immunol 37, 889-901 (2016). 3.Kennedy,M.et al.T Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem Cell Differentiation Cultures.Cell Reports 2,1722-1735(2012). 4.Sturgeon,C.M.,Ditadi,A.,Awong,G.,Kennedy,M.& Keller,G.Wnt Signaling Controls the Specification of Definitive and Primitive Hematopoiesis From Human Pluripotent Stem Cells.Nat Biotechnol 32,554-561(2014). 5.Chang,C.-W.,Lai,Y.-S.,Lamb,L.S.& Townes,T.M.Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells.PLoS One 9,(2014). 6.Nishimura,T.et al.Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation.Cell Stem Cell 12,114-126(2013). 7.Themeli,M.et al.Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.Nat Biotechnol 31,928-933(2013). 8.Vizcardo,R.et al.Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+T Cells.Cell Stem Cell 12,31-36(2013). 9.Montel-Hagen,A.et al.Organoid-induced differentiation of conventional T cells from human pluripotent stem cells.Cell Stem Cell 24,376-389.e8(2019). 10.Guo,R.et al.Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors.Cell Research 30,21-33(2020). 11.Nagano,S.et al.High Frequency Production of T Cell-Derived iPSC Clones Capable of Generating Potent Cytotoxic T Cells.Molecular Therapy-Methods & Clinical Development 16,126-135(2020). 12.Iriguchi,S.et al.A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.Nature Communications 12,430(2021).

Claims

1. 1. A method for preparing a population of cells of erythroid lineage, comprising: enriching CD34+ cells from the differentiated pluripotent stem cell (PSC) population to prepare a CD34+ enriched population; inducing endothelial-to-hematopoietic transformation of the CD34+ enriched cell population for at least 2 days but not more than 12 days to produce a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs); differentiating said population comprising HSCs and / or HSPCs into an erythroid lineage population.

2. 2. The method of claim 1, wherein the PSC population is a human iPSC population derived from erythroblasts, lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or primary human tissue.

3. 3. The method of claim 2, wherein the iPSC population is derived from CD34+ enriched cells isolated from peripheral blood.

4. 3. The method of claim 2, wherein the iPSCs are homozygous for one or more HLA class I and / or HLA class II genes.

5. The method of claim 4, wherein the iPSCs are homozygous for HLA-DRB1.

6. The method of claim 4, wherein the iPSCs are homozygous for both HLA-B and HLA-C.

7. 3. The method of claim 2, wherein the iPSCs have been gene-edited to delete one or more HLA class I genes, one or more class II genes, and / or one or more genes that govern HLA or MHC expression or presentation capacity.

8. The method of claim 7, wherein the iPSCs comprise a deletion of HLA-A.

9. The method of claim 7 or 8, wherein the iPSCs comprise a deletion of HLA-DPB1 and / or HLA-DQB1.

10. The method according to claim 7, wherein the one or more genes controlling the ability to express or present HLA or MHC are β2-microglobulin and / or CIITA.

11. 10. The method of any one of claims 2 to 9, wherein the iPSCs comprise a deletion of HLA-A and are homozygous for both HLA-B and HLA-C, and comprise a deletion of HLA-DPB1 and HLA-DQB1 and are homozygous for HLA-DRB1.

12. 12. The method of any one of claims 1 to 11, wherein the HSCs and / or HSPCs have been gene-edited to encode a hyperresponsive EPO receptor.

13. The method of any one of claims 1 to 12, wherein CD34+ enrichment and endothelial-hematopoietic transition are induced between days 8 and 15 of iPSC differentiation.

14. 14. The method of claim 13, wherein the endothelial-to-hematopoietic conversion generates a population of HSCs comprising one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem progenitor cells.

15. 14. The method of claim 13, wherein the CD34+ cells are harvested from a culture undergoing endothelial-hematopoietic transformation, including harvesting of CD34+ floating and / or adherent cells.

16. The method of claim 14, wherein the HSC population comprises long-term hematopoietic stem cells (LT-HSCs).

17. The method of claim 13, wherein the induction of endothelial-hematopoietic transition comprises increasing the expression or activity of dnmt3b.

18. The method of claim 13, wherein the induction of endothelial-to-hematopoietic transition comprises applying cyclic stretch to the CD34-enriched cells.

19. 19. The method of claim 18, wherein the cyclic stretching is 2D, 3D, or 4D cyclic stretching.

20. The method of claim 13, wherein the induction of endothelial-hematopoietic transition comprises Piezo1 activation.

21. 21. The method of claim 20, wherein the Piezo1 activation is by contacting the CD34+ enriched cells or a fraction thereof, optionally with one or more Piezo1 agonists selected from Yoda1, Jedi1, Jedi2, ssRNA40, or analogs or derivatives thereof.

22. The method of claim 13, wherein the induction of endothelial-hematopoietic transition comprises Trpv4 activation.

23. 23. The method of claim 22, wherein said Trpv4 activation is by contacting said CD34+ enriched cells, optionally with one or more Trpv4 agonists selected from GSK1016790A, 4α-PDD, or analogs or derivatives thereof.

24. 19. The method of any one of claims 1 to 18, wherein the erythroid lineage is selected from erythroid progenitor cells, proerythroblasts, granulocyte-macrophage progenitor cells (GMP), and megakaryocytic erythroid progenitor cells (MEP), and erythroid cells.

25. 25. The method of any one of claims 1 to 24, wherein the HSCs or a fraction thereof or progeny thereof are cultured with EPO, IL-3, and SCF to prepare a committed erythroid lineage.

26. 26. The method of claim 24 or 25, wherein the method gives rise to (i) hematopoietic colonies with a high percentage of burst-forming unit-erythroid (BFU-E) and / or (ii) colony-forming unit-erythroid (CFU-E) cells.

27. The method comprises:

27. The method of claim 25 or 26, further comprising culturing the erythroid lineage under culture condition(s) sufficient to produce enucleated red blood cells, said culturing optionally comprising one or more of SCF, EPO, and IL-3.

28. 28. The method of claim 27, wherein the progeny (i) differentiate into cells characterized by CD36+ / CD45+ markers and (ii) further differentiate over time into cells characterized by CD36+ / CD45- markers, wherein at least 80% of the cells in (ii) are identified as CD36+.

29. The red blood cell system (i) a pH of about 4.0 to about 7.9; (ii) a level of oxygen that is less than about 75% of atmospheric oxygen; and 29. The method of claim 27 or 28, wherein the cells are cultured under one or more culture conditions selected from the group consisting of (iii) mechanical stress.

30. 30. The method of claim 29, wherein the pH is from about pH 7.0 to about pH 7.

9.

31. 31. The method of claim 29 or 30, wherein the level of oxygen comprises less than about 50% and / or 15% or less dissolved oxygen of atmospheric oxygen.

32. The method according to any one of claims 29 to 31, wherein the mechanical stress is generated in a bioreactor.

33. 33. The method of claim 32, wherein the mechanical stress is controlled by adjusting the speed of a bioreactor impeller.

34. 34. The method of claim 33, wherein the mechanical stress can be applied continuously or continuously throughout the red blood cell / reticulocyte culture protocol.

35. 35. The method of claim 34, wherein the erythroid growth medium is supplemented with one or more of: (i) a piezo1 agonist, (ii) a Trpv4 agonist, (iii) a phosphodiesterase inhibitor, or (iv) a GSK3 inhibitor.

36. 36. The method of claim 35, wherein the erythroid growth medium is supplemented with one or more of: (i) stem cell factor (SCF), (ii) insulin growth factor I (IGF1), (iii) IL3, (iv) IL11, and (v) EPO.

37. 37. The method of claim 36, wherein the erythroid expansion medium is supplemented with (i) Flt3 ligand and / or (ii) bone morphogenetic protein 4 (BMP4).

38. A population of cells of erythroid lineage prepared by the method of any one of claims 1 to 37.

39. 34. A method for treating a patient having a genetic or acquired red blood cell disorder, a bone marrow failure disorder, a high altitude-associated physiological and pathological condition, anemia, an red blood cell enzyme deficiency, an red blood cell membrane disorder, a hemoglobinopathy, a hemolytic anemia, a nutritional anemic disorder of heme production, or hemochromatosis, comprising administering to the patient the red blood cell lineage of claim 33.

40. A hematopoietic stem cell (HSC) composition or an erythroid progenitor cell composition, wherein the HSC or erythroid progenitor cell expresses an erythropoietin receptor (EPOR) that is hyperresponsive to EPO.

41. 41. The composition of claim 40, wherein the composition comprises HSCs, including at least 0.0001% LT-HSCs.

42. The composition is at least about 10 ２ pieces, at least about 10 ３ or at least about 10 ４ or at least about 10 ５ or at least about 10 ６ 42. The composition of claim 40 or 41, comprising HSCs or erythroid progenitor cells.

43. The composition of any one of claims 40 to 42, wherein the EPOR has a truncation mutation.

44. The composition of claim 43, wherein the EPOR lacks the SHP-1 inhibitory domain.

45. 44. The composition of any one of claims 40 to 43, wherein the EPOR comprises one or more missense or frameshift mutations resulting in hyperresponsiveness, optionally due to mutations in the SHP-1 inhibitory domain.

46. 46. ​​The composition of any one of claims 40 to 45, wherein the HSCs or erythroid progenitor cells are differentiated from induced pluripotent stem cells (iPSCs).

47. HSC or erythroid progenitor cells are HLA-A ｎｅｇ , homozygous for both HLA-B and HLA-C, and HLA-DPB1 ｎｅｇ and HLA-DQB1 ｎｅｇ 47. The composition of claim 46, wherein: In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

48. 48. The composition of claim 46 or 47, wherein the iPSCs have been gene-edited to encode the hyperresponsive EPO receptor.

49. The composition of any one of claims 46 to 48, wherein iPSCs are differentiated into HSCs by a process comprising increasing the expression or activity of dnmt3b or Gimap6.

50. 50. The composition of any one of claims 46 to 49, wherein the HSCs are prepared from iPSCs in a process comprising applying cyclic stretch.

51. 51. The composition of claim 50, wherein the cells are subjected to 2D or 3D cyclic stretch, and the cells subjected to cyclic stretch are optionally selected from one or more of iPSCs, endothelial cells, hemogenic endothelial cells (HECs), and hematopoietic stem cells (HSCs).

52. 52. The composition of claim 51, wherein iPSCs are differentiated into endothelial cells or HECs, and cyclic stretch is applied to the endothelial cells or HECs.

53. 53. The composition of claim 52, wherein CD34+ cells are enriched from iPSCs or embryonic bodies (EBs) prepared therefrom, and cyclic stretching is applied to the CD34+ cells or a subpopulation thereof.

54. 54. The composition of any one of claims 46 to 53, wherein the HSCs are prepared from iPSCs in a process comprising stimulation by Piezo1 activation or by Trpv4 activation.

55. 55. The composition of claim 54, wherein the cells have undergone Piezo1 activation, and the cells that have undergone Piezo1 activation are optionally selected from one or more of iPSCs, ECs, HECs, and HSCs.

56. 56. The composition of claim 55, wherein CD34+ cells are enriched from iPSCs or embryonic bodies (EBs) prepared therefrom, and Piezo1 activation is applied to the CD34+ cells or a subpopulation thereof.

57. 57. The composition of claim 55 or 56, wherein the Piezo1 activation is achieved by contacting a pluripotent stem cell, or a cell derived therefrom or differentiated therefrom, with one or more Piezo1 agonists optionally selected from Yoda1, Jedi1, Jedi2, ssRNA40, or an analog thereof.

58. 58. The composition of claim 57, wherein the effective amount of the Piezo1 agonist is in the range of 0.1 to 500 μM, or in the range of 0.1 to 100 μM.

59. 59. The composition of any one of claims 40 to 58, wherein the composition further comprises a pharmaceutically acceptable carrier suitable for injection into a patient.

60. A method for producing an HSC population, comprising preparing a population of iPSCs having an EPOR gene encoding EPOR that is hyperresponsive to EPO, and differentiating the iPSCs into HSCs, including LT-HSCs.

61. 61. The method of claim 60, wherein the EPOR has a truncation mutation.

62. 62. The method of claim 61, wherein the EPOR lacks the SHP-1 inhibitory domain.

63. 61. The method of claim 60, wherein the EPOR comprises one or more missense or frameshift mutations that result in hyperresponsiveness, optionally due to mutations in the SHP-1 inhibitory domain.

64. 64. The method of any one of claims 60 to 63, wherein the HSCs are differentiated from induced pluripotent stem cells (iPSCs).

65. 65. The method of claim 64, wherein the iPSCs are derived from universally compatible donor cells.

66. 66. The method of claim 64 or 65, wherein the iPSCs have been gene-edited to encode the hyperresponsive EPO receptor.

67. 67. The method of any one of claims 64 to 66, wherein iPSCs are differentiated into HSCs by a process comprising increasing the expression or activity of dnmt3b or Gimap6.

68. 68. The method of any one of claims 60 to 67, wherein the HSCs are prepared from iPSCs in a process comprising applying cyclic stretch.

69. 69. The method of claim 68, wherein cells are subjected to 2D or 3D cyclic stretch, and the cells subjected to cyclic stretch are optionally selected from one or more of iPSCs, endothelial cells, hemogenic endothelial cells (HECs), and hematopoietic stem cells (HSCs).

70. 70. The method of claim 69, wherein iPSCs are differentiated into endothelial cells or HECs, and cyclic stretch is applied to the endothelial cells or HECs.

71. 70. The method of claim 69, wherein CD34+ cells are enriched from iPSCs or embryonic bodies (EBs) prepared therefrom, and cyclic strain is applied to the CD34+ cells or a subpopulation thereof.

72. 72. The method of any one of claims 60 to 71, wherein the HSCs are prepared from iPSCs in a process comprising stimulation by Piezo1 activation or by Trpv4 activation.

73. 73. The method of claim 72, wherein cells are subjected to Piezo1 activation, and the cells that have undergone Piezo1 activation are optionally selected from one or more of iPSCs, ECs, HECs, and HSCs.

74. 74. The method of claim 73, wherein CD34+ cells are enriched from iPSCs or embryonic bodies (EBs) prepared therefrom, and Piezo1 activation is applied to said CD34+ cells or a subpopulation thereof.

75. 75. The method of any one of claims 71 to 74, wherein the Piezo1 activation is by contacting the pluripotent stem cells or cells differentiated therefrom with one or more Piezo1 agonists optionally selected from Yoda1, Jedi1, Jedi2, ssRNA40, or analogs thereof.

76. 76. The method of claim 75, wherein the effective amount of the Piezo1 agonist is in the range of 0.1 to 500 μM, or in the range of 0.1 to 100 μM.

77. 77. The method of any one of claims 60 to 76, wherein the composition comprises at least 0.0001% LT-HSCs.

78. The composition is at least about 10 ２ pieces, at least about 10 ３ or at least about 10 ４ or at least about 10 ５ or at least about 10 ６ 78. The method of any one of claims 60 to 77, comprising HSCs.

79. A cell composition comprising a population of HSCs or erythroid progenitor cells carrying an erythropoietin receptor (EPOR) gene encoding a hyperreactive EPOR, wherein the HSC population is prepared by the method of any one of claims 60 to 78.

80. 80. A method of treating a subject in need of red blood cell production, comprising administering to said subject a composition of any one of claims 39-59 and 79.

81. 81. The method of claim 80, wherein the subject has anemia.

82. 82. The method of claim 81, wherein the subject has sickle cell anemia, aplastic anemia, anemia associated with bone marrow disease or failure, anemia from blood loss, or hemolytic anemia.

83. 83. The method of claim 82, wherein the subject has Fanconi anemia.

84. 83. The method of claim 82, wherein the subject has thalassemia.

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