Red lineage derived from pluripotent cells

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 413,439, filed on October 5, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted in XML format via EFS-Web and is hereby incorporated 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 Art

[0005] According to the American Red Cross, it is facing a national blood crisis - the worst blood shortage in more than a decade, posing alarming risks to patient care. During this crisis, physicians are forced to make difficult decisions about who receives a transfusion and who needs to wait until more product becomes available. Therefore, there is an urgent need to develop ready-made red blood cells (RBCs) or erythrocytes or their progenitors. Hematopoietic stem cells derived from induced pluripotent cells provide such an opportunity. However, current methods for generating RBCs from iPSCs have proven inadequate due to limitations in expansion of erythroid lineage cells or insufficient enucleation. Therefore, the successful in vitro generation of red blood cell products from iPSCs would fill a significant need. Summary of the Invention

[0006] The present disclosure provides in various aspects and embodiments a method for producing a hematopoietic lineage for cell therapy, including gene-edited hematopoietic stem cells (HSC), erythroid progenitors, progenitor erythroblasts, granulocyte-macrophage progenitors (GMP), megakaryocyte erythroblast progenitors (MEP) and erythroid cells. In various embodiments, the present invention provides an efficient in vitro method for developing such hematopoietic lineages (including but not limited to progenitor erythroblasts and erythroblast lineages) from human induced pluripotent stem cells (iPSC). The cells produced in various embodiments according to the present disclosure are functional and / or more closely resemble the corresponding lineages isolated from peripheral blood or bone marrow. The present invention also provides separated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

[0007] 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 highly responsive EPO receptor. These HSCs or erythroid progenitor cell populations can be used for more efficient in vitro red blood cell production, or in other aspects, can be used to deliver HSCs or erythroid progenitor cells to patients in need to reduce or eliminate the need for regular blood transfusions.

[0008] On the one hand, the present disclosure provides a method for preparing a cell colony of hematopoietic lineage. The method includes preparing pluripotent stem cell (PSC) colony, such as being differentiated into the induced pluripotent stem cell (iPSC) colony of embryoid body (EB), and enriching CD34+ cells, so as to prepare the colony of CD34+ enrichment. Inducing endothelial cells to hematopoietic cell transition (EHT) in the colony of CD34+ enrichment, so as to prepare hematopoietic stem cell (HSC) colony, optionally further enriching CD34+ cells afterwards. In certain embodiments, the HSC colony (or its fraction) gained in some embodiments can be differentiated into erythroid hematopoietic lineage (for example, erythroid progenitor cell). In various embodiments, hematopoietic lineage is selected from erythrocyte (i.e., red blood cell), progenitor cell erythroblast, granulocyte-macrophage progenitor cell (GMP) and megakaryocyte erythroid progenitor cell (MEP).

[0009] In various embodiments, HSCs and erythroid lineage cells derived therefrom are derived from iPSCs that have been gene-edited to one of the following: (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-, (vi) HLA-AB-C+DP-DR+DQ-. In some embodiments, HSCs and erythroid lineage cells are derived from iPSCs that have been gene-edited to one of the following: 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.

[0010] In some embodiments, the iPSCs disclosed herein are gene-edited to encode a reactive EPO receptor. Erythropoiesis is the process of red blood cell production. Erythropoietin (EPO) is a key hormone responsible for effective red blood cell production. Erythropoietin receptor (EPOR) is a protein encoded by the EPOR gene. The most confirmed function of EPOR is to promote proliferation and rescue erythroid (red blood cell) progenitor cells from apoptosis. Beneficial mutations of EPOR increase the number of red blood cells and improve oxygen delivery. For example, EPOR can be made highly responsive to EPO by truncation mutations (removing only the intracellular EPORC terminus that binds to negative regulatory factors).

[0011] These HSC populations or erythroid progenitor cells can be used for more efficient in vitro red blood cell production, or in other embodiments, can be used to deliver HSC or erythroid progenitor cells to patients in need to reduce or eliminate the need for regular blood transfusions. In addition, since such cells can be gene-edited to delete certain HLA genes (as described above), HSC populations and erythroid progenitor cells can be easily HLA-matched with recipients. According to the present disclosure, HSCs differentiate into various hematopoietic lineages (similar to bone marrow CD34+ cells) and are able to restore the recipient's hematopoietic system.

[0012] In some embodiments, iPSC differentiation is carried out 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 day 7 to day 14 (such as, for example, day 8, day 9, day 10, day 11, day 12, day 13 or day 14) of iPSC differentiation. EHT can be induced in CD34+ cells harvested from differentiated iPSCs by any known method. In some embodiments, EHT produces HSCs by endothelial cells or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological and / or genetic means (e.g., by stimulation, inhibition and / or genetic modification).

[0013] In some embodiments, the method comprises increasing the expression or activity of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) in PSCs, embryoid bodies, CD34+ enriched cells, ECs, HECs, or HSCs, which can be performed 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 a mechanosensitive receptor or mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol.

[0014] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from cultures undergoing an endothelial to hematopoietic transition and optionally expanded in culture. Hematopoietic stem cells (HSCs) that give rise to erythroid, myeloid, and lymphoid lineages can be identified and optionally sorted or enriched based on the expression of CD34+ and the absence of lineage-specific markers (referred to as Lin-).

[0015] In certain embodiments, HSC colony or its fraction is differentiated into erythrocyte or its progenitor cell or derivative.For example, HSC colony (or the cell separated therefrom) is cultured together with, for example, EPO, IL-3 and SCF (and / or other extracellular matrix components) and / or its combination, to produce a colony comprising erythroid progenitor cells or derivative cell colonies (e.g., erythrocytes). The HSC colony of the cell produces a high percentage of erythroid burst-forming unit (BFU-E) cells and erythroid colony-forming unit (CFU-E) cells, which are indicators of inducing erythropoiesis.

[0016] In other aspects, the present invention provides an erythroid 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 carrier. The pharmaceutical composition may comprise at least about 10 7 The pharmaceutical composition can 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.

[0017] In certain aspects, a HSC composition or erythroid progenitor cell having a high responsiveness EPOR is provided and can be produced by the methods described herein. In some embodiments, the HSC or erythroid progenitor cell is HLA-A neg , homozygous for both HLA-B and HLA-C), and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSC is further homozygous for HLA-DRB1. The cell composition of the present disclosure may further comprise a pharmaceutically acceptable carrier or excipient suitable for intravenous infusion or other routes of administration, and the composition may comprise a suitable antifreeze. An exemplary carrier is DMSO (e.g., about 10% DMSO). The cell composition can be provided in unit vials or pouches and stored frozen until use.

[0018] Cells produced according to the present disclosure can be administered or used, for example, in therapies for inherited or acquired red blood cell disorders, bone marrow failure disorders, altitude-related 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 anemias, nutritional anemias (e.g., iron deficiency anemia and folate deficiency), heme production disorders (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 materials to treat human diseases or physiological or pathological conditions.

[0019] In certain embodiments, the present disclosure provides compositions and methods for treating anemia. In an embodiment, the present disclosure provides hematopoietic stem cell (HSC) compositions or erythroid progenitor cell compositions that can provide long-lasting and effective anemia cell therapy. In an embodiment, HSC is overloaded with truncated and / or mutated EPORs, resulting in hyperresponsive EPORs. In an embodiment, the compositions and methods of the present disclosure can provide patients with enough red blood cells to supply healthy oxygenation levels.

[0020] In various embodiments, the present disclosure provides a method for treating a subject in need of red blood cell production, comprising administering to the subject an HSC or erythroid progenitor cell composition of the present disclosure. Thus, the HSC produced according to the present disclosure can be used to produce red blood cells in vivo in a durable and effective manner. In some embodiments, the recipient subject suffers from anemia. In some embodiments, the recipient subject suffers from sickle cell anemia, aplastic anemia, anemia associated with bone marrow disease or bone marrow failure, hemorrhagic anemia, or hemolytic anemia. In some embodiments, the recipient subject suffers from Fanconi anemia. In some embodiments, the subject suffers from thalassemia. In some embodiments, the HSC or erythroid progenitor cell is used to prepare a blood product for treating complications associated with blood, bone marrow, immune, metabolic, or mitochondrial disorders.

[0021] Other aspects and embodiments of the present disclosure will become apparent from the following detailed disclosure and working examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It was shown that ETV2 overexpression (OE) did not affect pluripotency. Figure 1FACS plots showing the transduction efficiency of iPSCs using adenoviral vectors overexpressing ETV2 and GFP sequences. ETV2 overexpression did not affect the stem cell properties of iPSCs, as shown by expression of the TRA-1-60 stem cell marker.

[0023] Figure 2 ETV2 overexpression (OE) was shown to increase the yield of hemogenic endothelial cells. Representative flow cytometric analysis and relative quantification of hemogenic endothelial cells (depicted as CD235a-CD34+CD31+) demonstrated that ETV2-OE enhanced the formation of hemogenic endothelial cells.

[0024] Figure 3 Figure 3: ETV2 overexpression (OE) enhances CD34+ cell formation during iPSC differentiation. Representative flow cytometric analysis of CD34+ cells, and relative quantification indicates that ETV2-OE enhances CD34+ cell formation.

[0025] Figure 4A and Figure 4B We show that iPSC-derived HSCs derived from EHTs of CD34+ cells (activated with Piezol in this example) undergo pro-T cell differentiation similar to bone marrow (BM)-HSCs. Figure 4A Figure 3 is a FACS graph showing the differentiation efficiency of bone marrow (BM) HSCs and iPSC-HSCs (activated with Piezol in this example) from EHTs that are CD34+ cells into 34+CD7+ pro-T cells. Figure 4B The numbers of CD34+CD7+ cells (%) derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation) are quantified. Figure 4B The mean of three experiments is shown.

[0026] Figure 5A and Figure 5B It was shown that iPSC-derived HSCs generated using EHT (activated with Piezol in this example) undergo T cell differentiation and can be activated by CD3 / CD28 beads similarly to BM-HSCs. Figure 5A Figure 3 is a FACS graph showing the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs (generated by activation with Piezol in this example). Figure 5B The quantification of CD3+CD69+ cells (%) derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation). Figure 5B The mean of three experiments is shown.

[0027] Figure 6It was shown that iPSC-derived HSCs generated by EHT of CD34+ cells (activated with Piezo1 in this example) can differentiate into functional T cells. IFNγ expression is a result of T cell activation after T cell receptor (TCR) stimulation via CD3 / CD28 beads. IFNγ expression in T cells differentiated from iPSC-derived HSCs (EHT of D8 34+ cells, including activation with Piezo1) enhances the ability of HSCs to further differentiate into functional hematopoietic lineages (in this case, T cells). Figure 6 The mean of three experiments is shown.

[0028] Figure 7A and Figure 7B Phenotypic analysis of HLA-edited (e.g., triple knockout) cells by FACS and immunofluorescence is shown. Figure 7A The overall expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface was shown, where HLA-edited cells were positive for overall HLA class I expression to a similar extent as wild-type cells (gHSCs). Figure 7B Cellular expression of HLA-A was shown via immunofluorescence, where HLA-A was not expressed in HLA-edited clones.

[0029] Figure 8 Clones showing HLA editing retained their pluripotency (maintained trilineage differentiation) as shown by immunofluorescence, where ectodermal differentiation was indicated by nestin-488 and PAX6-594 staining, mesodermal differentiation was indicated by GATA-488 staining, and endoderm differentiation was indicated by CXCR4-488 and FOX2A-594 staining.

[0030] Figure 9 The immunocompatibility of HLA-edited HSCs was shown. HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were co-cultured with HLA-B and HLA-C matched but HLA-A mismatched peripheral blood mononuclear cells (PBMCs), and PBMC-mediated cytotoxicity was measured by Annexin V staining assay.

[0031] Figure 10 To demonstrate the in vivo transplantation potential of HLA-edited HSCs, equal ratios of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed and used for competitive transplantation into mice, where bone marrow (BM) and peripheral blood samples were assessed by FACS to compare the relative amounts of each cell type present in the samples.

[0032] Figure 11A and 11B showed that the absence of HLA-A did not affect the presentation of class I peptides. Figure 11ASchematic diagram showing immunopeptidomic analysis. Figure 11B Results of immunopeptidomic analysis are shown, showing minimal differences in the number of peptides and representative proteins presented by class I molecules between WT and HLA-edited cells.

[0033] Figure 12A and 12B showed that the absence of HLA-DP and DQ did not affect the presentation of class II peptides. Figure 12A Shown is a graph of the immunopeptidomic analysis. Figure 12B showed that despite the loss of HLA-DP and DQ, cells still retained their ability to present a broad spectrum of peptides via HLA class II.

[0034] Figure 13 Schematic diagram of in vivo testing of antigen-mediated immune responses: delayed-type hypersensitivity assay (DTH), sensitization phase, and elimination phase.

[0035] Figure 14A and 14B HLA-edited HSCs were shown to reconstitute a functional immune system, as demonstrated by DTH responses in immunodeficient mice. Figure 14A Shown is a delayed-type hypersensitivity assay performed on transplanted mice, which involves the interaction of different types of immune cells. Mice were sensitized by subcutaneous injection of sheep red blood cells (antigen). A functional immune system resulted in swelling of the left paw, measured with a microcaliper. Figure 14A As can be seen in the figure, non-transplanted mice did not show any swelling of their left paw due to their immune deficiency. In contrast, mice transplanted with umbilical cord blood CD34+ cells showed tissue swelling, and the diameter of their left paw doubled. Figure 14B Yes Figure 14A Graphical evaluation of the results shown.

[0036] Figure 15 The potential of HSCs to differentiate into T cell subsets was demonstrated. After a 35-day differentiation period, naive T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. Figure 15 The differentiation potential of bone marrow-derived CD34+ cells, embryoid body CD34+ cells, and HSCs ("gHSCs") (eg, activated using Piezol) prepared according to the present invention were compared.

[0037] Figure 16 The extent of T cell-mediated cytotoxicity measured from co-cultures of HSC-derived T cells with CD19+ lymphoma cells in the presence of an anti-CD3 / CD-19 bispecific antibody is shown. According to the present disclosure, T cells prepared from gHSCs ("gHSCs") exhibit high levels of cytotoxicity against target cells.

[0038] Figure 17The ability of HSCs to develop into pro-T cells was shown as measured by their CD34-CD7+ markers.

[0039] Figure 18A and 18B It was shown that according to the present disclosure, the expression of T cell-specific transcription factors and thymic engraftment molecules is increased in proto-T cells derived from HSCs. Figure 18A showed TCF7 mRNA expression, Figure 18B CCR7 mRNA expression is shown.

[0040] Figure 19A and 19B showed that HSC-derived proto-T cells engrafted and differentiated in the thymus. Figure 19A Demonstrate implantation and analysis procedures. Figure 19B Shown is a FACS analysis of the CD3 cell population gated on the CD45+ cell population, demonstrating that HSC-derived proto-T cells have excellent engraftment and differentiation potential in the thymus.

[0041] Figure 20 Show that HSC-derived T cells can be activated in vitro. The top panel shows FACS analysis of activated T cells from different sources (including HSC prepared according to the present disclosure). The T cells of the present disclosure show comparable or better activation, as measured by increased expression of CD107. The bottom panel shows Dynabeads activation, in which the activated T cells express inflammatory cytokines. HSC-derived T cells express higher levels of inflammatory cytokines, as illustrated by the expression levels of TNF-α and interferon gamma.

[0042] Figure 21A and 21B We showed that both WT and HLA-edited HSCs could differentiate into the monocyte / macrophage lineage and also retained global expression of both class I and class II molecules, as identified by CD11b+CD14+ markers ( Figure 21A ). Figure 21B HLA-I and HLA-II analysis of cells gated on CD11b+CD14+ is shown.

[0043] Figures 22A to 22C The results showed that the loss of HLA-DQB1 and HLA-DPB1 did not affect the expression of other HLA class II molecules. Figure 22A Schematic diagram of HLA-edited iPSCs differentiating into macrophages. Figure 22B Immunofluorescence experiments confirmed the specific deletion of DPB1 and DQB1 molecules. Figure 22C The same cells were shown to retain expression of class II DRB1.

[0044] Figure 23The results show that HLA-edited HSCs can differentiate into megakaryocytes (MKs), which can further differentiate into platelets. The image on the left shows that the proportion of platelets in HSCs is increased by optical microscopy at 1000x magnification. The graph on the right shows that the proportion of platelets differentiated from HLA-edited HSCs is statistically significantly increased compared to BM CD34+ and iPSC-34+ cell populations.

[0045] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0046] The terms "wild type" (WT), "unedited," and "non-HLA edited" are used interchangeably herein to refer to the non-gene-edited cells of the present disclosure.

[0047] EB34+ cells refer to CD34+ cells derived from embryonic bodies. These include hematopoietic endothelial cells. DETAILED DESCRIPTION

[0048] The present disclosure provides in various aspects and embodiments a method for producing a hematopoietic lineage for cell therapy, including gene-edited hematopoietic stem cells (HSC), erythroid progenitors, progenitor erythroblasts, granulocyte-macrophage progenitors (GMP), megakaryocyte erythroblast progenitors (MEP) and erythroid cells. In various embodiments, the present invention provides an efficient in vitro method for developing such hematopoietic lineages (including but not limited to progenitor erythroblasts and erythroblast lineages) from human induced pluripotent stem cells (iPSC). The cells produced in various embodiments according to the present disclosure are functional and / or more closely resemble the corresponding lineages isolated from peripheral blood or bone marrow. The present invention also provides separated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

[0049] In other aspects and embodiments, the present disclosure provides HSCs derived from iPSCs that have been gene-edited to encode a highly responsive EPO receptor. These HSC populations can be used for more efficient in vitro red blood cell production, or in other aspects, can be used to deliver HSCs or erythroid progenitor cells to patients in need to reduce or eliminate the need for regular blood transfusions.

[0050] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to generate essentially unlimited pluripotent stem cells (PSCs) is used to generate an unlimited supply of hematopoietic cells, including but not limited to the production of therapeutic human erythrocytes or red blood cells ("RBCs") or their erythroid progenitors. The use of RBCs for therapeutic purposes is severely limited by their availability, cell number, and limited expansion potential. In addition, hiPSCs can be more easily genetically modified in vitro compared to primary cells, thereby providing improved cell targeting specificity, cell number, and bypassing issues such as HLA matching. In addition, fully engineered hiPSC clones can serve as a stable and safe source compared to primary cells (Nianias and Themeli, 2019). In addition, since hiPSCs, unlike human embryonic stem cells (hESCs), are of non-embryonic origin, they also have no ethical concerns. Therefore, the use of hiPSCs according to the present disclosure has several advantages over primary cells to produce therapeutic hematopoietic lineages (such as erythroid lineages).

[0051] On the one hand, the present disclosure provides a method for preparing a cell colony of hematopoietic lineage. The method includes preparing a pluripotent stem cell (PSC) colony, such as an induced pluripotent stem cell (iPSC) colony that is differentiated into an embryoid body, and enriching CD34+ cells, so as to prepare a colony enriched in CD34+. Inducing endothelial cells to hematopoietic cell transition (EHT) in a CD34+ enriched colony, so as to prepare a hematopoietic stem cell (HSC) colony, optionally further enriching CD34+ cells afterwards. In certain embodiments, gained HSC colony (or its fraction) can be differentiated into erythroid hematopoietic lineage. In various embodiments, hematopoietic lineage is selected from erythrocytes (i.e., red blood cells), progenitor cells into erythroblasts, granulocyte-macrophage progenitors (GMPs) and megakaryocyte erythroid progenitors (MEPs).

[0052] Traditionally, hematopoietic lineages are prepared by differentiating iPSCs into embryoid bodies until day 8 to harvest CD34+ cells. CD34 is commonly used as a marker for hematopoietic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it was found that endothelial to hematopoietic transition (EHT) of a CD34+ cell population was induced and that it can be derived from iPSC embryoid bodies, which can be used to generate advanced hematopoietic stem cells and hematopoietic lineages, including erythroid lineages, in vitro.

[0053] In certain embodiments, the present disclosure provides a method for producing a erythroblast colony (e.g., early erythroid progenitor cells, late erythroid progenitor cells, and morphologically identifiable erythroid precursors) or a derivative thereof. For example, the method includes producing a hematopoietic stem cell (HSC) colony comprising human long-term hematopoietic stem cells (LT-HSC) from iPSC (e.g., hiPSC). The HSC colony is obtained by inducing endothelial cells of CD34+ cells (e.g., CD34+ cells derived from embryoid bodies) to transform into hematopoietic cells. The HSC colony (or the cell separated therefrom) is cultured together with, for example, EPO, IL-3, and SCF (and / or other extracellular matrix components) and / or a combination thereof to produce a colony comprising erythroid progenitor cells or derivative cell colonies (e.g., erythrocytes). The HSC colony of the cell produces a high percentage of erythroid burst-forming unit (BFU-E) cells and erythroid colony-forming unit (CFU-E) cells, which are indicators of induced erythropoiesis.

[0054] Aspects and embodiments of the present disclosure can be used to produce reticulocytes and / or denucleated mature erythrocytes from cells differentiated or derived from hematopoietic stem cell (HSC) populations, comprising long-term hematopoietic stem cells (LT-HSC) derived from iPSC (e.g., hiPSC). For example, iPSC-derived HSC can be cultured under erythroid differentiation conditions in a bioreactor (e.g., a stirred bioreactor), and when cultured under optimal physical conditions (pH ranging from about 7.0 to about 7.5, about 25% to about 75% oxygen (e.g., about 50% oxygen), without gas bubbling, and mechanically stirred (impeller speed of about 300-450rev / min)), it will produce denucleated cells with a purity of about 80%. Supplementary compounds (such as cytokines and growth factors) can be added to erythroid differentiation conditions to promote the yield of denucleated cells.

[0055] In various embodiments, iPSC is prepared by reprogramming somatic cells. The term "induced pluripotent stem cell" or "iPSC" 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 certain embodiments, iPSC is produced by somatic cells, such as, but not limited to, fibroblasts or PBMCs (or cells separated therefrom). In certain embodiments, iPSC derives from lymphocytes (e.g., T cells, B cells, NK cells, etc.), cord blood cells, PBMCs, CD34+ cells or other human primary tissues. In certain embodiments, iPSC derives from CD34+ cells separated from peripheral blood, bone marrow or cord blood. In various embodiments, iPSC is autologous or allogeneic (e.g., HLA matching at one or more loci) for a recipient (needing a subject treated as described herein). In various embodiments, iPSC can be gene-edited to help HLA matching (such as making one or more HLA I class and / or II class alleles or their master regulators missing, including but not limited to beta-2-microglobulin (B2M), CIITA, etc.), or gene-edited to make other functions missing or express other functions. For example, iPSC can be gene-edited to make one or more of HLA-A, HLA-B and HLA-C missing, and one or more of HLA-DP, HLA-DQ and HLA-DR missing. In certain embodiments, iPSC retains the expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSC is homozygous for at least one retained class I and class II locus. In certain embodiments, iPSC derives from cord blood CD34+ cells or CD36+ into erythrocytes. iPSC can derive from CD34+ cells forming universal donor erythrocytes (i.e., O type). Other blood types can also be used.

[0056] In various embodiments, HSCs and erythroid lineage cells derived therefrom are derived from iPSCs that have been genetically edited to one of the following: (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-, (vi) HLA-AB-C+DP-DR+DQ-. For the retained HLA (e.g., HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or 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-.

[0057] In some embodiments, HSCs and erythroid lineage cells derived from iPSCs are gene-edited to express 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.

[0058] As used herein, the term "negative" (-) or "negative" for a specific HLA class or class II molecule indicates that both copies of the gene have been destroyed in a cell line or population, and therefore the cell line or population does not show significant functional expression of the gene. Such cells can be produced by total or partial gene deletion or destruction, or alternatively by other techniques such as siRNA production. As used herein, the term "deletion" in the context of genetic modification (i.e., gene editing) of a target gene refers to the abolition of functional expression of the corresponding gene product (i.e., corresponding polypeptide). Such gene editing includes total or partial gene deletion or destruction of a coding sequence, or the deletion of a key cis-acting expression control sequence.

[0059] In some embodiments, iPSC is gene-edited using a gRNA having a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more nucleotides. In some embodiments, the gRNA is included in a modification at or near the 5' end (e.g., 1 to 10, 1 to 5, or 1 to 2 nucleotides at the 5' end) and / or at or near the 3' end (e.g., 1 to 10, 1 to 5, or 1 to 2 nucleotides at the 3' end). In some embodiments, the modified gRNA exhibits increased resistance to nucleases. In some embodiments, the gRNA comprises two separate RNA molecules (i.e., "double gRNA"). Double gRNA comprises two separate RNA molecules: "crispr RNA" (or "crRNA") and "tracr RNA", and is well known to those skilled in the art.

[0060] Generally, various gene editing technologies are known and can be applied according to various embodiments of the present disclosure.Gene editing technologies include but are not limited to zinc fingers (ZFs), transcription activator-like effectors (TALEs), and the like. Fusion proteins containing one or more of these DNA binding domains and the cleavage domain of the Fok1 endonuclease can be used to generate double-strand breaks in desired regions of DNA in cells (see, e.g., U.S. Patent Application Publication No. US2012 / 0064620, U.S. Patent Application Publication No. US2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. US 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. US2011 / 0301073, U.S. Patent Application Publication No. US2011 / 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, all of which are hereby incorporated 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, US 8,697,359, US 8,906,616, and US 8,999,641, each of which is hereby incorporated 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 RNA-guided. The design of gRNAs that guide the 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,FEBS J. 2016 Sep;283(17):3232–3238. In other embodiments, non-canonical type II or type V Cas endonucleases with homology (albeit low primary sequence homology) to Streptococcus pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) may 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 other embodiments, gene editing employs base editing or primer editing to incorporate mutations without inducing double-strand breaks. See, e.g., Antoniou P et al., Base and Prime Editing Technologies for Blood Disorders , Front.Genome editor, January 28, 2021; Matsuokas IG, Prime Editing:Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA , Front. Genet., June 9, 2020. Various other gene editing processes are known, including the use of dead Cas (dCas) systems (e.g., Cas fusion proteins) to direct DNA-modifying enzymes to desired targets and the use of dCas as a guide RNA guidance system. Brezgin S, Dead Cas Systems:Types,Principles,and Applications ,IntJ Mol Sci. 2019 Dec;20(23):6041.

[0061] Base editors that can install precise genomic changes without producing double-stranded DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., designing gene therapy vectors). Base editors essentially contain catalytically disabled nucleases, such as Cas9 nickelase (nCas9), which cannot produce DSBs and are fused to nucleobase deaminases and, in some cases, to DNA glycosylase inhibitors. Currently, there are two main base editors, cytidine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G conversions. Base editors can be delivered by, for example, HDAd5 / 35++ vectors to efficiently edit promoters and enhancers, thereby activating or inactivating 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. Primer editors comprising a reverse transcriptase conjugated (e.g., fused) to a Cas endonuclease and a polynucleotide conjugated (e.g., fused) to a guide RNA used as a template for DNA synthesis are also contemplated, as described in WO 2020 / 191153.

[0062] Exemplary vectors that can be used for genome editing applications include, but are not limited to, 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, coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses (including herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, Cytomegalovirus) and poxvirus (e.g., canarypox virus, vaccinia virus or modified vaccinia virus)). The vector comprising the nucleic acid molecule of interest can be delivered to cells (e.g., iPS cells, endothelial cells, hematopoietic endothelial cells, HSC (ST-HSC or LT-HSC)) by any method known in the art, including but not limited to transduction, transfection, infection and electroporation. Any of these vectors can include a transposable element (such as a piggyback transposon or a sleeping beauty transposon). The transposon inserts a specific DNA sequence into the genome of the vertebrate. Once excised from the transposon, the gene can be integrated into the genome of the mammalian cell by catalyzing the cutting of a similar excision site present in the nuclear genome by a transposase.

[0063] To improve efficiency, in some embodiments, Cas and gRNA can be combined before being delivered to cells. The Cas-gRNA complex is called a ribonucleoprotein (RNP). Many methods have been developed to deliver RNP directly to cells. For example, RNP can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nuclear transfection protocol can be used, and this procedure allows RNP to quickly enter the cell nucleus so that genome cleavage can begin immediately. See, for example, Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9ribonucleoprotein for CRISPR / Cas9 genome editing .Theranostics. 2021 Jan 1; 11(2): 614-648, which is hereby incorporated by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNPs.

[0064] Typically, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage and is typically found 3 to 4 nucleotides downstream of the cleavage site. A PAM is a short DNA sequence (typically 2 to 6 base pairs in length) that is located after the region of DNA 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 of the HLA locus does not include four Gs, four Cs, GC repeats, or a combination thereof.

[0065] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed by designing a single gRNA targeting each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example), using gRNA as described herein. In order to perform gene knockout, gRNA targets the Cas9 protein to a suitable site for editing. Next, the Cas9 protein can perform double-strand breaks (DSBs), wherein DNA is repaired by a non-homologous end joining (NHEJ) mechanism, which produces indels that cause frameshift mutations and terminates the function of the resulting protein. However, off-target gene modification can occur and change the function of other complete genes. For example, even in the presence of a certain degree of mismatch, the Cas9 endonuclease can still produce DSBs at unwanted off-target positions. This off-target activity can produce genomic instability events, such as point mutations and genomic structural variations. In various embodiments, the sgRNA targeting HLA-A can target the chromosome 6 region 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.

[0066] gRNA can be used to develop cloned iPSCs. Such iPSC lines can be evaluated for (i) on-target editing, (ii) off-target editing, and (iii) translocation editing, for example, using sequencing, as described herein. Specifically, such an assay can be performed by multiplex PCR, which utilizes primers designed to target and enrich the region of interest, followed by next-generation sequencing (e.g., amplicon sequencing, AMP-seq). The on-target group and the translocation group can amplify the expected editing region, allowing the selection of iPSC clones with the expected editing, which do not have chromosomal translocations caused by unexpected DSB cleavage site fusions. The off-target group can enrich any potential off-target regions identified by sequencing, and allows the selection of iPSC clones with negligible off-target mutations. In summary, these assays enable the screening of iPSC clones to select clones with the desired editing while excluding potential CRISPR / Cas9-related genomic integrity issues.

[0067] In certain embodiments, in order to further ensure the genomic stability and integrity of the reprogrammed and edited iPSC, genetic and genomic assays can be performed to select clones that have not undergone translocation and mutation events and have not integrated episomal vectors. For example, whole genome sequencing (WGS) is performed on CD34+ cells and iPSC clones after reprogramming, wherein the differences in genomes due to editing are compared. These analyses provide an assessment of which iPSC clone genomes are different from the CD34+ starting material, enabling the judicious selection of iPSC clones that do not produce mutations during reprogramming.

[0068] In some embodiments, karyotyping using a system such as the KARYOSTAT assay is used to select iPSC clones that do not generate indels and 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 ,” described in Data Brief. 2021 May 15;37:107140, which is hereby incorporated by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal aberrations with a resolution similar to G-banded karyotyping. Structural abnormalities >2 Mb in size can be detected for chromosome gains and >1 Mb in size for chromosome losses. The KARYOSTAT array is functionalized for balanced whole-genome coverage by low-resolution DNA copy number analysis, with the assay covering all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay is capable of detecting aneuploidies, submicroscopic aberrations, and mosaic events.

[0069] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that do not develop copy number aberrations (CNAs) during reprogramming, e.g., as described in Wiesner et al. Molecular Techniques ”, edited by Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, “Pathology of Melanocytic Tumors,” Elsevier, 2019, pp. 364-373, ISBN 9780323374576; and Hussein SM, et al. “Copynumber variation and selection during reprogramming to pluripotency,” Nature. 2011 Mar 3; 471(7336): 58-62, which are hereby incorporated by reference in their entirety. aCGH is a technique for analyzing the entire genome of CNA by comparing sample DNA to reference DNA.

[0070] In some embodiments, targeted heme malignancy NGS panels are used to select iPSC clones that do not acquire hematologic malignancy mutations during reprogramming. For example, a targeted heme malignancy NGS panel can focus on genes associated with myeloid leukemia, lymphoma, and / or other hematologic malignancies to generate a smaller, more manageable dataset than a broader approach. Targeted heme malignancy NGS panels involve the use of highly multiplexed PCR to amplify regions associated with hematologic malignancies, followed by next-generation sequencing.

[0071] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that do not integrate additional vectors and have been passaged enough to perform additional vector removal. As described herein, iPSC reprogramming of CD34+ cells can be achieved by delivering additional vectors encoding reprogramming factors. However, although rare, additional vectors can be randomly integrated into the cell genome, which may disrupt developmental processes, homeostasis, etc. Therefore, the ddPCR method can be used to detect residual additional vectors in iPSC cultures, and it is possible to select iPSC clones that do not integrate additional vectors.

[0072] In some embodiments, after evaluating the selected clones for the absence of editing-related genomic aberrations, the clones can be additionally tested for spontaneous mutations that may have occurred during expansion. For example, mutations affecting hematological malignancy genes, indels, translocations, quantitative aberrations, for example, as described for pre-edited reprogrammed clones. Analysis of spontaneous mutations may include whole genome sequencing (WGS), KARYOSTAT analysis, array comparative genomic hybridization (aCGH) analysis, targeted heme malignancy NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0073] Somatic cells can be reprogrammed by expressing reprogramming factors selected from Sox2, Oct3 / 4, c-Myc, Nanog, Lin28 and klf4. In some embodiments, reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28 and klf4. In some embodiments, reprogramming factors are Sox2, Oct3 / 4, c-Myc and klf4. In some embodiments, 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 in, for example, U.S. Patent No. 10,676,165; U.S. Patent No. 9,580,689; U.S. Patent No. 10,221,395, and U.S. Patent No. 9,376,664, which are hereby incorporated by reference in their entirety. In various embodiments, well-known viral vector systems such as slow virus, Sendai virus or measles virus systems are used to express reprogramming factors. Alternatively, reprogramming factors can be expressed by importing the mRNA encoding reprogramming factors into somatic cells. In addition, iPSC can be produced by introducing a non-integrated episomal plasmid expressing reprogramming factors, that is, for producing iPSC without transgenic and virus-free. Known episomal plasmids can be used, which have limited replication capacity and are therefore lost after several generations of cells. In certain embodiments, the method includes using a vector (for example, a viral vector or an episomal vector) expressing POU5F1 / OCT4, SOX2, KLF4 and c-MYC as a polycistronic unit to reprogram CD36+ basophils into erythroblasts to obtain pluripotency. In certain embodiments, the method includes reprogramming PBMC with Oct4, Sox2, Lin28, Klf4 and L-myc.

[0074] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are gene-edited. Gene editing can include, but is not limited to, modification of HLA genes (e.g., deletion of one or more HLA class I and / or class II genes), deletion of β2 microglobulin (β2M), and deletion of CIITA.

[0075] In some embodiments, the iPSCs disclosed herein are gene-edited to encode a reactive EPO receptor. Erythropoiesis is the process by which red blood cells are produced. Erythropoietin (EPO) is a key hormone responsible for effective erythropoiesis. Erythropoietin receptor (EPOR) is a protein encoded by the EPOR gene. EPOR is a 52kDa peptide with a monosaccharide chain, resulting in the formation of a protein of approximately 56 to 57kDa on the surface of EPO-responsive cells. The most confirmed function of EPOR is to promote proliferation and rescue erythroid (red blood cell) progenitor cells from apoptosis.

[0076] Beneficial mutations have been reported in EPOR, where an increase in the number of red blood cells allows for improved oxygen delivery during endurance sports without significant adverse effects on the athlete's health. de La Chapelle et al., PNAS 1993;90.10:4495-4499. It has been reported that, in general, truncating mutations that remove only the portion of the intracellular C-terminus of EPOR that binds to negative regulators are associated with primary erythrocytosis, with decreased EPO levels and increased hemoglobin levels. These mutations render EPOR hyperresponsive to EPO, with the side effect of increased 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 binding to the receptor through 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 Jak2kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1 .Molecular and Cellular Biology, 1996; 16(12), 6985–6992.

[0077] Thus, in various embodiments, the HSC populations of the present disclosure express EPORs with truncating mutations. In some embodiments, the HSC populations of the present disclosure express EPORs that lack or have a mutated SHP-1 inhibitory domain. In some embodiments, the HSC populations of the present disclosure express EPORs with one or more missense or frameshift mutations that result in hyperresponsiveness, optionally achieved by mutation or deletion of the SHP-1 inhibitory domain.

[0078] These HSC populations can be used for more efficient in vitro red blood cell production, or in other embodiments, can be used to deliver HSC or erythroid progenitor cells to patients in need to reduce or eliminate the need for regular blood transfusions. In addition, because such cells can be gene-edited to delete certain HLA genes (as described above), HSC populations and erythroid progenitor cells can be easily HLA-matched with recipients. According to the present disclosure, HSCs differentiate into various hematopoietic lineages (similar to bone marrow CD34+ cells) and are able to restore the recipient's hematopoietic system.

[0079] In various embodiments, iPSCs are prepared and expanded using conventional culture systems. Amplified iPSCs can be recovered from the culture to produce embryoid bodies (EBs). The EBs produced by iPSC differentiation are three-dimensional aggregates of iPSCs and contain three (or two or one) embryonic germ layers based on the differentiation method. For example, the preparation of EBs is described in US2019 / 0177695, which is hereby incorporated by reference in its entirety. In some embodiments, EBs prepared by differentiation of iPSCs are expanded in bioreactors, such as Abecasis B. et al., 3D人类诱导多能干细胞的扩增 生物反应器中的聚集体:生物过程强化与放大 方法 .J.of Biotechnol.246(2017)81-93. EBs can be used to generate any desired cell type. Other methods for the expansion or differentiation of EBs, including 3D suspension culture, are described in WO 2020 / 086889, which is hereby incorporated by reference in its entirety.

[0080] In some embodiments, the method according to each aspect may comprise generating CD34+ enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial cell to hematopoietic cell differentiation. HSCs comprising a relatively high frequency of LT-HSCs may be generated from a cell population using various stimuli or factors, including mechanical, biochemical, metabolic, and / or topographical stimulation, 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.

[0081] In some embodiments, the method includes preparing endothelial cells with hematopoietic potential from pluripotent stem cells before inducing EHT. In some embodiments, the method includes overexpressing the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSC. ETV2 can be expressed by introducing a non-integrating episomal plasmid encoding for constitutive or inducible expression of ETV2, and for producing transgenic-free hematopoietic EC. In some embodiments, ETV2 is expressed by mRNA introduced into iPSC. mRNA can be introduced using any available method, including electroporation or lipofection. Differentiation of cells expressing ETV2 can include adding VEGF-A. See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 withmRNA. Sci. Adv. Vol. 6 (2020). According to embodiments of the present disclosure, cells produced in this manner can be used to produce CD34+ cells and induce EHT.

[0082] Following CD34+ enrichment, HSCs are generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification.

[0083] In some embodiments, iPSC differentiation is carried out 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 the 7th to 14th day (such as, for example, the 8th day, the 9th day, the 10th day, the 11th day, the 12th day, the 13th day or the 14th day) of iPSC differentiation. In some embodiments, CD34+ cells are harvested on about the 8th day. The differentiation of iPSC can be carried out according to known techniques. In some embodiments, iPSC differentiation involves the following 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, hPSC is differentiated using feeder-free, serum-free and / or GMP-compatible materials (such as pomalidomide or lenalidomide). In some embodiments, hPSCs are co-cultured with mouse bone marrow-derived feeder cells (such as OP9 or MS5 cell lines) in a serum-containing medium. The culture may contain growth factors and cytokines to support differentiation of embryoid bodies or monolayer systems. The OP9 co-culture system can be used to generate multipotent HSPCs, which can further differentiate into several hematopoietic lineages, including T lymphocytes, B lymphocytes, megakaryocytes, monocytes or macrophages, and erythrocytes. See Netsrithong R. et al., 多谱系分化 的潜力 源自人类诱导的 多能干细胞的造血内皮祖细胞 , Stem Cell Research & Therapy 11, 481 (2020). Alternatively, a stepwise process using defined conditions with specific signals can be used. For example, the expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs favors direct differentiation into CD34+ / CD45+ progenitor cells with multilineage potential. In addition, the expression of factors such as HOXB4, CDX4, SCL / TAL1, or RUNX1a supports the hematopoietic program in human PSCs. See Doulatov S. et al., 通过 重编程 从人类多能 干细胞诱导多能造血祖细胞 谱系受限的前体细胞 , CellStemCell. 2013 Oct 3; 13(4).

[0084] Induction of EHT can be performed using any known method. In some embodiments, induction of EHT produces a hematopoietic stem cell (HSC) population comprising LT-HSC. In some embodiments, EHT produces HSC through endothelial cells or hematopoietic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological and / or genetic means (e.g., by stimulation, inhibition and / or genetic modification). In some embodiments, EHT produces a stem cell population comprising one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC) and hematopoietic stem cell progenitors.

[0085] In some embodiments, the method includes increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34+ enriched cells, ECs, HECs or HSCs, which can be performed by mechanical, genetic, biochemical or pharmacological means. In some embodiments, the method includes increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in cells. See WO 2019 / 236943 and WO 2021 / 119061, which are hereby incorporated by reference in their entirety. In some embodiments, the induction of EHT includes increasing the expression or activity of dnmt3b.

[0086] In some embodiments, the cell is contacted with an effective amount of a mechanosensitive receptor or mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. An exemplary Piezol agonist is Yodal. 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, 机械转导通道Piezo1的化学激活 的 Yoda1类似物(Dooku1)拮抗Yoda1诱导的Piezo1激活和主动脉舒张 .eLife (2015). Yoda 1 has the following structure:

[0087]

[0088] Derivatives of Yodal can be used in various embodiments. For example, in some embodiments, derivatives containing a 2,6-dichlorophenyl nucleus are used. Exemplary agonists are disclosed in Evans EL, et al., 一种杠杆样 转导, British J. of Pharmacology 175(1744-1759):2018. Other Piezo1 agonists include Jedi1, Jedi2, single-stranded (ss) RNA (e.g., ssRNA40) and their derivatives and analogs. See Wang Y et al., 途径,用于机械敏感的Piezo1通道的长距离化学和机械门控 肠道Piezo1的RNA传感对于全身血清素合成至关重要 镰状细胞 病: Nature Communications (2018) 9:1300; Sugisawa et al., 何时以及如何输血 , Cell, Volume 182, Issue 3, 2020, Pages 609-624, incorporated herein by reference in its entirety. These Piezol agonists are commercially available. In various embodiments, the effective amount of the Piezol agonist or derivative is in 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, in the range of about 25 μM to about 150 μM, or about 25 μM to about 100 μM, or about 25 μM to about 50 μM.

[0089] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34+ enriched cells). In certain embodiments, pharmacological Piezo1 activation can be further applied to iPSCs, embryoid bodies, ECs, hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineages. In certain embodiments, Piezo1 activation is applied to at least iPSCs generated from iPSCs, EBs, or CD34+ cells isolated from EBs, and / or a combination thereof, which, according to various embodiments, allows for better generation of erythroid progenitor cells compared to other methods for inducing EHT.

[0090] In some embodiments, Piezol activation is not used during induction of EHT.

[0091] In certain embodiments, the expansion of hematopoietic cells may further comprise contacting the cells with an immunomodulatory compound (e.g., a TNF-α inhibitory compound) for a period of time and in an amount sufficient to significantly increase the proliferation of the hematopoietic cells within a given time period (compared to an equivalent number of hematopoietic cells not contacted with the immunomodulatory compound). See, for example, U.S. Patent No. 7,498,171, the disclosure of which is hereby incorporated 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'-aminoisoindolin-1'-one)-1-piperidine-2,6-dione; 4-(amino)-2-(2,6-dioxo(3-piperidinyl))-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.

[0092] Alternatively or additionally, the activity or expression of Dnmt3b can be increased directly in cells (e.g., in cells enriched for CD34). For example, Dnmt3b mRNA expression can be increased by delivering transcripts encoding Dnmt3b to cells, or by introducing a transgene encoding Dnmt3b, or by non-transgenic methods (including but not limited to introducing non-integrating episomes into cells). In some embodiments, gene editing is used to introduce genetic modifications to Dnmt3b expression elements in cells, such as, but not limited to, increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0093] In some embodiments, the method comprises increasing the activity or expression of Gimap6 in a cell, alone or in combination with Dnmt3b and / or other genes that are upregulated or downregulated upon cyclic strain or piezoelectric activation. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cell, or a non-transgenic approach can be used, 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 used to introduce genetic modifications to Gimap6 expression elements in the cell (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or affect RNA splicing).

[0094] In the embodiment of the present disclosure using mRNA to be delivered to cells, known chemical modifications can be used to avoid the innate immune response in cells. For example, synthetic RNAs comprising only standard nucleotides can bind to pattern recognition receptors and can trigger an effective immune response in cells. This reaction can lead to translational blockade, secretion of inflammatory cytokines and cell death. RNAs comprising certain unconventional nucleotides can escape detection by the innate immune system and can be efficiently translated into protein. Referring to US 9,181,319, which is hereby incorporated by reference, particularly with respect to nucleotide modifications to avoid innate immune responses.

[0095] In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into the cell, which can guide the desired overexpression level (with different promoter strengths or other options for expression control elements). The transgene can be introduced using various viral vectors or transfection reagents known in the art (including lipid nanoparticles). In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by a transgene-free method (e.g., episomal delivery). In some embodiments, gene editing techniques are used to increase the expression or activity of Dnmt3b and / or Gimap6 or other genes disclosed herein, for example, to introduce one or more modifications to increase promoter strength, ribosome binding, or RNA stability.

[0096] In some embodiments, the method includes applying cyclic 2D, 3D or 4D stretching to the cells. In various embodiments, the cells subjected to periodic 2D, 3D or 4D stretching are selected from one or more of CD34-enriched cells, iPSCs, ECs and HECs. For example, a cell colony is introduced into a bioreactor that provides periodic strain biomechanical stretching, as described in WO 2017 / 096215, which is hereby incorporated by reference in its entirety. Periodic strain biomechanical stretching can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, mechanical means applies a tensile force to the cells or a cell culture surface on which cells (e.g., ECs or HECs) are cultured. For example, a computer-controlled vacuum pump system or other components (e.g., FlexCell) for providing tensile forces attached to flexible biocompatible and / or biomimetic surfaces. TMTension system, Cytostretcher system) can be used to apply in vitro periodic 2D, 3D or 4D stretching to cells under limited and controlled periodic strain conditions. For example, the periodic stretching applied can be a periodic strain of about 1% to about 20% (e.g., a periodic strain of about 6%) for several hours or several days (e.g., about 7 days). In various embodiments, the periodic strain is applied for at least about one hour, at least about two hours, at least about six hours, at least about eight 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.

[0097] Alternatively or additionally, EHT is stimulated by Trpv4 activation. Trpv4 activation can be achieved by contacting cells (eg, CD34-enriched cells, ECs, or HECs) with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0098] When a cell colony is described herein as having a certain phenotype, it should be understood that the phenotype represents a significant portion of the cell colony, 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 colony. In addition, at each step, the cell colony can be enriched for cells of the desired phenotype, and / or cells of unwanted phenotypes can be removed so that the cell colony comprises 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 using a fluorescence activated cell sorter or magnetic beads that bind cells to certain cell surface antigens based on cell surface antigens (including those described herein). Negative selection columns can be used to remove cells expressing unwanted cell surface markers. In some embodiments, enriched cells are used for CD34+ cells (before and / or after experiencing EHT). In some embodiments, the cell colony is cultured under conditions that promote the expansion of CD34+ cells, thereby producing an expanded stem cell colony.

[0099] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from a culture undergoing endothelial cells to hematopoietic cell transition. In various embodiments, the cells enriched in HSC or CD34 are further expanded. For example, the cells enriched in HSC or CD34 can be expanded according to the methods disclosed in the following documents: US 8,168,428; US 9,028,811; US10,272,110; and US10,278,990, which are hereby incorporated by reference in their entirety. In certain embodiments, the in vitro expansion of the cells enriched in HSC or CD34 adopts prostaglandin E2 (PGE2) or PGE2 derivatives. In some embodiments of the present disclosure, HSC includes at least about 0.01% LT-HSC, or at least about 0.05% LT-HSC, or at least about 0.1% LT-HSC, or at least about 0.5% LT-HSC, or at least about 1% LT-HSC.

[0100] The hematopoietic stem cells (HSC) that produce erythroid, myeloid and lymphoid lineages can be identified based on the expression of CD34+ cells and the lack of lineage-specific markers (called Lin-). In certain embodiments, the stem cell colony comprising HSC is enriched, for example, as described in US 9,834,754, which is hereby incorporated by reference in its entirety. For example, the method can include sorting cell colonies based on the expression of one or more of CD34, CD90, CD38 and CD43 (HSC markers) or CD71 (early erythroid progenitor cell markers) or CD235a (mature erythroid progenitor cell markers). One or more fractions of CD34+, CD90+, CD38- and CD43- can be selected for further differentiation. In certain embodiments, the stem cell colony for differentiation into hematopoietic lineages is at least about 80% CD34+ or at least about 90% CD34+ or at least about 95% CD34+.

[0101] In some embodiments, stem cell populations, or CD34-enriched cells or fractions thereof, or derived cell populations are expanded as described in US2020 / 0308540, which is hereby incorporated by reference in its entirety. For example, 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.

[0102] In some embodiments, compounds that promote CD34+ cell expansion include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, which is hereby incorporated by reference).

[0103] In some embodiments, stem cell populations or CD34-enriched cells are further enriched for cells expressing periostin and / or platelet-derived growth factor receptor alpha (pdgfra), or are modified to express periostin and / or pdgfra, as described in WO 2020 / 205969 (which is hereby incorporated by reference in its entirety). Such expression can be carried out by delivering the encoded transcript to the cell, or by introducing an encoded transgene, or by a transgenic-free method (not limited to introducing a non-integrated episome into the cell). In some embodiments, gene editing is used to introduce genetic modifications to expression elements in the cell, such as to modify promoter activity or strength, ribosome binding, RNA stability, or to affect RNA splicing.

[0104] In other embodiments, stem cell populations or CD34-enriched cells are cultured with an inhibitor of the histone methyltransferase EZH1. Alternatively, EZH1 is partially or completely deleted or inactivated or temporarily silenced in the stem cell population. Inhibition of EZH1 can guide bone marrow progenitor cells (e.g., CD34+CD45+) to the erythroblast lineage. In other embodiments, EZH1 is overexpressed in the stem cell population.

[0105] In certain embodiments, HSC colony or its fraction is differentiated into erythrocyte or its progenitor cell or derivative.For example, HSC colony (or the cell separated therefrom) is cultivated together with EPO, IL-3 and SCF (and / or other extracellular matrix components) and / or its combination, to produce a colony comprising erythroid progenitor cells or derivative cell colony (for example, erythrocyte). The HSC colony of cell produces high percentage of erythroid burst-forming unit (BFU-E) cell and erythroid colony-forming unit (CFU-E) cell, which are indicators for inducing erythropoiesis. These cells can be further enriched and / or expanded.

[0106] For example, generating an iPS cell line from a peripheral blood sample may include the following initial steps: erythroblast enrichment and iPSC initiation. In the erythroblast enrichment step, cells are reprogrammed, for example, with POU5F1 / OCT4, SOX2, KLF4 and c-MYC transfection (or as otherwise described herein). In certain embodiments, the enriched erythroblast colony exceeds 80%. Once the pluripotency of the 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 iPSC is cultured under culture conditions to produce embryoid bodies (EBs). Between the 8th and 14th days of iPSC differentiation, CD34+ cells (as previously described) are separated / enriched from the dissociated EBs. After inducing EHT in CD34+ cells (optionally, further enriching CD34+ cells and / or enriching with other erythroid progenitor cell markers), the cells are cultured under conditions of erythroid differentiation.

[0107] In some embodiments, an HSC population or fraction thereof is differentiated into erythroid cells or progenitor cells or derivatives thereof without relying on 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. Therefore, in some embodiments, CD34+ cells are enriched from a differentiated pluripotent stem cell population to prepare a CD34+ enriched population. The CD34+ enriched cell population is induced to undergo endothelial cell to hematopoietic cell transformation for at least two days, but no more than 12 days, wherein the use of agonists of mechanosensitive receptors or mechanosensitive channels (such as Yoda1, jedi1, jedi2, ssRNA40) is optional. HSC and / or HSPC are differentiated into a progenitor erythroid cell population or an erythroid cell population.

[0108] In some embodiments, the endothelial to hematopoietic transition of the CD34+ enriched cell population is induced for at least two days, and 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. Typically, the induction of EHT lasts no more than 12 days.

[0109] During differentiation, the proliferative progenitor stage of erythroid committed cells can be assessed by assessing their colony-forming ability in semi-solid culture medium. For example, harvested cells can produce hematopoietic colonies with a high percentage of erythroid burst-forming units (BFU-E) and erythroid colony-forming units (CFU-E). The Hb content of a cell can often be used to determine its developmental stage, for example by assessing the conversion of gamma to beta globin in vitro.

[0110] In certain embodiments, the method utilizes serum-free, xeno-free protocols that comply with good manufacturing practices (GMP). In certain embodiments, the method utilizes feeder layers (such as OP9) or co-cultures with other cells (such as stromal cells as an example). In certain embodiments, the method utilizes small molecules, such as StemRegenin (SR1, RasGAP and ERK1 / 2 dual inhibitors), Yoda1, Jedi1, Jedi2, ssRNA 40 or its analogs or derivatives, BIO (prototype GSK3b inhibitor), CHIR99021 (GSK3b inhibitor), IBMX (non-specific inhibitor of cAMP and cGMP phosphodiesterase) and A-A014418 (GSK3b inhibitor VIII) as a substitute for growth factors or various cytokines, to reduce side effects and culture medium costs.

[0111] In various embodiments and aspects of the present invention, the use of bioreactors, the modification of the microenvironment with macrophages and small molecules, and the use of genetic alterations to enhance the survival of mature RBCs, improve the rate of enucleation, and promote hemoglobin conversion are also contemplated. As an example of using a bioreactor to produce enucleated cells, HSCs or erythroid progenitor cells can be cultured in a bioreactor under one or more maturation conditions. Maturation conditions can include: (i) a predetermined pH; (ii) a predetermined or specific (dissolved) oxygen level; and (iii) mechanical stress.

[0112] The predetermined pH can be selected from a pH of about 4.0 to about 7.9. For example, the ripening conditions can include a pH in the 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 (such as about 7.4 to about 7.5). The ripening conditions can include one or more of the pH values ​​listed above; for example, the ripening conditions can be adjusted between different pH levels, such as between a first pH value and a second pH value.

[0113] Ripening conditions can utilize an oxygen level that is 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, the methods of the present invention can utilize an oxygen level that is less than about 50% of atmospheric oxygen (e.g., about 25% to about 50% of atmospheric oxygen). Ripening conditions can utilize a dissolved oxygen (i.e., oxygen dissolved in the culture medium) level of about 2% to about 29%, such as about 5% to about 20%, or about 5% to about 15% (e.g., about 11%).

[0114] The speed of the bioreactor impeller tip through the cell culture can be controlled to produce mechanical stress levels. About 50 to about 500rpm, for example, about 100 to about 450rpm or about 150 to about 300rpm or about 200 to about 250rpm impeller tip speeds can be used. The mechanical stress level used can be regulated between two or more predetermined mechanical stress levels. In this case, and when using a bioreactor to apply necessary mechanical stress, variable or regulated mechanical stress can be produced via selecting (and using) one or more different impeller speeds. One or more mechanical stress levels can be applied for any suitable time period. For example, one or more mechanical stress levels can be applied for several minutes (for example, 10 to 60 minutes), one or several hours (for example, one to ten hours) or one or several days (for example, one to ten days). For example, one or more mechanical stress levels can be applied continuously or intermittently over the entire time period.

[0115] In an embodiment, an erythroid expansion medium comprising at least EPO and optionally comprising cytokines and growth factors is used, which can be supplemented with one or more small molecule compounds selected from the following: (i) piezo1 agonists, (ii) Trpv4 agonists, (iii) phosphodiesterase inhibitors or (iv) GSK 3 inhibitors, including the compounds and concentrations described herein.

[0116] Another key question for the clinical application of hESC-derived RBCs is whether they can be enucleated in vitro. Under the conditions disclosed herein, RBCs undergo a differentiation event, including a gradual decrease in size and an increase in glycophorin A expression (a marker of mature RBCs) and chromatin / nuclear condensation, which results in the extrusion of pyknotic nuclei to form enucleated erythrocytes with a diameter of 6 to 8 μm, which is similar to normal RBCs.

[0117] Events associated with enucleation can be assessed by examining multiple features associated with the erythrocyte maturation process. For example, before enucleation occurs, cell size and nuclear-cytoplasmic ratio (N / C) gradually decrease, and the size and N / C of these cells decrease significantly over time, indicating that substantial nuclear condensation has occurred during this process. In addition, during this period, cells express high levels of CD71 (early erythroblast marker) and reduce their expression over time. Although they show negligible levels of CD235a (glycophorin A) protein (mature erythrocyte marker) at the beginning, their expression increases dramatically as they mature. Benzidine staining can be used to show the gradual accumulation of hemoglobin in cells and the reduction of cell size over time.

[0118] In some embodiments, erythrocytes or red blood cells or their precursors (eg, iPSCs) can be genetically engineered to carry various useful substances to specific locations in the body.

[0119] In other aspects, the present invention provides a red blood 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 carrier. The pharmaceutical composition may comprise at least about 10 5 or at least about 10 6 or at least about 10 7 The pharmaceutical composition can 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.

[0120] In some aspects, the HSC composition is provided with a highly reactive EPOR (as described). The HSC composition of the present disclosure comprises at least 0.0001% LT-HSC. In some embodiments of the present disclosure, the HSC comprises at least about 0.05% LT-HSC, or at least about 0.1% LT-HSC, or at least about 0.5% LT-HSC, or at least about 1% LT-HSC. In other aspects, the present invention provides a cell colony produced by the methods described herein or a pharmaceutically acceptable composition thereof. In various embodiments, a composition for RBC therapy is prepared comprising a cell colony and a pharmaceutically acceptable excipient. The pharmaceutical composition may comprise at least about 10 LT-HSCs 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 109 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, a pharmaceutical composition is administered that contains HSCs (e.g., with a high EPOR response) in the range of about 100,000 to about 400,000 cells per kilogram (e.g., about 200,000 cells / kg). In other embodiments, a pharmaceutical composition is administered that contains HSCs (e.g., with a high EPOR response) in the range of about 100,000 to about 400,000 cells per kilogram (e.g., about 200,000 cells / kg). 5 to about 5×10 5 cells (e.g., approximately 2.55 × 10 5 cells / kg), or about 10 per kilogram 6 to about 5×10 6 cells (e.g., approximately 2.5 × 10 6 cells / kg), or about 5×10 6 to about 10 7 cells (e.g., approximately 5 × 10 6 cells / kg), or about 10 per kilogram 7 to about 10 8 cells (e.g., approximately 5 × 10 7 cells / kg), or about 10 per kilogram 8 to about 10 9 cells (e.g., approximately 5 × 10 8 cells / kg), or about 10 per kilogram 9 to about 10 10 cells, or about 10 per kilogram 10 to about 10 11 or about 10 11 to about 10 12 cells, or about 10 per kilogram 12 to about 10 13 cells, or about 10 per kilogram of recipient body weight 13 to about 10 14 RBCs are administered to each cell.

[0121] In some embodiments (as described), the HSC is 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.

[0122] The cell compositions of the present disclosure may further comprise a pharmaceutically acceptable carrier or excipient suitable for intravenous infusion or other routes of administration, and the composition may comprise a suitable antifreeze. An exemplary carrier is DMSO (e.g., about 10% DMSO). The cell compositions may be provided in unit vials or pouches and stored frozen until use.

[0123] Cells produced according to the present disclosure can be administered or used, for example, in therapies for inherited or acquired red blood cell disorders, bone marrow failure disorders, altitude-related 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 anemias, nutritional anemias (e.g., iron deficiency anemia and folate deficiency), heme production disorders (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 materials to treat human diseases or physiological or pathological conditions.

[0124] In certain embodiments, the present disclosure provides compositions and methods for treating anemia. In an embodiment, the present disclosure provides hematopoietic stem cell (HSC) compositions or erythroid progenitor cell compositions that can provide lasting and effective anemia cell therapy. In some embodiments, HSC is overloaded with truncated and / or mutated erythropoietin receptors (EPORs), resulting in high-responsiveness EPORs. In an embodiment, the compositions and methods of the present disclosure can provide patients with enough red blood cells to supply healthy oxygenation levels. In an embodiment, the compositions and methods of the present disclosure effectively utilize the potential of induced pluripotent stem cells (iPSCs) to produce HSC populations comprising a large number of long-term (LT)-HSCs for therapy, thereby providing lasting production of red blood cells in vivo.

[0125] Anemia is a condition in which a person lacks enough healthy red blood cells to carry enough oxygen to the body's tissues. The main symptoms of anemia include feeling tired and weak. There are many forms of anemia, such as sickle cell anemia, aplastic anemia, and anemia associated with bone marrow disease or failure. For example, sickle cell disease is a group of inherited red blood cell disorders in which a person has a mutation in the beta globin gene, resulting in an abnormal hemoglobin called hemoglobin S. Hemoglobin S causes flexible red blood cells to become rigid, sickle-shaped. Sickle cell anemia is the most common and severe form of sickle cell disease, in which red blood cells die prematurely, leaving a shortage of healthy red blood cells.

[0126] Currently, treatment for sickle cell disease and other types of anemia includes blood transfusions, as well as blood and bone marrow transplants. Patients undergoing existing treatments still face significant difficulties and complications. For example, patients treated with long-term transfusion therapy (e.g., to increase oxygen-carrying capacity and reduce the ratio of sickle hemoglobin (HbS) to hemoglobin A (HbA)) face a significant burden, including the need for regular hospitalization and often the need for iron chelation therapy. Howard J. 无关供体干细胞移植联合移植后环磷酰胺预防严重镰状细胞病患者移植物抗宿主病的结果 Disease Method .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 blood-forming stem cells with those from a donor) still 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. Results Figure 1 Figure 1 Figure 2 .Biology of Blood and Marrow Transplantation. 2018; 24(2), 413–417.

[0127] The HSC or erythroid progenitor cells produced using the methods described herein are administered to a subject (recipient) by, for example, intravenous infusion or intramedullary transplantation. The method can be performed after a myeloablative, non-myeloablative, or immunotoxin-based (e.g., anti-c-Kit, anti-CD45, etc.) conditioning regimen. In certain embodiments, the method is performed without a myeloablative, non-myeloablative, or immunotoxin-based (e.g., anti-c-Kit, anti-CD45, etc.) conditioning regimen.

[0128] 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 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 efficient manner.

[0129] 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 bone marrow failure, hemorrhagic anemia, or hemolytic anemia. In some embodiments, the recipient subject has Fanconi anemia. In some embodiments, the subject has thalassemia. In some embodiments, HSCs or erythroid progenitor cells are used to prepare blood products for treating complications associated with blood, bone marrow, immune, metabolic, or mitochondrial disorders.

[0130] As used herein, the term "about" means ±10% of the associated numerical value.

[0131] Certain aspects and embodiments of the present disclosure are further described with reference to the following examples.

[0132] Examples

[0133] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances the formation of CD34+ cells during iPSC differentiation without affecting pluripotency.

[0134] Figure 2

[0135] iPSCs were developed from hCD34+ cells by episomal reprogramming 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 body and hemogenic endothelial differentiation were essentially as described in R. Sugimura et al., Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438, (2017); CM 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).

[0136] In brief, hiPSC is dissociated and resuspended in the culture medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holotransferrin, monothioglycerol, BMP4 and Y-27632. Next, cells are seeded in 10cm culture dishes (EZSPHERE or low attachment flat plate) for EB formation. At the 1st day, bFGF and BMP4 are added to the culture medium. At the 2nd day, the culture medium is replaced with the culture medium containing SB431542, CHIR99021, bFGF and BMP4. At the 4th day, the cell culture medium is replaced with the culture medium supplemented with VEGF and bFGF. At the 6th day, the cell culture medium is replaced with the culture medium supplemented with bFGF, VEGF, interleukin (IL) -6, IGF-1, IL-11, SCF and EPO. Cells are maintained in an incubator of 5% CO2, 5% O2 and 95% humidity. To harvest CD34+ cells, EBs were dissociated on day 8, cells were filtered through a 70 μm filter, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0137] Figure 3

[0138] Induced pluripotent stem cells (iPSCs) were transduced using an adenoviral vector containing 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 pluripotency characteristics of iPSCs, as shown by the expression of the stemness marker TRA-1-60 ( Figure 3 ). Method Shown are FACS graphs representing the transduction efficiency of iPSCs with adenoviral vectors overexpressing ETV2 and GFP sequences.

[0139] Next, ETV2-OE-iPSCs (as well as control iPSCs transduced with a vector carrying the GFP sequence but without ETV2) were differentiated into embryoid bodies and subsequently into hemogenic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 promoted the formation of hemogenic endothelial cells, as evidenced by the expression of CD34+ and CD31+ markers within the CD235a- population ( Results ). Specifically, Figure 4A Representative flow cytometric analysis of hemogenic endothelial cells (defined here as CD235a-CD34+CD31+) is shown, and relative quantification demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells compared to controls.

[0140] Furthermore, the results showed that ETV2-OE enhanced the formation of CD34+ cells ( Figure 4B ).Figure 5A Representative flow cytometric analysis of CD34+ cells is shown, and relative quantification indicates that ETV2-OE enhances the formation of CD34+ cells.

[0141] Overall, these data indicate that overexpression of ETV2 in iPSCs does not affect their pluripotency and promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation.

[0142] Example 2 - iPSC-derived HSCs generated using Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs.

[0143] Figure 5B

[0144] To analyze EHT, EB-derived CD34+ cells were suspended in a culture medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. After approximately 4 to 18 hours of cell adhesion to the bottom of the wells (by visual inspection), Yoda1 was added to the cultures. After 4 to 7 days, the cells were harvested for analysis.

[0145] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells from iPSC-derived embryoid bodies were harvested and cultured for another 5 to 7 days to induce endothelial cell to hematopoietic cell (EHT) transformation. CD34+ cells were then harvested from EHT cultures between day 5 and day 7 for further hematopoietic lineage differentiation.

[0146] CD34+ cells harvested from EHT cultures between days 5 and 7 (or days 13 to 21 total for iPSC differentiation) were seeded into 48-well plates pre-coated with rhDL4 and recombinant human fibrin fragments. T-lineage differentiation was induced in a medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid 2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1a, and SB203580.

[0147] 80% of the medium was changed every other day from day 2 to day 6. On day 7, cells were transferred to new coated plates and analyzed for the presence of primary T cells (CD34+CD7+CD5+ / -).

[0148] 80% of the medium was replaced every other day from day 8 to day 13. On day 14, 100,000 cells / well were transferred to a new coated plate and analyzed for the presence of pre-T cells (CD34-CD7+CD5+ / -).

[0149] 80% of the medium was replaced every other day from day 15 to day 20. Cells were harvested on day 21 and analyzed by FACS for expression of CD3, CD8, CD5, CD7, TCRab as a surrogate for T cells, and / or activated using CD3 / CD28 beads to assess their functional properties.

[0150] After 21 days of differentiation, cells were harvested and replated at approximately 80,000 cells in a new 96-well plate in RPMI 1640 (without L-glutamine and phenol red) supplemented with FBS, L-glutamine, and IL-2. Cells were then activated with a 1:1 CD3 / CD28 bead mixture. 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 by RT-qPCR. Supernatants were analyzed by ELISA.

[0151] Figure 6

[0152] Table 1: Cloned HSCs and HLA knockout. We show that iPSC-derived HSCs derived from EHTs at day 8 34+ cells (activated with Piezo1 in this example) undergo pro-T cell differentiation similar to bone marrow (BM)-HSCs. Table 2: Exemplary gRNA sequences and Figure 9 We show that iPSC-derived HSCs generated using EHT of D8 34+ cells (activated with Piezo1 in this case) undergo T cell differentiation and can be activated by CD3 / CD28 beads similarly to BM-HSCs. Figure 9 We show that iPSC-derived HSCs generated by EHT of D8 34+ cells (activated with Piezo1 in this case) can differentiate into functional T cells, as confirmed by the expression of INFγ after CD3 / CD28 bead stimulation. Taken together, these results indicate that EHT of 34+ cells from differentiated iPSCs enhances the ability of HSCs to further differentiate into hematopoietic lineages in vitro, such as progenitor T cells and functional T cells.

[0153] Example 3 – Assessing off-target editing in HLA-depleted HSCs

[0154] HLA typing of triple knockout (HLA-edited) HSC clones was performed to check for unwanted edits and to ensure that no major editing events, such as deletions, occurred in other regions of chromosome 6. Sequencing methods and analyses were performed to evaluate the extent of gRNA off-target activity and to select gRNAs that represent a low risk of affecting non-target HLA genes.

[0155] By connecting the full-length P5 sequencing adapter to the DSB prepared at the end, sequencing is performed using in situ breakage tags in fixed and permeabilized cells. Genomic DNA is extracted, fragmented, end-prepared, and connected using a chemically modified semi-functional P7 adapter. The resulting DNA library contains a mixture of functional DSB-marked fragments (P5:P7) and non-functional genomic DNA fragments (P7:P7). Subsequently, DNA sequencing is performed on the DNA library rich in DNA-marked fragments to remove all external, non-functional DNA. Since the library preparation does not contain PCR, each sequencing read obtained is equivalent to the DSB end of a single tag from the cell. This produces DNA breakage reads, making it possible to directly detect and quantify genomic DSBs by sequencing without the need for error correction, and to draw a clear list of off-target mutations.

[0156] Table 1 below summarizes the results of the editing strategy for two representative clones relative to wild-type cells.

[0157] ​ ​

[0158]

[0159]

[0160] Table 2 provides non-limiting examples of gRNAs used in experiments that can be used to knock out the expression of the indicated HLA genes.

[0161] ​

[0162]

[0163]

[0164] The results showed that the editing strategy successfully selectively targeted the HLA-A, DPB1, and DQB1 genes without affecting other HLA genes or introducing major deletions elsewhere.

[0165] Phenotypic analysis of HLA-edited clones by FACS and immunofluorescence confirmed these results. ​ A and ​ As shown in Figure B, HLA-edited cells tested positive for overall expression of HLA-I class molecules compared to wild-type cells. Specific expression of HLA-A by immunofluorescence confirmed that HLA-A was not expressed in HLA-edited cells, confirming the finding that the gene editing strategy successfully deleted only the HLA-A gene. Specifically, Figure 7AThe cells that showed HLA editing were all positive for class I HLA to the same extent as wild-type (WT) (i.e., non-HLA-edited) cells. This result indicates that despite the loss of HLA-A, other class I molecules such as HLA-B and C are expressed and are not affected by the gene editing strategy.

[0166] To confirm the HLA-A gene deletion, the specific expression of HLA-A was analyzed by immunofluorescence. Figure 7B As can be seen in the figure, HLA-A is not expressed in the HLA-edited clones, indicating that the gene editing strategy is efficient only in specifically deleting the HLA-A gene. This strategy, which retains full class I expression and HLA-A deletion, will facilitate patient matching while avoiding NK cell-mediated rejection.

[0167] Example 4 - Evaluation of the Pluripotency and Immunocompatibility of HLA-Edited HSCs

[0168] The ability of HLA-edited cells to retain pluripotency was evaluated. Figure 8 As shown, immunofluorescence evaluation of HLA-edited iPSC clones indicated 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 endoderm differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0169] HLA class I molecules are expressed on the surface of all nucleated cells, and if between donor and recipient, HLA class I molecules do not match, then cells can be recognized and killed by CD8+T cells. In addition, HLA mismatch may cause cytokine release syndrome (CRS) and graft-versus-host disease (GVD). On the contrary, by B2M KO, HLA-I molecules are completely missing, and cells will be made to become the target of NK cell-mediated cytotoxicity. Retaining all I classes to express and HLA-A deletions can promote patient matching while preventing NK cell-mediated rejection. Therefore, by co-culturing with peripheral blood mononuclear cells (PBMC) to test the immune compatibility of the HSC edited by HLA, to evaluate whether immune cells will reject the transplant of HSC.

[0170] Wild-type (WT) and HLA-edited HSCs were co-cultured with PBMCs matched for HLA-B and HLA-C markers but mismatched for HLA-A. B2M KO HSCs, which lack expression of HLA class I molecules, and CIITA KO HSCs, which lack expression of HLA class II molecules, were used as controls to compare the extent of cytotoxicity mediated by HLA-null and HLA-mismatched PBMCs, respectively. Figure 9Figure 2 shows the results of PBMC-mediated cytotoxicity assays in co-cultures measured by Annexin V staining. The results show that the absence of HLA-A in HLA-edited HSCs protects cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO are susceptible to PBMC-mediated cytotoxicity. HSCs co-cultured with sorted CD8+ T cells from the same PBMC donor protected HLA-edited and B2M KO HSCs from CD8+ T cell cytotoxicity. In contrast, HSCs co-cultured with sorted NK cells only protected WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0171] In summary, immune compatibility results show that the CD8+T cells present in the PBMC samples are responsible for killing cells with mismatched HLA molecules (WT and CIITA KO), while the NK cells present in the PBMCs are responsible for killing HLA-invalid cells (B2M KO). However, HLA-edited HSCs are protected from CD8+T cell-mediated cytotoxicity (because mismatched HLA-A has been knocked out) and are protected from NK cell-mediated cytotoxicity (because the expression of HLA class I molecules is retained to a great extent).

[0172] Example 5 - Evaluation of the in vivo transplantation potential of HLA-edited HSCs

[0173] To evaluate the transplantation potential of HLA-edited HSCs, the in vivo engraftment capacity of the cells was assessed by competitive transplantation against WT HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs were mixed and transplanted into mice, where bone marrow (BM) and peripheral blood samples were recovered and evaluated by FACS to compare the relative amounts of each cell type present in the samples. Figure 10 As shown, both HLA-edited HSCs and WT 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 WT HSCs in their engraftment and reconstitution potential. Therefore, it is expected that the properties of WT (unedited, parental) HSCs are consistent with those of the HLA-edited HSCs of the present disclosure for generating T cell lineages.

[0174] Example 6 – Differentiation of HLA-edited HSCs into CD4+ / CD8+ T cells

[0175] 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 develop into antigen-specific CTLs. Similarly, HLA class I molecules are expressed on the surface of all nucleated cells and display peptide fragments of intracellular proteins to CD8+ CTLs. CTLs induce cytotoxic killing of target (infected) cells after recognizing HLA-I-peptide complexes expressed on the cell surface. Therefore, studies were conducted to determine whether the absence of HLA-A affects the presentation of class I peptides by edited HSCs. Figure 11A and 11B As shown in Figure 2, immunopeptidomic analysis revealed that loss of HLA-A did not affect the overall presentation of class I peptides. HLA-A edited cells displayed comparable peptide and protein presentation when compared to wild-type (non-HLA edited) HSCs. Figure 12A and 12B As shown, loss of HLA-DQB1 and HLA-DPB1 does not affect the overall presentation of class II peptides by macrophages differentiated from HSCs. Taken together, these data indicate that despite the loss of HLA-A, HLA-DQ, and HLA-DP molecules, these cells (and their derived lineages) retain their ability to present a broad spectrum of class I and class II peptides.

[0176] Example 7 - In vivo testing of antigen-mediated immune responses.

[0177] Figure 13 This is a schematic diagram of a delayed-type hypersensitivity reaction, showing the sensitization and induction stages of antigen presentation. Briefly, after antigen injection, it is processed by antigen-presenting cells (APCs) and presented by MHC class II molecules on the APC surface. CD4+ T cells recognize the peptide-MHC on the APCs. When challenged with an antigen, CD4+ helper T cells are activated, and cytokines recruit macrophages and other immune cells, inducing tissue swelling.

[0178] Transplanted mice were assayed for delayed-type hypersensitivity reactions. Specifically, mice were sensitized by subcutaneous injection of sheep red blood cells as an antigen. If the mice have a functional immune system, APCs process the antigen and present the peptide antigen to CD4+ T cells. Next, the mice were challenged by subcutaneous injection of the same antigen into their left paw. At this point, T cells are activated and secrete cytokines, which recruit macrophages and other immune cells at the site of antigen injection, causing tissue swelling. In this assay, a functional immune system results in swelling of the left paw, as measured with a microcaliper.

[0179] like Figure 14A and 14BAs can be seen in the figure, control (untransplanted) mice did not develop swelling in their left paws due to their immune deficiency. In contrast, mice transplanted with cord blood CD34+ cells showed tissue swelling, and the diameter of their left paws doubled. Similar immune system responses were found in mice transplanted with both WT (unedited HSCs) and HLA-edited HSCs.

[0180] Example 8 - Assessment of differentiation and maturation of HSC-derived T cells (pro-T cells)

[0181] Next, the ability of HSC-derived T cells (primary T cells) to differentiate into mature T cells was tested. After a 35-day differentiation period, the presence of CD4+, CD8+, and AB+ T cell populations in the primary T cells was assessed by cell sorting. Figure 15 As shown, naive 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.

[0182] Next, to test functional properties, each T cell population was co-cultured with a CD19+ lymphoma cell line and an anti-CD3 / CD-19 bispecific antibody. In this experimental model, the bispecific antibody acts simultaneously on the CD3 receptor on T cells and the CD19 cell surface receptor on lymphoma cells, triggering T cell activation. The degree of activation was assessed by measuring the subsequent T cell-mediated cytotoxicity compared to a pan-T cell positive control. Figure 16 As shown, naive T cells exhibited statistically significant superiority in cytotoxicity compared with BM CD34+ T cells and EB CD34+ T cells.

[0183] Example 9 - Assessment of the properties of HSCs developing into pro-T cells.

[0184] The ability of HSCs to develop into proto-T cells was assessed by measuring the CD34-CD7+ markers on proto-T cells. Figure 17 As shown, FACS analysis showed that HSCs generated according to the present disclosure successfully differentiated into CD34-CD7+ pro-T cells compared with bone marrow-derived CD34+ cells or EB-derived CD34+ cells.

[0185] Next, the expression of T cell-specific transcription factors and thymic engraftment molecules was measured. Figure 18A showed that TCF7 expression was increased in HSC-derived primary T cells of the present disclosure, and Figure 18B showed that CCR7 expression was increased. Figure 19A showed that HSC-derived proto-T cells engrafted and differentiated in the thymus. Figure 19BFACS analysis of the CD3 cell population gated on the CD45+ cell population is shown, demonstrating that HSC-derived primary T cells have excellent engraftment and differentiation potential in the thymus. The primary T cells of this example were prepared from HSCs using Piezol activation as described above.

[0186] like Figure 20 As shown, in vitro activation of HSC-derived T cells was also measured. Figure 20 The top panel shows FACS analysis of activated T cells from different sources, including HSCs of the present disclosure (e.g., prepared using Piezo1 activation). T cells prepared from HSCs of the present disclosure exhibit comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, wherein the activated T cells express inflammatory cytokines. According to the present disclosure, HSC-derived T cells (e.g., prepared using Piezo1 activation) express higher levels of inflammatory cytokines, as exemplified by expression levels of TNF-α and interferon gamma.

[0187] Example 10: HLA-edited HSCs differentiate into hematopoietic lineages, i.e., promonocytes / macrophages

[0188] Experiments were performed to determine whether HLA deletion affects the ability of HSCs to differentiate into different types of immune cells. Using methods essentially as described in Example 2, HLA-edited HSCs were differentiated into promonocytes / macrophages. It was determined that HLA-edited HSCs were able to differentiate into the monocyte / macrophage lineage comparable to WT (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 ), indicating that HLA-edited HSCs also retained global expression of both class I and class II molecules.

[0189] The overall expression of other class II molecules in HLA-DQB1 and HLA-DPB1 supported by edited HSCs was evaluated by evaluating their expression in macrophages differentiated from HSCs. The study design is schematically shown in Figure 22A It was found that the loss of HLA-DQB1 and HLA-DPB1 did not affect the expression of other HLA class II molecules ( Figure 22B For example, HLA-DR is equally expressed in both WT and HLA-edited cells ( Figure 22C ).exist Figure 22B and 22C CIITA-KO was used as a positive control.

[0190] Example 11: HLA-edited HSCs differentiate into proplatelets

[0191] It has been determined that HLA-edited HSCs can differentiate into megakaryocytes (MKs) and further differentiate into platelets. Differentiation was compared between bone marrow (BM)-derived CD34+ cells and iPSC-CD34+ cells. Figure 23 As shown, HLA-edited HSCs exhibited a statistically significant increase in platelet content compared to BM CD34+ and iPSC-34+ cell populations. Therefore, HLA-edited HSCs can differentiate into megakaryocytes (MKs), which can further support differentiation into platelets.

[0192] References

[0193] 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).

[0194] 2.Brauer,PM,Singh,J.,Xhiku,S.& JCT Cell Genesis:InVitro Veritas Est? Trends Immunol 37,889–901(2016).

[0195] 3.Kennedy, M. et al. Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem CellDifferentiation Cultures. Cell Reports 2, 1722–1735 (2012).

[0196] 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).

[0197] 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).

[0198] 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).

[0199] 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).

[0200] 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).

[0201] 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).

[0202] 10. Guo, R. et al. Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors. Cell Research 30, 21–33 (2020).

[0203] 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).

[0204] 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. A method for preparing a cell population of erythroid lineage, the method comprising: Enriching CD34+ cells from a differentiated pluripotent stem cell (PSC) population to prepare a CD34+ enriched population; Inducing the CD34+ enriched cell population to undergo endothelial to hematopoietic cell transition for at least two days but no more than 12 days to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitor cells (HSPCs); and The population comprising HSCs and / or HSPCs is differentiated into an erythroid lineage population.

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 human primary tissue.

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

4. The method of claim 2, wherein the iPSCs are homozygous for one or more HLA class I and / or class II genes. The method of claim 4 , wherein the iPSCs are homozygous for HLA-DRB1. The method of claim 4 , wherein the iPSCs are homozygous for both HLA-B and HLA-C.

7. The method of claim 2, wherein the iPSCs are gene-edited to delete one or more HLA class I genes, to delete one or more class II genes, and / or to delete one or more genes governing HLA or MHC expression or presentation capabilities.

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 iPSC comprises a deletion of HLA-DPB1 and / or HLA-DQB1.

10. The method of claim 7, wherein the one or more genes governing HLA or MHC expression or presentation ability are β2-microglobulin and / or CIITA.

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

12. The method of any one of claims 1 to 11, wherein the HSCs and / or HSPCs are gene-edited to encode a highly responsive EPO receptor. 13 . The method according to claim 1 , wherein CD34+ enrichment and endothelial-to-hematopoietic cell transition are induced on day 8 to day 15 of iPSC differentiation.

14. The method of claim 13, wherein the endothelial cell to hematopoietic cell transition produces an HSC population comprising one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem cell progenitors.

15. The method of claim 13, wherein harvesting CD34+ cells from the culture undergoing endothelial cell to hematopoietic cell transition comprises harvesting CD34+ floating cells and / or adherent cells.

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

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

18. The method of claim 13, wherein the inducing endothelial cell to hematopoietic cell transition comprises applying cyclic stretch to the CD34-enriched cells. The method according to claim 18 , wherein the periodic stretching is 2D, 3D or 4D periodic stretching.

20. The method of claim 13, wherein the induction of endothelial cell to hematopoietic cell transition comprises Piezol activation.

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

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

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

24. The method of any one of claims 1 to 18, wherein the erythroid lineage is selected from erythroid progenitors, progenitor erythroblasts, granulocyte-macrophage progenitors (GMPs), and megakaryocyte erythroid progenitors (MEPs) and erythroid cells.

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

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

27. The method according to claim 25 or 26, further comprising: The erythroid lineage is cultured under culture conditions sufficient to produce enucleated erythrocytes, wherein the culture optionally comprises one or more of SCF, EPO, and IL-3.

28. The method of claim 27, wherein the progeny differentiate into cells characterized by (i) 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 method of any one of claims 27 or 28, wherein the erythroid lineage is cultured under one or more culture conditions selected from the group consisting of: (i) a pH of about 4.0 to about 7.9; (ii) an oxygen level of less than about 75% of atmospheric oxygen; and (iii) Mechanical stress.

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

9.

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

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

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

34. The method of claim 33, wherein the mechanical stress is applied continuously or constantly throughout the erythrocyte / reticulocyte culture protocol.

35. The method of claim 34, wherein the erythroid expansion medium is supplemented with one or more of the following: (i) a piezo1 agonist, (ii) a Trpv4 agonist, (iii) a phosphodiesterase inhibitor, or (iv) a GSK 3 inhibitor.

36. The method of claim 35, wherein the erythroid expansion medium is supplemented with one or more of: (i) stem cell factor (SCF); (ii) insulin growth factor 1 (IGF1); (iii) IL3; (iv) IL11; and (v) EPO.

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 cell population of the erythroid lineage prepared by the method of any one of claims 1 to 37.

39. A method for treating a patient suffering from an inherited or acquired red blood cell disorder, a bone marrow failure disorder, altitude-related physiological and pathological conditions, anemia, red blood cell enzyme deficiency, red blood cell membrane disorder, hemoglobinopathy, hemolytic anemia, nutritional anemia, a heme production disorder, or hemochromatosis, the method comprising administering to the patient the red blood cell lineage of claim 33.

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

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

42. The composition of claim 40 or 41, wherein the composition comprises at least about 10 2 , at least about 10 3 , or at least about 10 4 , or at least about 10 5 , or at least about 10 6 HSCs or erythroid progenitor cells.

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

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

45. 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 achieved by mutation of the SHP-1 inhibitory domain.

46. ​​The composition of any one of claims 40 to 45, wherein the HSC or erythroid progenitor cell is differentiated from an induced pluripotent stem cell (iPSC).

47. The composition of claim 46, wherein the HSC or erythroid progenitor cell is 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.

48. The composition of claim 46 or 47, wherein the iPSCs are gene-edited to encode a highly responsive EPO receptor.

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

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

51. The composition of claim 50, wherein cells are subjected to 2D or 3D cyclic stretching, and the cells subjected to cyclic stretching are optionally selected from one or more of iPSCs, endothelial cells, hematopoietic endothelial cells (HECs), and hematopoietic stem cells (HSCs). 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. The composition according to claim 52 , wherein CD34+ cells are enriched from iPSCs or embryoid bodies (EBs) prepared therefrom, and cyclic stretch is applied to the CD34+ cells or a subpopulation thereof.

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

55. The composition of claim 54, wherein cells are subjected to Piezol activation, and the cells subjected to Piezol activation are optionally selected from one or more of iPSCs, ECs, HECs, and HSCs. The composition according to claim 55 , wherein CD34+ cells are enriched from iPSCs or embryoid bodies (EBs) prepared therefrom, and Piezol activation is applied to the CD34+ cells or a subpopulation thereof.

57. The composition according to claim 55 or 56, wherein the Piezol activation is performed by contacting pluripotent stem cells or cells derived or differentiated therefrom with one or more Piezol agonists, optionally selected from Yoda1, Jedi1, Jedi2, ssRNA40 or analogs thereof.

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

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

60. A method for preparing a HSC population, the method comprising: An iPSC population carrying an EPOR gene encoding a gene highly responsive to EPO is prepared, and the iPSCs are differentiated into HSCs including LT-HSCs.

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

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

63. The method of claim 60, wherein the EPOR comprises one or more missense or frameshift mutations resulting in hyperresponsiveness, optionally achieved by mutation of the SHP-1 inhibitory domain.

64. The method of any one of claims 60 to 63, wherein the HSC is differentiated from an induced pluripotent stem cell (iPSC).

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

66. The method of claim 64 or 65, wherein the iPSCs are gene-edited to encode the highly responsive EPO receptor.

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

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

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

73. The method of claim 72, wherein cells are subjected to Piezol activation, and the cells subjected to Piezol activation are optionally selected from one or more of iPSCs, ECs, HECs, and HSCs. The method according to claim 73 , wherein CD34+ cells are enriched from iPSCs or embryoid bodies (EBs) prepared therefrom, and Piezol activation is applied to the CD34+ cells or a subpopulation thereof.

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

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

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

78. The method of any one of claims 60 to 77, wherein the composition comprises at least about 10 2 , at least about 10 3 , or at least about 10 4 , or at least about 10 5 or at least about 10 6 HSC.

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

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

81. The method of claim 80, wherein the subject is anemic.

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

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

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

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