Pluripotent stem cell-derived megakaryocytes and platelets

Abstract

The present disclosure provides efficient in vitro methods for producing megakaryocytes and / or platelets from human induced pluripotent stem cells (iPSCs). In various embodiments, the cells and platelets produced in accordance with the present disclosure are functional and / or more closely similar to corresponding lineages isolated from peripheral blood, bone marrow, or other tissue. The present invention provides, in some aspects, isolated cells / platelets and compositions produced by the methods disclosed herein, as well as methods for use in 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 / 413,337, 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-013PC_Sequence_Listing.xml and is 30,048 bytes in size. Background Art

[0005] In addition to playing a major role in physiological hemostasis, thrombosis and wound healing, platelets can also make a significant contribution to host inflammation and immune responses to infection and injury. Many patients with hematopoietic diseases or who are undergoing intensive chemotherapy require platelet transfusion using platelet concentrates obtained through blood / platelet donation. However, platelets obtained from voluntary blood donors for transfusion are a limited resource because, for example, the blood donation industry is facing an ongoing crisis due to the frequent demand for blood products, particularly platelets, that exceeds supply. Therefore, the development of large-scale, readily available megakaryocytes and / or platelets, such as those from hematopoietic stem cells (HSCs), is an attractive tool in regenerative medicine. Summary of the Invention

[0006] In various aspects and embodiments, the present disclosure provides methods for producing hematopoietic lineages for cell therapy, including megakaryocyte lineages, such as megakaryocyte-erythroid progenitors (MEP), CFU-Me (pluripotent hematopoietic stem cells or hematoblasts), megakaryocytes, promegakaryocytes and megakaryocytes, and proplatelets, proplatelets or platelets derived therefrom. In various embodiments, the present invention provides efficient in vitro methods for developing megakaryocytes and / or platelets from human induced pluripotent stem cells (iPSC) (including gene-edited iPSC). In various embodiments, megakaryocytes and / or proplatelets, proplatelets or platelets produced according to the present disclosure are functional and / or more closely resemble corresponding natural lineages separated from bone marrow or blood or corresponding natural platelets separated from blood. The present invention also provides separated cells and compositions produced by the methods disclosed herein, and methods for therapy.

[0007] In other aspects and embodiments, the present disclosure provides HSCs derived from iPSCs that have been gene-edited to be immunocompatible with a large portion of the population. These HSC populations can be used for more efficient in vitro platelet production, or in other aspects, can be used to deliver HSCs or megakaryocytes, platelets, or their progenitors 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 comprising megakaryocytes. The megakaryocytes of the present disclosure can further produce functional platelets. The method of the present disclosure 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, thereby preparing a colony enriched in CD34+. Inducing endothelial cells to hematopoietic cell transition (EHT) in a CD34+ enriched colony, thereby preparing a colony comprising hematopoietic stem cells (HSC) and / or hematopoietic stem cell progenitor cells (HSPC). The resulting cell colony (or part thereof) can be differentiated into megakaryocytes, and is optionally used to produce platelets.

[0009] In various embodiments, iPSC is prepared by reprogramming somatic cells. In certain embodiments, iPSC derives from CD34+ cells separated from peripheral blood. In various embodiments, iPSC is autologous or allogeneic (for example, HLA matching at one or more loci) for receptors (needing the subject treated as described herein). In various embodiments, iPSC can be gene-edited to help HLA matching. For example, iPSC can be gene-edited to make one or more of HLA-A, HLA-B and HLA-C lack, and one or more of HLA-DP, HLA-DQ and HLA-DR lack. 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 class I and class II loci retained. In certain embodiments, iPSC is gene-edited to 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 various embodiments, iPSC is prepared and expanded using a culture system. Amplified iPSC can be recovered from the culture and used to produce embryoid bodies (EBs). The EBs produced by iPSC differentiation are three-dimensional aggregates of iPSC and contain three (or two or one) embryonic germ layers based on the differentiation method. In certain embodiments, the method according to each aspect can include producing CD34+ cells from pluripotent stem cells (e.g., EBs) and inducing endothelial cells to differentiate into hematopoietic cells. The following HSCs can be produced from a cell population using various stimuli or factors, including mechanical, biochemical, metabolic and / or topographical stimuli, and factors such as induction of extracellular matrix, niche factors, cell-extrinsic factors, and intrinsic properties of the cells; and including pharmacological and / or genetic means.

[0011] In some embodiments, iPSC differentiation is performed until the cells are at least about 20% CD34+ or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced on day 7 to day 14 of iPSC differentiation. iPSC differentiation can be performed according to known techniques. In some embodiments, iPSC differentiation involves a combination of factors such as, but not limited to, bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1.

[0012] Induction of EHT can be carried out with any known method. In certain embodiments, the induction of EHT produces a hematopoietic stem cell (HSC) colony comprising LT-HSC. In certain embodiments, EHT produces HSC by endothelial cells or hematopoietic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological and / or genetic means (for example, by stimulation, inhibition and / or genetic modification). In certain embodiments, EHT produces a stem cell colony comprising one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC) and hematopoietic stem cell progenitor cells. In certain embodiments, EHT is induced in culture for about 5 days to about 7 days. In an embodiment, EHT is carried out using a culture medium comprising one or more growth factors and cytokines, the cytokine being selected from TPO, SCF, Flt3L, IL3, IL-6, IL7, IL-11, IGF, bFGF and IL15. The culture medium may optionally contain one or more of VEGF, bFGF, a BMP activator, a Wnt pathway activator, or a ROCK inhibitor (e.g., thiazolinone or Y27632). In some embodiments, the HSC and / or HSPC population or a portion thereof is differentiated independently of the use of a mechanosensitive receptor or a mechanosensitive channel agonist such as Yoda1. In some embodiments, the use of a mechanosensitive receptor or a mechanosensitive channel agonist (e.g., Yoda1) is optional.

[0013] 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, including the megakaryocyte lineage responsible for platelet production. In certain embodiments, Piezo1 activation is applied to at least EBs generated from iPSCs and / or CD34+ cells isolated from EBs, which, according to various embodiments, allows for better generation of megakaryocytes compared to other methods for inducing EHT.

[0014] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from cultures undergoing endothelial cell to hematopoietic cell transition from day 10 to day 20 of iPSC differentiation, such as from day 12 to day 17. In some embodiments, CD34+ cells include non-adherent cells. In various embodiments, HSCs or CD34+ enriched cells are further expanded.

[0015] HSCs and / or HSPCs that produce megakaryocytes and / or platelets can be identified based on the expression of CD34 and the absence of a lineage-specific marker called Lin-. In some embodiments, a stem cell population for differentiation into a hematopoietic lineage is at least about 80% CD34. + , or at least about 90% CD34 + , or at least about 95% CD34 + .

[0016] In various embodiments, HSCs and / or HSPC populations or fractions thereof are differentiated in vitro into megakaryocytes, from which platelets can be generated.

[0017] In certain embodiments, the step of being differentiated into megakaryocyte by the colony comprising HSC and / or HSPC includes cultivating with thrombopoietin (TPO).Culture can further include one or more additional cell factors or growth factors, such as those selected from IL-1, IL-3, IL-6, IL-9, IL-11, SCF, SDF-1 and PDGF-BB.Cytokines and growth factors can be selected, including TPO, to further expand megakaryocyte.In certain embodiments, such additional cell factors or growth factors for expanding megakaryocyte can be selected from stem cell factor (SCF), FMS sample tyrosine kinase 3 ligands (Flt3L), IL-6, IL-9 and erythropoietin (EPO).

[0018] As known in the art, mature megakaryocytes can form proplatelets and platelets, such as by culturing in the presence of fibroblast growth factor 4 (FGF4) and stromal cell-derived factor 1 (SDF 1). In certain embodiments, megakaryocytes or proplatelets are cultured in a bioreactor so that the cells are subjected to fluid shear stress. For example, platelets can be produced under static 2D, serum-free, cytokine-dependent conditions. Alternatively, platelets can be produced in a three-dimensional (3D) microenvironment. In various embodiments, platelets will have a phenotype of CD41+CD42b+. The platelets recovered can be activated by thrombin. Before infusion therapy, platelets can be recovered and gamma irradiated.

[0019] The compositions of the present disclosure (e.g., comprising platelets prepared according to the present disclosure) may further comprise a pharmaceutically acceptable carrier. Such carrier solutions may also contain buffers, diluents, and other suitable additives. The cell or platelet compositions may be provided in an implantable device (e.g., a stent) or in a bag or in a vial, test tube, or container in an appropriate volume and stored frozen until use. In various embodiments, the composition comprises at least about 10% by volume per 50 mL or 100 mL. 9 platelets, or at least about 10 10 platelets, or at least about 10 11 platelets, or at least about 10 12 Platelets.

[0020] In other aspects, the present invention provides a method for platelet therapy, the method comprising administering platelets (prepared as described herein) or a pharmaceutically acceptable composition thereof to a human subject in need thereof. In various embodiments, the methods described herein are used to treat subjects suffering from following thrombocytopenia, such as ACTN1-related thrombocytopenia, amegakaryocytic thrombocytopenia with radial-ulnar fusion, ANKRD26-related thrombocytopenia, autosomal dominant thrombocytopenia, congenital amegakaryocytic thrombocytopenia, CYCS-related thrombocytopenia, FYB-related thrombocytopenia, idiopathic thrombocytopenic purpura or X-linked thrombocytopenia. In some embodiments, platelets are administered to a subject suffering from bleeding.

[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 4 FACS analysis showing megakaryocyte differentiation, indicating commitment of HSCs to the megakaryocyte lineage.

[0026] Figure 5 showed that megakaryocytes derived from iPSC-derived HSCs can be expanded in vitro.

[0027] Figure 6 Immunofluorescence analysis showed platelet differentiation of iPSC-derived HSCs, indicating that the derived megakaryocytes were phenotypically similar to BM CD34+-derived megakaryocytes and were able to release platelets.

[0028] Figure 7 showed thrombogenicity of platelets generated from iPSC-derived HSCs, indicating that these platelets, once activated, are able to clot and promote thrombosis.

[0029] Figure 8A and Figure 8B Phenotypic analysis of HLA-edited (e.g., triple knockout) cells by FACS and immunofluorescence is shown. Figure 8A The overall expression of HLA-I class molecules (HLA-A, HLA-B, and HLA-C) on the cell surface is shown, where HLA-edited cells are positive for overall HLA-I class expression to a similar extent as wild-type cells. Figure 8B Cellular expression of HLA-A was shown via immunofluorescence, where HLA-A was not expressed in HLA-edited clones.

[0030] Figure 9Clones 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.

[0031] Figure 10 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.

[0032] Figure 11 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.

[0033] Figure 12 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 those platelets differentiated from bone marrow (BM) CD34+ cells and CD34+ cell populations isolated from differentiated iPSCs (e.g., EBs).

[0034] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0035] 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.

[0036] EB34+ cells refer to CD34+ cells derived from embryonic bodies. These include hematopoietic endothelial cells. DETAILED DESCRIPTION

[0037] In various aspects and embodiments, the present disclosure provides methods for producing hematopoietic lineages for cell therapy, including megakaryocyte lineages, such as megakaryocyte-erythroid progenitors (MEP), CFU-Me (pluripotent hematopoietic stem cells or hematoblasts), megakaryocytes, promegakaryocytes and megakaryocytes, and proplatelets, proplatelets or platelets derived therefrom. In various embodiments, the present invention provides efficient in vitro methods for developing megakaryocytes and / or platelets from human induced pluripotent stem cells (iPSC) (including gene-edited iPSC). In various embodiments, megakaryocytes and / or proplatelets, proplatelets or platelets produced according to the present disclosure are functional and / or more closely resemble corresponding natural lineages separated from bone marrow or blood or corresponding natural platelets separated from blood. The present invention also provides separated cells and compositions produced by the methods disclosed herein, and methods for therapy.

[0038] In other aspects and embodiments, the present disclosure provides HSCs derived from iPSCs that have been gene-edited to be immunocompatible with a large portion of the population. These HSC populations can be used for more efficient in vitro platelet production, or in other aspects, can be used to deliver HSCs or megakaryocytes, platelets, or their progenitors to patients in need to reduce or eliminate the need for regular blood transfusions.

[0039] 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 exploited to generate an unlimited supply of hematopoietic cells, including but not limited to therapeutic lineages that generate megakaryocytes, which in turn release proplatelets and platelets. The use of platelets in therapy has been limited because volunteer blood donors are a limited resource. Because donor-derived platelets must be stored at room temperature, their shelf life is limited to about 5 days, during which time they are susceptible to bacterial growth and lack consistency between donor-derived batches. In addition, platelet transfusions typically carry several risks to the recipient, including allergic reactions and febrile non-hemolytic reactions. See, for example, Kaufman, Richard M, et al. " 血小板输注:来自 the AABBAnnals of Internal Medicine 162, No. 3 (2015): 205-213. In addition, hiPSCs can be more easily genetically modified in vitro than primary cells, providing the opportunity to improve cell / platelet numbers and circumvent issues such as HLA matching. In addition, fully engineered hiPSC clones can serve as a stable and safe source compared to primary cells. Furthermore, because hiPSCs, unlike human embryonic stem cells (hESCs), are of non-embryonic origin, they are also free of ethical concerns and have consistent quality. Therefore, the use of hiPSCs according to the present disclosure has several advantages over primary cells to generate therapeutic hematopoietic lineages, such as megakaryocyte lineages, including as megakaryocytes and platelets (including their progenitors).

[0040] On the one hand, the present disclosure provides a method for preparing a cell colony comprising megakaryocytes. Megakaryocytes of the present disclosure can further produce functional platelets. The method of the present disclosure 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 to prepare a colony enriched in CD34+. Endothelial cells are induced to transition to hematopoietic cells (EHT) in a CD34+ enriched colony to prepare a colony comprising hematopoietic stem cells (HSC) and / or hematopoietic stem cell progenitor cells (HSPC). In certain embodiments, CD34+ cells are enriched from a colony comprising HSC and / or HSPC. In various embodiments, EHT is induced for at least 2 days, and at most 12 days. The resulting cell colony (or part thereof) can be differentiated into megakaryocytes, and is optionally used to produce platelets.

[0041] 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 induced CD34+ cell populations (which can be derived from iPSC-embryoid bodies) can be used to generate advanced hematopoietic stem cells and hematopoietic lineages in vitro, such as megakaryocyte lineages, which are also capable of producing functional platelets.

[0042] In some embodiments, CD34+ cells (i.e., recovered from EB dissociation) are contacted with an effective amount of a mechanosensitive receptor or a mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. Exemplary Piezol agonists include Yoda1, Jedi1, and Jedi2 or their analogs. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Other methods for inducing EHT can be used (alternatively or additionally) and are described herein. In some embodiments, after inducing EHT, cells (comprising a population of HSCs and / or HSPCs) differentiate into a megakaryocyte lineage, such as a cell population comprising megakaryocytes capable of producing or secreting platelets.

[0043] In various embodiments, iPSC is prepared by reprogramming somatic cells. The term "induced pluripotent stem cell" or "iPSC" refers to a cell derived from a somatic cell, such as a skin or blood cell that has been reprogrammed back to an embryonic-like pluripotent state. In certain embodiments, iPSC is produced by a somatic cell, such as, but not limited to, a fibroblast or PBMC (or a cell separated therefrom). In certain embodiments, iPSC derives from lymphocytes, granulocyte / macrophage lineage restricted progenitor cells (GMPs), cord blood cells, PBMCs, CD34+ cells or other human primary tissues. In certain embodiments, iPSC derives from CD34+ cells separated from peripheral 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 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 edited to make other functions missing or express other functions. For example, iPSC can be 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 I class and at least one HLA II class complex. In certain embodiments, iPSC is homozygous for at least one retained Class I and Class II loci.

[0044] In various embodiments, HSC and megakaryocyte lineage cells are derived from iPSCs that are 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-. For retained HLAs (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-.

[0045] In some embodiments, 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.

[0046] 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.

[0047] 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.

[0048] 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-related 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 guiding 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. September 2016; 283(17): 3232–3238. In other embodiments, non-canonical type II or type V Cas endonucleases with homology (although low primary sequence homology) to Streptococcus pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) can be employed. Many such non-canonical Cas endonucleases are known in the art. Nidhi S, et al. Novel CRISPR–Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives, Int J Mol Sci. 2021 Apr; 22(7): 3327.In other embodiments, gene editing uses base editing or primer editing to combine mutations without causing double-strand breaks. See, for example, Antoniou P, et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Editing, January 28, 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-StrandBreaks or Donor DNA, Front. Genet., June 9, 2020. Various other gene editing processes are known, including using dead Cas (dCas) systems (e.g., Cas fusion proteins) to direct DNA modification enzymes to desired targets, using dCas as a guide RNA guide system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications, Int J Mol Sci. December 2019; 20(23): 6041.

[0049] 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.

[0050] 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.

[0051] In order to improve efficiency, in some embodiments, Cas and gRNA can be combined before being delivered into the cell. The Cas-gRNA complex is referred to as ribonucleoprotein (RNP). Many methods for delivering RNP directly to cells have been developed. 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 program allows RNP to quickly enter the nucleus, so genome cutting can begin immediately. See, for example, Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics. January 1, 2021; 11 (2): 614-648, which is hereby incorporated by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSC and / or HSC as RNP.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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,” Data Brief. 2021 May 15;37:107140, which is hereby incorporated by reference in its entirety. The KARYOSTAT assay allows for visualization of chromosomal aberrations at a resolution similar to that of G-banded karyotyping. For chromosome gains, structural abnormalities >2 Mb in size can be detected, and for chromosome losses, structural abnormalities >1 Mb in size can be detected. 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 aneuploidy, submicroscopic aberrations, and mosaic events.

[0057] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that do not generate copy number aberrations (CNAs) during reprogramming, such as described in Wiesner et al. "Molecular Techniques," Editor(s): Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, "Pathology of Melanocytic Tumors," Elsevier, 2019, pp. 364-373, ISBN 9780323374576; and Hussein SM, et al. "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 for CNAs by comparing sample DNA to reference DNA.

[0058] 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.

[0059] 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.

[0060] 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).

[0061] Somatic cells can be reprogrammed by expressing reprogramming factors selected from Sox2, Oct3 / 4, c-Myc, Nanog, Lin28 and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28 and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc and klf4. Methods for preparing iPSCs are described in, for example, U.S. Patent No. 10,676,165; U.S. Patent No. 9,580,689; 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 lentivirus, Sendai virus or measles virus systems are used to express reprogramming factors. Alternatively, reprogramming factors can be expressed by introducing mRNA encoding reprogramming factors into somatic cells. In addition, iPSCs can be produced by introducing non-integrated episomal plasmids expressing reprogramming factors, that is, for producing transgenic-free and virus-free iPSCs. Known episomal plasmids may be employed which have limited replication capacity and are therefore lost after a few cell generations.

[0062] In some embodiments, human pluripotent stem cells (e.g., iPSC) are gene-edited. Gene editing may 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), deletion of CIITA, deletion of receptor genes, or addition. Alternatively, engineered iPSCs with one or more HLA knockouts and TCR knockouts may be placed in a bioreactor and differentiated without a feeder layer and serum under GMP-grade conditions to produce fully functional megakaryocytes and resulting platelets.

[0063] In various embodiments, iPSCs are prepared and expanded using a culture system. 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 a bioreactor, such as Abecasis B. et al., 3D人诱导多能干细胞在生物反应器中的聚集物的扩增:生物过程强化和放大 方法 稳健的 .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.

[0064] In some embodiments, the method according to each aspect may comprise generating CD34+ 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.

[0065] In some embodiments, the method comprises preparing endothelial cells with hematopoietic potential from pluripotent stem cells before inducing EHT. In some embodiments, the combined overexpression of GATA2 / ETV2, GATA2 / TAL1 or ER71 / GATA2 / SCL can result in the formation of endothelial cells with hematopoietic potential from PSC sources. In some embodiments, the method comprises 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 generating transgene-free hematopoietic EC. In some embodiments, ETV2 is expressed by mRNA introduced into iPSC. Any available method can be used to introduce mRNA, including electroporation or lipofection. Differentiation of cells expressing ETV2 can include adding VEGF-A. See Wang K, et al., 人多能干细胞通过 ETV2的mRNA的时间调制分化为 内皮细胞 多谱系 .Sci.Adv.Volume 6 (2020). According to embodiments of the present disclosure, cells generated in this manner can be used to generate CD34+ cells and induce EHT.

[0066] Following CD34+ enrichment, HSCs and / or HSPCs are generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification.

[0067] 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. The differentiation of iPSC can be carried out according to known technology. 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. Serum-free cultures typically include a mixture of cytokines / growth factors / small molecules.

[0068] In some embodiments, hPSCs are co-cultured with feeder cells derived from mouse bone marrow, such as OP9, STO mouse fibroblasts, or peripheral blood mononuclear cells (PBMCs) derived from blood, or mesenchymal stem cells derived from umbilical cord blood, or feeder layers of cancer cell lines derived from lymphocytes in a serum-containing culture medium. The culture may contain growth factors and cytokines to support the differentiation of embryoid bodies or monolayer systems. The feeder cell co-culture system can be used to produce multipotent HSPCs, which can be further differentiated into several hematopoietic lineages, including monocytes or macrophages, dendritic cells, neutrophils, NK cells, T lymphocytes, B lymphocytes, megakaryocytes, and erythrocytes. See Netsrithong R. et al., 源自人诱导多能干细胞的造血内皮祖细胞的分化潜能 通过谱系受限前体的重新指定从人多能干细胞诱导多能造血祖细胞 化学激活 机械转导通道Piezo , Stem Cell Research & Therapy Vol. 11 No. 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., Yoda1类似物(Dooku1),其 拮抗Yoda1诱导的Piezo1激活和主动脉舒张 一种杠杆样 ,Cell Stem Cell. 2013 Oct 3;13(4).

[0069] Differentiation of iPSCs (eg, to EBs) can employ WNT agonists, such as CHIR99021. WNT agonists are molecules that mimic or increase WNT signaling. Non-limiting examples of WNT agonists include small molecules CHIR-99021 (CAS 252917-06-9), 2-amino-4,6-disubstituted pyrimidines such as BML 284 (CAS 853220-52-7), SKL2001 (CAS 909089-13-0), WAY 262611 (CAS 1123231-07-1), WAY 316606 (CAS 915759-45-4), SB 216763 (CAS 280744-09-4), IQ 1 (CAS 331001-62-8), QS 11 (CAS 944328-88-5), deoxycholic acid (CAS 83-44-3), BIO (CAS 667463-62-9), kemperolone (CAS 915759-45-4), 142273-20-9) or (hetero)arylpyrimidine. In some embodiments, the WNT agonist is an agonist antibody or a functional fragment thereof or an antibody-like polypeptide.

[0070] Differentiation of iPSC (for example, to differentiation of EB) can use ROCK inhibitor.Exemplary ROCK inhibitor for setting up and differentiating iPSC includes but is not limited to: Thiazolidinedion, Y27632, Fasudil, AR122-86, RevitaCell.TM. supplement, H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N '-(2,4,6-trichlorophenyl) urea, 3-(4-pyridyl)-1H-indole and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, H-100 and ROCK inhibitor, it is disclosed in U.S. Patent number 8,044,201, it is hereby incorporated by reference in its entirety.

[0071] Induction of EHT can be carried out by any known method. In certain embodiments, the induction of EHT produces a hematopoietic stem cell (HSC) colony comprising LT-HSC. In certain embodiments, EHT produces HSC by 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 certain embodiments, EHT produces a stem cell colony comprising one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC) and hematopoietic stem cell progenitor cells. In various embodiments, EHT can be induced in culture for 2 to 12 days, such as from about 4 to about 8 days (e.g., about 4 days, about 5 days, about 6 days, about 7 days or about 8 days). In certain embodiments, EHT is induced in culture for about 5 to about 7 days. In an embodiment, EHT is performed using a culture medium comprising one or more growth factors and cytokines selected from the group consisting of TPO, SCF, Flt3L, IL3, IL-6, IL7, IL-11, IGF, bFGF, and IL 15. The culture medium may optionally contain one or more of VEGF, bFGF, a BMP activator, a Wnt pathway activator, or a ROCK inhibitor (e.g., thiazolinone or Y27632).

[0072] In some embodiments, HSC and / or HSPC populations or portions thereof are differentiated independently of the use of mechanosensitive receptors or mechanosensitive channel agonists such as Yoda1. In some embodiments, the use of mechanosensitive receptors or mechanosensitive channel agonists (e.g., Yoda1) is optional. Thus, in some embodiments, CD34+ cells are enriched from a differentiated pluripotent stem cell population to prepare a CD34+ enriched population. The endothelial cell to hematopoietic cell transition of the CD34+ enriched cell population is induced for at least two days, but no more than 12 days, optionally using a mechanosensitive receptor or mechanosensitive channel agonist, such as Yoda1, Jedi1, Jedi2, or ssRNA40. In some embodiments, the endothelial to hematopoietic transition of the CD34+ enriched cell population is induced for at least two days and further for about 4 hours, or about 8 hours, or about 12 hours, or about 16 hours, or about 20 hours, or about 24 hours, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days. Total EHT differentiation is performed for no more than 12 days.

[0073] In some embodiments, the method includes increasing the expression or activity of dnmt3b in PSC, embryoid bodies, CD34+ enriched cells, EC, HEC or HSC, 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 WO2021 / 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.

[0074] 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 Yoda1. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Yoda1 (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, 转导 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 .eLife(2015).

[0075] Derivatives of Yodal can be used in various embodiments. For example, in some embodiments, derivatives containing a 2,6-dichlorophenyl nucleus are employed. Exemplary agonists are disclosed in Evans EL, et al., 芳烃受体 拮抗剂 , 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., ​ ​ ​ ​ .Nature Communications(2018)9:1300;Sugisawa, et al., ​, Cell, Vol. 182, No. 3, 2020, pp. 609-624, which is hereby incorporated by reference in its entirety. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of the Piezo1 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. Alternatively, single-stranded (ss) RNA (e.g., ssRNA40) and its derivatives and analogs can be used for Piezo1 activation.

[0076] 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, including the megakaryocyte lineage responsible for platelet production. In certain embodiments, Piezo1 activation is applied to at least EBs generated from iPSCs and / or CD34+ cells isolated from EBs, which, according to various embodiments, allows for better generation of megakaryocytes compared to other methods for inducing EHT.

[0077] Alternatively or additionally, the activity or expression of Dnmt3b can be increased directly in cells (e.g., in CD34+ enriched cells). 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.

[0078] 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).

[0079] 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.

[0080] 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.

[0081] 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.

[0082] Alternatively or additionally, EHT is stimulated by Trpv4 activation. Trpv4 activation can be performed by contacting cells (e.g., CD34+ enriched cells, ECs, or HECs) with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0083] 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.

[0084] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from cultures undergoing endothelial to hematopoietic cell transition between day 10 and day 20 of iPSC differentiation, such as from day 12 to day 17. In some embodiments, the CD34+ cells include non-adherent cells.

[0085] In various embodiments, HSC or CD34+ enriched cells are further expanded. For example, HSC or CD34+ enriched cells can be expanded according to the method disclosed in the following documents: US 8,168,428; US 9,028,811; US ​​10,272,110; and US10,278,990, which are hereby incorporated by reference in their entirety. In certain embodiments, the in vitro expansion of HSC or CD34+ enriched cells uses prostaglandin E2 (PGE2) or PGE2 derivatives. In some embodiments of the present disclosure, HSC comprises 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.

[0086] HSCs and / or HSPCs that produce megakaryocytes and / or platelets can be identified based on the expression of CD34 and the absence of a lineage-specific marker known as Lin-. In some embodiments, a stem cell population comprising HSCs and / or HSPCs 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 the cell population based on the expression of one or more of CD34, CD90, CD38, and CD43. CD34 can be selected. + 、CD90 + 、CD38 - and CD43 - In some embodiments, the stem cell population for differentiation into hematopoietic lineages is at least about 80% CD34 + , or at least about 90% CD34 + , or at least about 95% CD34 + .

[0087] In some embodiments, the stem cell population, 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, the cells are expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or a derivative of SR1. See also Wagner et al., Cell Stem Cell 2016; 18(1): 144-55 and Boitano A., et al., ​ ​ Promote the Expansion of Human Hematopoietic Stem Cells .Science 2010 Sep 10;329(5997):1345–1348.

[0088] In some embodiments, CD34 is promoted +Cell-expanding compounds include pyrimidine indole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, which is hereby incorporated by reference).

[0089] 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 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 encoding transcript to the cell, or by introducing an encoding 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.

[0090] In other embodiments, stem cell populations or CD34+ enriched cells are cultured with histone methyltransferase EZH1 inhibitors. 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 lymphoid lineage. See WO 2018 / 048828, which is hereby incorporated by reference in its entirety. In other embodiments, EZH1 is overexpressed in the stem cell population.

[0091] In various embodiments, HSCs and / or HSPC populations or fractions thereof are differentiated in vitro into megakaryocytes, from which platelets can be generated.

[0092] In some embodiments, a colony comprising HSC and / or HSPC or its offspring can be cultured in vitro with Notch ligands (partial or complete), SHH, extracellular matrix components and / or combinations thereof to differentiate cells. In addition, according to known methods, feeder layers of xenogeneic OP9-DL1 or STO mouse fibroblasts or peripheral blood mononuclear cells (PBMCs) of blood origin or mesenchymal stem cells of umbilical cord blood origin or cancer cell line cells of lymphocyte origin are often used for the differentiation of hematopoietic cells. The OP9-DL1 co-culture system uses a bone marrow stromal cell line (OP9) transduced with Notch ligand δ-like 1 (DLL1) to support T cell development from stem cell sources. The OP9-DL1 system limits the potential for clinical application of cells. It is necessary to be able to produce hematopoietic cells from hiPSC for clinical application without feeder layer cell systems, and in some embodiments, the present invention achieves this purpose. In a non-limiting example, in order to produce megakaryocytes, iPSC expansion is performed for 6 days, followed by embryoid body formation, which requires about 8 days. The cells are further cultured for approximately 5 days to allow HSCs to develop from the CD34+ hemogenic endothelial cells from which they originate. HSCs are then cultured in a specific culture medium for differentiation into megakaryocytes and / or further differentiation into platelets.

[0093] As used herein, the term "Notch ligand" refers to a ligand that is capable of binding to a Notch receptor polypeptide present in the membrane of a hematopoietic stem cell or progenitor T cell. Notch receptors include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically have a DSL domain (D-δ, S-serrated, and L-Lag2), which comprises 20 to 22 amino acids at the amino terminus and 3 to 8 EGF repeats on the extracellular surface. In various embodiments, Notch ligands comprise δ-like-1 (DLL1), δ-like-4 (DLL4), SFIP3, δ 最大 (disclosed in PCT / US2020 / 041765 and PCT / US2020 / 030977, which are incorporated herein by reference in their entireties), Jagged 1 (JAG1), Jagged 2 (JAG2), Delta-like ligand 3 (DLL3) and X-δ2, or a functional portion thereof.

[0094] As used herein, "Notch ligands" also include complete (full-length), partial (truncated form) or modified (comprising one or more mutations, such as conservative mutations) Notch ligands, as well as Notch ligands of any species or fragments thereof that retain at least one activity or function of the full-length Notch ligand. Also included are peptides that mimic notch ligands. Notch ligands can be "canonical notch ligands" or "non-canonical notch ligands". Canonical notch ligands are characterized by an extracellular domain that typically includes an N-terminal (NT) domain, followed by a δ / serrated / LAG-2 (DSL) domain and multiple tandemly arranged epidermal growth factor (EGF)-like repeats. Canonical ligand binding to Notch typically requires a DSL domain and flanking NT domains and the first two EGF repeats containing δ and OSM-11-like protein (DOS) motifs. The intracellular domains of some canonical ligands contain a carboxyl-terminal PSD-95 / Dlg / ZO-1-ligand (PDZL) motif, whose effects are independent of Notch signaling.

[0095] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody that can bind to and participate in Notch signaling. In some embodiments, the antibody is a monoclonal antibody (including human or humanized antibodies), a single-chain antibody (scFv), a nanobody, or other antibody fragments or antigen binding molecules capable of activating the Notch signaling pathway.

[0096] In some embodiments, the Notch ligand is a δ family Notch ligand. In some embodiments, the δ family ligand is δ-1 (Genbank accession number AF003522, Homo sapiens), δ-like 1 (DLL1, Genbank accession number NM_005618 and NP_005609, Homo sapiens; Genbank accession number X80903, 148324, Mus musculus), δ-4 (Genbank accession number AF273454, BAB18580, Mus musculus; Genbank accession number AF279305, AAF81912, Homo sapiens) and / or δ-like 4 (DLL4; Genbank accession number Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank accession number NM 019454, Mus musculus). Notch ligands are commercially available or can be produced, for example, by recombinant DNA technology.

[0097] In some embodiments, the Notch ligand comprises an amino acid sequence that is at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97% identical (e.g., about 100% identical) to a human DLL1 or DLL4 Notch ligand. Functional derivatives of Notch ligands (including fragments or portions thereof) will be able to bind to and activate Notch receptors. Binding to Notch receptors can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cell signaling assays.

[0098] In some embodiments, the Notch ligand is a DLL4 with one or more affinity-enhancing mutations with respect to hDLL4, such as one or more (or all) of the following: G28S, F107L, I143F, H194Y, L206P, N257P, T271L, F280Y, S301R, and Q305P. See Gonzalez-Perez, et al., Affinity-matured DLL4ligands as broad-spectrum modulators of Notch signaling ,Nature Chemical Biology(2022).

[0099] In various embodiments, the Notch ligand is soluble and is optionally immobilized on microparticles or nanoparticles, which are optionally paramagnetic to allow magnetic enrichment or concentration processes. In other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally together with other adhesion molecules such as VCAM-1. See US2020 / 0399599, which is hereby incorporated by reference in its entirety. In other embodiments, the beads or particles are polymers (e.g., polystyrene or PLGA), gold, iron dextran, or are composed of biomaterials, such as particles formed by lipids and / or proteins. In various embodiments, the particles have a diameter or maximum size from about 0.01 μm (10 nm) to about 500 μm (e.g., about 1 μm to about 7 μm). In other embodiments, a polymer scaffold with a conjugated ligand can be used, as described in WO 2020 / 131582, which is hereby incorporated by reference in its entirety. For example, the scaffold can be made of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivative, gelatin, collagen, agarose, hyaluronic acid, poly (lysine), polyhydroxybutyrate, poly-ε-caprolactone, polyphosphazene, poly (vinyl alcohol), poly (alkylene oxide), poly (ethylene oxide), poly (allylamine), poly (acrylate), poly (4-aminomethylstyrene), pluronic polyol, poloxamer, poly (uronic acid), poly (anhydride), poly (vinyl pyrrolidone) and any combination thereof. In some embodiments, the scaffold comprises a pore with a diameter between about 1 μm and 100 μm.

[0100] In some embodiments, the C-terminus of the Notch ligand is conjugated to a selected support. In some embodiments, this may include adding a sequence at the C-terminus of the Notch ligand that can be enzymatically conjugated to the support, for example, via a biotin molecule. In another embodiment, a Notch ligand-Fc fusion is prepared such that the Fc fragment can be immobilized by binding to protein A or protein G that is conjugated to the support. Of course, any known protein conjugation method can be used.

[0101] Therefore, in various embodiments, Notch ligands are immobilized, functionalized and / or embedded in 2D or 3D culture systems. Notch ligands can be incorporated together with components of the extracellular matrix, such as one or more selected from fibronectin, recombinant human fibrin fragments and laminin. In some embodiments, Notch ligands and / or components of the extracellular matrix are embedded in an inert material that provides 3D culture conditions. Exemplary materials include but are not limited to cellulose, alginate and combinations thereof. In some embodiments, Notch ligands, components of the extracellular matrix or a combination thereof are contacted with culture conditions to provide cells with a morphological pattern and / or texture (e.g., roughness) that contributes to differentiation and / or amplification.

[0102] In certain embodiments, the step of being differentiated into megakaryocyte by the colony comprising HSC and / or HSPC includes cultivating with thrombopoietin (TPO).Culture can further include one or more additional cell factors or growth factors, such as those selected from IL-1, IL-3, IL-6, IL-9, IL-11, SCF, SDF-1 and PDGF-BB.Cytokines and growth factors can be selected, including TPO, to further expand megakaryocyte.In certain embodiments, such additional cell factors or growth factors for expanding megakaryocyte can be selected from stem cell factor (SCF), FMS sample tyrosine kinase 3 ligands (Flt3L), IL-6, IL-9 and erythropoietin (EPO).

[0103] In certain embodiments, immature megakaryocytes are produced, and they can be identified as CD34-CD41+CD61+CD42b-. The maturation of megakaryocytes can be promoted by cultivating one or more of the cytokines and growth factors selected from stem cell factor (SCF), IL-6 and IL-9. In certain embodiments, erythropoietin (EPO) and / or IL-8 are not included. Megakaryocyte maturation relates to the increase of cytoplasm volume, α and the increase of dense granule quantity, the formation of dense tubular network and / or the formation of the open tubule system for granule release. In certain embodiments, mature megakaryocytes contain granules. In certain embodiments, mature megakaryocyte portion contains granules.

[0104] Megakaryocyte ploidy can be correlated with platelet production. For example, in vivo, bone marrow with higher ploidy megakaryocytes produces larger and more heterogeneous platelets. In some embodiments, mature megakaryocytes produced according to the present disclosure comprise a ploidy of at least 8N. In some embodiments, mature megakaryocytes comprise an average ploidy of at least about 8N, about 16N, about 32N, or about 64N.

[0105] As known in the art, mature megakaryocytes can form proplatelets and platelets, such as by culturing in the presence of fibroblast growth factor 4 (FGF4) and stromal cell-derived factor 1 (SDF 1). In certain embodiments, megakaryocytes or proplatelets are cultured in a bioreactor so that the cells are subjected to fluid shear stress. For example, platelets can be produced under static 2D, serum-free, cytokine-dependent conditions. Alternatively, platelets can be produced in a three-dimensional (3D) microenvironment. In various embodiments, platelets will have a phenotype of CD41+CD42b+. The platelets recovered can be activated by thrombin. Before infusion therapy, platelets can be recovered and gamma irradiated.

[0106] The compositions of the present disclosure (e.g., comprising platelets prepared according to the present disclosure) may further comprise a pharmaceutically acceptable carrier. Such carrier solutions may also contain buffers, diluents, and other suitable additives. A buffer refers to a solution or liquid whose chemical composition neutralizes an acid or base without significantly changing the pH. Examples of buffers contemplated by the present invention include, but are not limited to, normal / physiological saline (0.9% NaCl), 5% glucose in water (D5W), Dulbecco's phosphate-buffered saline (PBS), and Ringer's solution. The composition may comprise an 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). Other carriers may include dimethoxyethane (DME), N-dimethylformamide (DMF), or dimethylacetamide, including mixtures or combinations thereof. The cell or platelet composition may be provided in an implantable device (e.g., a stent) or in a bag or in a vial, test tube, or container in an appropriate volume and stored frozen until use.

[0107] In various embodiments, the composition comprises at least about 10 9 platelets, or at least about 10 10 platelets, or at least about 10 11 platelets, or at least about 10 12 Platelets.

[0108] The pharmaceutical composition used for the disclosed method may also contain other therapeutic agents for treating specific target diseases. For example, the pharmaceutical composition may also include cytokines and growth factors (interleukins, interferons, FGF, VEGF, PDGF, PIGF, STAT, etc.). Such additional factors and / or agents may be included in the pharmaceutical composition to produce the advantages of the methods disclosed herein, that is, to provide improved therapeutic effects and reduced systemic toxicity.

[0109] In other aspects, platelets are used to enrich platelet-rich plasma (PRP) from subjects in need of PRP therapy. Here, a combination of whole platelets or lysates extracted therefrom can be used to enrich platelet-rich plasma from other sources, such as donor-derived PRP. Optionally, it can include growth factors, cytokines, or other agents from other sources that supplement the PRP-based therapeutic application in the patient.

[0110] In other aspects, the present invention provides a method for platelet therapy, the method comprising administering platelets (prepared as described herein) or a pharmaceutically acceptable composition thereof to a human subject in need thereof. In various embodiments, the methods described herein are used to treat subjects suffering from following thrombocytopenia, such as ACTN1-related thrombocytopenia, amegakaryocytic thrombocytopenia with radial-ulnar fusion, ANKRD26-related thrombocytopenia, autosomal dominant thrombocytopenia, congenital amegakaryocytic thrombocytopenia, CYCS-related thrombocytopenia, FYB-related thrombocytopenia, idiopathic thrombocytopenic purpura or X-linked thrombocytopenia. In some embodiments, platelets are administered to a subject suffering from bleeding.

[0111] As used herein, the term "about" means ±10% of the associated numerical value.

[0112] Certain aspects and embodiments of the present disclosure are further described with reference to the following examples.

[0113] Examples

[0114] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances the formation of CD34+ cells during iPSC differentiation without affecting pluripotency.

[0115] method

[0116] iPSCs were developed from hCD34+ cells by episomal reprogramming known in the art and essentially as described by Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells ,Science318,1917-1920,(2007); and J.Yu,et al. Human induced pluripotent stem cells free of vector and transgene sequences .Science 324,797-801,(2009). Embryoid bodies and hemogenic endothelial differentiation were basically as described in the following literature: R.Sugimura, et al., Haematopoietic stem and progenitor cells from human pluripotent stem cells .Nature 545,432-438,(2017);CMSturgeon,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).

[0117] 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.

[0118] result

[0119] 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 indicated by the expression of the stem cell marker TRA-1-60 ( Figure 1 ). Figure 1 Shown are FACS graphs representing the transduction efficiency of iPSCs with adenoviral vectors overexpressing ETV2 and GFP sequences.

[0120] 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, such as CD235a - CD34 in the population + and CD31 + The expression of markers was confirmed ( Figure 2 ). Specifically, Figure 2 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.

[0121] Furthermore, the results showed that ETV2-OE enhanced CD34 + Cell formation ( Figure 3 ). Figure 3 Representative flow cytometric analysis of CD34+ cells is shown, and relative quantification indicates that ETV2-OE enhances the formation of CD34+ cells.

[0122] 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.

[0123] Example 2 - iPSC-derived HSCs generated using Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs.

[0124] method

[0125] To analyze EHT, EB-derived CD34+ cells were suspended in culture medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. After the cells had adhered to the bottom of the wells for approximately 4 to 18 hours (by visual inspection), Yoda1 was added to the culture for some experiments. After 4 to 7 days, the cells were harvested for analysis.

[0126] 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 (with or without Yoda1). Then, CD34+ cells were harvested from EHT cultures between day 5 and day 7 for further hematopoietic lineage differentiation.

[0127] result

[0128] Figure 4 FACS analysis showing megakaryocyte differentiation, indicating commitment of cells to the megakaryocyte lineage. HSC populations generated by inducing EHT in CD34+ cells differentiated from iPSCs (D8+7 iPSC-CD34+, with or without Yoda 1 "Y") outperformed CD34+ cells isolated from differentiated iPSCs (D8 iPSC-CD34+) and bone marrow (BM) CD34+ cells in supporting megakaryocyte differentiation. Figure 5 We show that megakaryocytes derived from D8+7 iPSC-CD34+ cells (with or without Y) are capable of in vitro expansion and outperform megakaryocytes differentiated from D8 iPSC-CD34+ cells and are similar to megakaryocytes differentiated from BM CD34+ cells.

[0129] Figure 6 Immunofluorescence analysis showing platelet differentiation of megakaryocytes derived from D8+7 iPSC-CD34+ cells (with or without Y) and bone marrow-derived CD34+ cells, indicating that D8+7 iPSC-CD34+-derived megakaryocytes are phenotypically similar to BM CD34+-derived megakaryocytes and are able to release platelets.

[0130] Figure 7 Thrombus formation is shown from D8+7 iPSC-CD34(+Y)-derived platelets, indicating that these platelets are able to clot and promote thrombus formation once activated.

[0131] Example 3 – Assessing off-target editing in HLA-depleted HSCs

[0132] 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.

[0133] 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.

[0134] Table 1 below summarizes the results of the editing strategy for two representative clones relative to wild-type cells.

[0135] Table 1: Clonal HSC HLA knockouts.

[0136]

[0137]

[0138] 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.

[0139] Table 2: Exemplary gRNA sequences

[0140]

[0141]

[0142] 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.

[0143] Phenotypic analysis of HLA-edited clones by FACS and immunofluorescence confirmed these results. Figure 8A and Figure 8B As shown, HLA-edited cells tested positive for overall expression of HLA-I class molecules compared to the overall expression of HLA-I class molecules in 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 8AThe 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.

[0144] To confirm the HLA-A gene deletion, the specific expression of HLA-A was analyzed by immunofluorescence. Figure 8B 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.

[0145] Example 4 - Evaluation of the Pluripotency and Immunocompatibility of HLA-Edited HSCs

[0146] The ability of HLA-edited cells to retain pluripotency was evaluated. Figure 9 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.

[0147] 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 (GVHD). 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 HLA edits and the transplant of wild-type HSC (gHSC).

[0148] Wild-type (gHSC) and HLA-edited HSC were co-cultured with PBMC matched for HLA-B and HLA-C markers but mismatched for HLA-A. B2M KO HSC, which lack expression of HLA class I molecules, and CIITA KO HSC, 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 10Figure 2 shows the results of a PBMC-mediated cytotoxicity assay 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 CIITAKO 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.

[0149] In summary, immune compatibility results showed that CD8+T cells present in PBMC samples were responsible for killing cells with mismatched HLA molecules (WT) and CIITA KO, while NK cells present in PBMC were responsible for killing HLA-ineffective cells (B2M KO). However, HLA-edited HSCs were protected from CD8+T cell-mediated cytotoxicity (because mismatched HLA-A had been knocked out) and from NK cell-mediated cytotoxicity (because the expression of HLA class I molecules was retained to a large extent).

[0150] Example 5 - Evaluation of the in vivo transplantation potential of HLA-edited HSCs

[0151] 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 (gHSCs) 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 11 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.

[0152] Example 6: HLA-edited HSCs differentiate into pro-megakaryocytes / pro-platelets

[0153] 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 12As 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.

[0154] References

[0155] 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).

[0156] 2.Brauer,PM,Singh,J.,Xhiku,S.& JCT Cell Genesis:InVitro Veritas Est? Trends Immunol 37,889–901(2016).

[0157] 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).

[0158] 4. Sturgeon, CM, 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).

[0159] 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).

[0160] 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).

[0161] 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).

[0162] 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).

[0163] 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).

[0164] 10. Guo, R. et al. Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors. Cell Research 30, 21–33 (2020).

[0165] 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).

[0166] 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 comprising megakaryocytes, the method comprising: Enriching CD34+ cells from a differentiated pluripotent stem cell (PSC) population to prepare a CD34+ enriched population; inducing endothelial to hematopoietic transition (EHT) of the CD34+ enriched population to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitor cells (HSPCs), and optionally harvesting cells that have undergone endothelial to hematopoietic transition; The HSC population is differentiated into megakaryocytes and optionally platelets.

2. The method of claim 1, wherein EHT is induced for at least 2 days and no more than 12 days.

3. The method of claim 1 or 2, wherein the population comprising HSCs and / or HSPCs comprises non-adherent cells. 4 . The method according to claim 1 , wherein the PSC population is a human iPSC population derived from lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells or human primary tissue. The method of claim 4 , wherein the iPSC population is derived from CD34+ enriched cells isolated from peripheral blood.

6. The method of any one of claims 1 to 5, wherein the iPSCs are homozygous for one or more HLA class I and / or class II genes. The method of claim 6 , wherein the iPSCs are homozygous for HLA-DRB1.

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

9. The method according to any one of claims 1 to 8, 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 ability.

10. The method of claim 9, wherein the iPSCs comprise a deletion of HLA-A.

11. The method of claim 9 or 10, wherein the iPSC comprises a deletion of HLA-DPB1 and / or HLA-DQB1.

12. The method according to any one of claims 2 to 11, wherein the iPSCs are: HLA-A neg , homozygous for both HLA-B and HLA-C, HLA-DPB1 neg and HLA-DQB1 neg , and optionally are homozygous for HLA-DRB1.

13. The method of claim 9, wherein the one or more genes governing HLA or MHC expression or presentation ability are β2-microglobulin and / or CIITA. 14 . The method according to claim 1 , wherein CD34+ enrichment and endothelial cell to hematopoietic cell transition are induced on day 8 to day 15 of iPSC differentiation.

15. The method of claim 14, 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.

16. The method of claim 14 or 15, wherein harvesting CD34+ cells from a culture undergoing endothelial to hematopoietic transition comprises harvesting CD34+ floating cells and / or adherent cells.

17. The method of claim 10 or 11, wherein the population comprising HSCs and / or HSPCs comprises long-term hematopoietic stem cells (LT-HSCs).

18. The method of any one of claims 1 to 17, wherein inducing endothelial cell to hematopoietic cell transition comprises increasing the expression or activity of dnmt3b.

19. The method of any one of claims 1 to 18, wherein the induction of endothelial cell to hematopoietic cell transition comprises applying cyclic stretch to the CD34-enriched cells.

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

21. The method of claim 18, wherein the induction of endothelial cell to hematopoietic cell transition comprises activation of Piezol.

22. The method of claim 21, 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 or analogs or derivatives thereof, or single-stranded RNA agonists.

23. The method of claim 22, 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.

24. The method of any one of claims 1 to 23, wherein the induction of endothelial cell to hematopoietic cell transition comprises Trpv4 activation.

25. The method of claim 24, 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.

26. The method according to any one of claims 1 to 25, wherein the step of differentiating the population comprising HSCs and / or HSPCs into megakaryocytes comprises culturing with thrombopoietin (TPO).

27. The method of claim 26, wherein the step of differentiating the population comprising HSCs and / or HSPCs into megakaryocytes further comprises culturing with one or more additional cytokines or growth factors selected from the group consisting of IL-1, IL-3, IL-6, IL-9, IL-11, SCF, SDF-1, and PDGF-BB.

28. The method of claim 26 or 27, further comprising the step of expanding the megakaryocytes, optionally by culturing with thrombopoietin (TPO), optionally one or more additional cytokines or growth factors selected from the group consisting of stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L), IL-6, IL-9, and erythropoietin (EPO).

29. The method according to any one of claims 26 to 28, further comprising the step of promoting maturation of the megakaryocytes, optionally by culturing with one or more of stem cell factor (SCF), IL-6 and IL-9.

30. The method of claim 29, wherein the step of promoting megakaryocyte maturation does not include culturing in the presence of erythropoietin (EPO) and / or IL-8.

31. The method according to any one of claims 1 to 30, wherein proplatelets and platelets are formed from the megakaryocytes by culturing in the presence of fibroblast growth factor 4 (FGF4) and stromal cell-derived factor 1 (SDF-1).

32. The method of claim 31, wherein the megakaryocytes or proplatelets are cultured in a bioreactor and the cells are subjected to fluid shear stress.

33. The method of any one of claims 31 or 32, wherein the platelets are capable of being activated by thrombin.

34. A composition comprising platelets and a carrier, said platelets being prepared by the method of any one of claims 1 to 33.

35. The composition of claim 24 comprising at least about 10 9 platelets, or at least about 10 10 platelets, or at least about 10 11 platelets, or at least about 10 12 Platelets.

36. A method of treating a subject in need of platelet transfusion comprising administering to the subject a platelet composition according to claim 34 or 35.

37. The method of claim 36, wherein the subject has thrombocytopenia.

38. The method of claim 37, wherein the subject has amegakaryocytic thrombocytopenia with radioulnar synostosis, ANKRD26-related thrombocytopenia, autosomal dominant thrombocytopenia, congenital amegakaryocytic thrombocytopenia, CYCS-related thrombocytopenia, FYB-related thrombocytopenia, idiopathic thrombocytopenic purpura, or X-linked thrombocytopenia.

39. The method of any one of claims 36 to 38, wherein the subject suffers from bleeding.

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