Megakaryocytes and platelets derived from pluripotent stem cells

Abstract

The present disclosure provides an efficient ex vivo process for generating megakaryocytes and / or platelets from human induced pluripotent stem cells (iPSCs). In various embodiments, the cells and platelets produced according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood, bone marrow, or other tissues. The invention in some aspects provides isolated cells / platelets and compositions produced by the methods disclosed herein, as well as methods for treatment.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 413,337, filed October 5, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in XML format and is incorporated herein by reference in its entirety. The XML copy, created on September 26, 2023, is named GRU-013PC_Sequence_Listing.xml and is 30,048 bytes in size. [Background technology]

[0003] In addition to playing a key role in physiological hemostasis, thrombus formation, and wound healing, platelets can also significantly contribute to host inflammation and immune responses to infection and injury. Many patients with hematopoietic disorders or undergoing aggressive chemotherapy require platelet transfusions using platelet concentrates obtained through blood / platelet donations. However, platelets for transfusion obtained from volunteer donors are a limited resource, for example, as the blood donation industry faces an ongoing crisis as demand for blood products, particularly platelets, frequently exceeds supply. Therefore, the development of large-scale, commercially available megakaryocytes and / or platelets, such as those derived from hematopoietic stem cells (HSCs), is an attractive tool in regenerative medicine. Summary of the Invention [Means for solving the problem]

[0004] In various aspects and embodiments, the present disclosure provides methods for generating hematopoietic lineages for cell therapy, including megakaryocyte-erythroid progenitors (MEPs), CFU-Me (pluripotent hematopoietic stem cells or hemoblasts), megakaryoblasts, promegakaryocytes, and megakaryocytes, as well as proplatelets, proplatelets, or platelets derived therefrom. In various embodiments, the present disclosure provides an efficient ex vivo process for developing megakaryocytes and / or platelets from human induced pluripotent stem cells (iPSCs), including gene-edited iPSCs. In various embodiments, the megakaryocytes and / or proplatelets, proplatelets, or platelets generated according to the present disclosure are functional and / or more closely resemble the corresponding native lineages isolated from bone marrow or blood, or the corresponding native platelets isolated from blood. The present disclosure also provides isolated cells and compositions produced by the methods disclosed herein, as well as methods for treatment.

[0005] In other aspects and embodiments, the present disclosure provides HSCs derived from iPSCs that have been gene-edited to be immunocompatible with a substantial portion of the population. These HSC populations can be used for more efficient ex vivo platelet production, or in other aspects, to deliver HSCs, or megakaryocytes, platelets, or their progenitors, to patients in need of reducing or eliminating the need for regular blood transfusions.

[0006] In one aspect, the present disclosure provides a method for preparing a cell population comprising megakaryocytes. The megakaryocytes of the present disclosure can further generate functional platelets. The method of the present disclosure includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population differentiated into embryoid bodies, and enriching for CD34+ cells, thereby preparing a CD34+-enriched population. Endothelial-hematopoietic transition (EHT) is induced in the CD34+-enriched population, thereby preparing a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs). The resulting cell population (or a fraction thereof) can be differentiated into megakaryocytes and, optionally, used to produce platelets.

[0007] In various embodiments, iPSCs are prepared by reprogramming somatic cells. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to a recipient (a subject in need of a treatment described herein). In various embodiments, iPSCs can be gene-edited to support HLA matching. For example, iPSCs can be gene-edited to delete one or more of HLA-A, HLA-B, and HLA-C, and to delete one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, iPSCs are homozygous for 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.

[0008] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be recovered from the culture and used to generate embryoid bodies (EBs). EBs, generated by differentiation of iPSCs, are three-dimensional aggregates of iPSCs that contain three (or alternatively, two or one) embryonic germ cell layers depending on the differentiation method(s). In some embodiments, processes according to each aspect can include generating CD34+ cells from pluripotent stem cells (e.g., EBs) and inducing endothelial-hematopoietic transformation. HSCs can be generated from cell populations using a variety of stimuli or factors, including mechanical, biochemical, metabolic, and / or local stimuli, as well as factors such as extracellular matrix, niche factors, cell-extrinsic factors, induction of cell-intrinsic properties, and including pharmacological and / or genetic means.

[0009] In some embodiments, iPSC differentiation proceeds 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 days 7-14 of iPSC differentiation. Differentiation of iPSCs can be by known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, a combination of bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1.

[0010] Induction of EHT can be performed using any known process. In some embodiments, induction of EHT generates a hematopoietic stem cell (HSC) population, including LT-HSC. In some embodiments, EHT generates HSCs through endothelial or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population, including one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), and hematopoietic stem progenitor cells. In some embodiments, EHT is induced in culture for about 5 to about 7 days. In some embodiments, EHT is performed using a medium containing one or more growth factors and cytokines selected from TPO, SCF, Flt3L, IL3, IL-6, IL-7, IL-11, IGF, bFGF, and IL-15. The medium may optionally include one or more of VEGF, bFGF, a BMP activator, a Wnt pathway activator, or a ROCK inhibitor (e.g., thiazovivin or Y27632). In some embodiments, the HSC and / or HSPC population or fraction thereof is differentiated independent of the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as Yoda1. In some embodiments, the use of an agonist of a mechanosensitive receptor or mechanosensitive channel (e.g., Yoda1) is optional.

[0011] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34+ enriched cells). In certain embodiments, pharmacological Piezo1 activation may be further applied to hematopoietic lineage(s), including iPSCs, embryoid bodies, ECs, hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and the megakaryocytic lineage involved in the generation of platelets. 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 superior generation of megakaryocytes compared to other methods for inducing EHT.

[0012] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition on days 10-20 of iPSC differentiation, such as days 12-17. In some embodiments, the CD34+ cells include non-adherent cells. In various embodiments, the HSCs or CD34+ enriched cells are further expanded.

[0013] HSCs and / or HSPCs that give rise to megakaryocytes and / or platelets can be identified based on the expression of CD34 and the absence of lineage-specific markers (termed Lin-). In some embodiments, stem cell populations for differentiation into hematopoietic lineages are characterized by at least about 80% CD34 expression. + , or at least about 90% CD34 + , or at least about 95% CD34 + is.

[0014] In various embodiments, the HSC and / or HSPC population or a fraction thereof can be differentiated ex vivo into megakaryocytes, which can then generate platelets.

[0015] In some embodiments, differentiating the population comprising HSCs and / or HSPCs into megakaryocytes comprises culturing with thrombopoietin (TPO). The culture may further comprise one or more additional cytokines 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, including TPO, can be selected to further expand megakaryocytes. In some embodiments, such additional cytokines or growth factors for expanding megakaryocytes may be selected from stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L), IL-6, IL-9, and erythropoietin (EPO).

[0016] Mature megakaryocytes can be cultured in the presence of fibroblast growth factor 4 (FGF4) and stromal cell-derived factor 1 (SDF1) to form proplatelets and platelets, as known in the art. In some embodiments, megakaryocytes or proplatelets are cultured in a bioreactor that subjects the cells to hydrodynamic shear stress. For example, platelets can be produced in static 2D, serum-free, cytokine-dependent conditions. Alternatively, platelets can be produced in a three-dimensional (3D) microenvironment. In various embodiments, the platelets will have a phenotype that is CD41+CD42b+. Recovered platelets can be activated by thrombin. Platelets can be collected and subjected to gamma irradiation prior to transfusion therapy.

[0017] 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 composition may be provided in an appropriate volume within an implantable device (e.g., a scaffold), or within a bag, or within a vial, tube, or container, and may be stored frozen until use. In various embodiments, the composition contains at least about 10 per 50 or 100 mL volume. 9 platelets, or at least about 10 10 platelets, or at least about 10 11 platelets, or at least about 10 12 Contains platelets.

[0018] In another aspect, the present invention provides methods for platelet therapy, 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 a subject with thrombocytopenia, such as ACTN1-associated thrombocytopenia, amegakaryocytic thrombocytopenia with radioulnar synostosis, ANKRD26-associated thrombocytopenia, autosomal dominant thrombocytopenia, congenital amegakaryocytic thrombocytopenia, CYCS-associated thrombocytopenia, FYB-associated thrombocytopenia, idiopathic thrombocytopenic purpura, or X-linked thrombocytopenia. In some embodiments, the platelets are administered to a subject experiencing bleeding.

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

[0020] [Figure 1] Figure 1 shows that ETV2 overexpression (OE) does not affect pluripotency. Figure 1 shows a FACS plot showing the transduction efficiency of iPSCs using an adenoviral vector to overexpress ETV2 and GFP sequences. ETV2 overexpression does not affect iPSC stemness, as indicated by the expression of the TRA-1-60 stemness marker. [Figure 2] ETV2 overexpression (OE) increases the yield of hemogenic endothelial cells. Representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (described as CD235a-CD34+CD31+) demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells. [Figure 3] Figure 1 shows that ETV2 overexpression (OE) enhances CD34+ cell formation during iPSC differentiation. Representative flow cytometry analysis and relative quantification of CD34+ cells demonstrates that ETV2-OE enhances CD34+ cell formation. [Figure 4] 1 shows FACS analysis of megakaryocytic differentiation demonstrating commitment of HSCs to the megakaryocytic lineage. [Figure 5]We show that megakaryocytic cells derived from iPSC-derived HSCs can be expanded ex vivo. [Figure 6] 1 shows immunofluorescence analysis of platelet differentiation of iPSC-derived HSCs, demonstrating that the derived megakaryocytes are phenotypically similar to BM CD34+-derived megakaryocytes and are capable of releasing platelets. [Figure 7] 1 shows thrombus formation by platelets produced from iPSC-derived HSCs, indicating that these platelets can clot and promote thrombus formation upon activation. [Figure 8A]

[0023] Figure 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence, demonstrating the overall expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface, with HLA-edited cells being positive for overall HLA class I expression to a similar extent as wild-type cells. [Figure 8B] Figure 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence. Cellular expression of HLA-A via immunofluorescence is shown, where HLA-A is not expressed in HLA-edited clones. [Figure 9] We show that the HLA-edited clones retain their pluripotency (maintain tri-lineage differentiation) as illustrated by immunofluorescence, where ectodermal differentiation is indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation is indicated by GATA-488 staining, and endodermal differentiation is indicated by CXCR4-488 and FOX2A-594 staining. [Figure 10] Immunocompatibility of HLA-edited HSCs: HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were cocultured with peripheral blood mononuclear cells (PBMCs) bearing HLA-B and HLA-C matched but mismatched HLA-A, and PBMC-mediated cytotoxicity was measured by Annexin V staining assay. [Figure 11]Figure 1 shows the in vivo engraftment potential of HLA-edited HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed for competitive transplantation into mice, and bone marrow (BM) and peripheral blood samples were assessed by FACS to compare the relative amounts of each cell type present in the samples. [Figure 12] This shows that HLA-edited HSCs can differentiate into megakaryocytes (MKs), which can further differentiate into platelets. The image on the left shows an increase in the percentage of platelets among HSCs by light microscopy at 1000x magnification. The graph on the right shows a statistically significant increase in the percentage of platelets differentiated from HLA-edited HSCs compared to bone marrow (BM) CD34+ cells and CD34+ cell populations isolated from differentiated iPSCs (e.g., EBs). DETAILED DESCRIPTION OF THE INVENTION

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

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

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

[0024] In various aspects and embodiments, the present disclosure provides methods for generating hematopoietic lineages for cell therapy, including megakaryocyte-erythroid progenitors (MEPs), CFU-Me (pluripotent hematopoietic stem cells or hemoblasts), megakaryoblasts, promegakaryocytes, and megakaryocytes, as well as proplatelets, proplatelets, or platelets derived therefrom. In various embodiments, the present disclosure provides an efficient ex vivo process for developing megakaryocytes and / or platelets from human induced pluripotent stem cells (iPSCs), including gene-edited iPSCs. In various embodiments, the megakaryocytes and / or proplatelets, proplatelets, or platelets generated according to the present disclosure are functional and / or more closely resemble the corresponding native lineages isolated from bone marrow or blood, or the corresponding native platelets isolated from blood. The present disclosure also provides isolated cells and compositions produced by the methods disclosed herein, as well as methods for treatment.

[0025] In other aspects and embodiments, the present disclosure provides HSCs derived from iPSCs that have been gene-edited to be immunocompatible with a substantial portion of the population. These HSC populations can be used for more efficient ex vivo platelet production, or in other aspects, to deliver HSCs, or megakaryocytes, platelets, or their progenitors, to patients in need of reducing or eliminating the need for regular blood transfusions.

[0026] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to 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 in turn give rise to proplatelets and megakaryocytes, which release platelets. The use of platelets in therapy is 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 approximately five days, they are prone to bacterial growth during storage, and they lack consistency between donor-derived batches. Furthermore, platelet transfusions are often associated with several risks to recipients, including allergic reactions and febrile nonhemolytic reactions. See, for example, Kaufman, Richard M., et al., “Platelet transfusion: a clinical practice guideline from the AABB,” Annals of Internal Medicine 162, no. 3 (2015): 205-213. Furthermore, compared to primary cells, hiPSCs are more easily genetically modified in vitro, thereby providing opportunities for improved cell / platelet numbers and avoiding, for example, HLA matching issues. Additionally, fully engineered hiPSC clones can serve as a stable and safe source compared to primary cells. Furthermore, unlike human embryonic stem cells (hESCs), hiPSCs are of non-embryonic origin, eliminating ethical concerns and ensuring consistent quality. Therefore, the use of hiPSCs according to the present disclosure offers several advantages over primary cells for generating therapeutic hematopoietic lineages, such as the megakaryocytic lineage, including megakaryocytes and platelets (including their progenitors).

[0027] In one aspect, the present disclosure provides a method for preparing a cell population comprising megakaryocytes. The megakaryocytes of the present disclosure can further generate functional platelets. The method of the present disclosure includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population differentiated into embryoid bodies, and enriching for CD34+ cells, thereby preparing a CD34+ enriched population. Endothelial-hematopoietic transition (EHT) is induced in the CD34+ enriched population, thereby preparing a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs). In some embodiments, CD34+ cells are enriched from the population comprising HSCs and / or HSPCs. In various embodiments, EHT is induced for at least 2 days and up to 12 days. The resulting cell population (or a fraction thereof) can be differentiated into megakaryocytes and, optionally, used to produce platelets.

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

[0029] In some embodiments, CD34+ cells (i.e., recovered from EB dissociation) are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. Exemplary Piezol agonists include Yoda1, Jedi1, and Jedi2, or analogs thereof. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Other modalities for inducing EHT can be used (alternatively or in addition) and are described herein. In some embodiments, after inducing EHT, the cells (populations including HSCs and / or HSPCs) are differentiated into the megakaryocytic lineage, e.g., into a cell population including megakaryocytes capable of producing or secreting platelets.

[0030] In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cells" or "iPSCs" refers to cells derived from somatic cells, such as skin or blood cells, that have been reprogrammed back to an embryonic-like pluripotent state. In some embodiments, iPSCs are generated from somatic cells, such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from lymphocytes, granulocyte / macrophage lineage-restricted progenitor cells (GMPs), umbilical cord blood cells, PBMCs, CD34+ cells, or other primary human tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to the recipient (the subject in need of the treatment described herein). In various embodiments, iPSCs can be genetically edited to support HLA matching (such as deletion of one or more HLA class I and / or HLA class II alleles or their master regulators, including, but not limited to, beta-2-microglobulin (B2M), CIITA, etc.), or to delete or express other functions. For example, iPSCs can be genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C, and one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus.

[0031] In various embodiments, the HSCs and megakaryocytic lineage cells are derived from iPSCs that have been gene-edited to be one of: (i) HLA-A-B+C+DP-DR+DQ+, (ii) HLA-A-B+C+DP+DR+DQ-, (iii) HLA-A-B+C+DP-DR+DQ-, (iv) HLA-AB-C+DP-DR+DQ+, (v) HLA-AB-C+DP+DR+DQ-, or (vi) HLA-AB-C+DP-DR+DQ-. For retained HLA (e.g., HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or can retain only a single copy of the gene. For example, the modified cells are identified as at least (a) HLA-C+ and HLA-DR+, and optionally as one or more of (b) HLA-B-, (c) HLA-DP-, and (d) HLA-DQ-. In an exemplary embodiment, the modified cells are HLA-B+, HLA-DP-, and HLA-DQ-.

[0032] In some embodiments, the iPSCs are HLA-A neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0033] As used herein, the term "neg," (-), or "negative" with respect to a particular HLA class I or HLA class II molecule indicates that both copies of the gene have been disrupted in a cell line or population, and therefore the cell line or population does not exhibit significant functional expression of the gene. Such cells can be generated by complete or partial gene deletion or disruption, or alternatively by other techniques such as siRNA. As used herein, the term "deletion" in the context of genetic modification (i.e., gene editing) of a targeted gene refers to the elimination of functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletion or disruption of a coding sequence or deletion of a critical cis-acting expression control sequence.

[0034] In some embodiments, iPSCs are gene-edited using gRNAs that are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more nucleotides in length. In some embodiments, the gRNAs comprise modifications at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and / or at or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end). In some embodiments, the modified gRNAs exhibit increased resistance to nucleases. In some embodiments, the gRNAs comprise two separate RNA molecules (i.e., "duplex gRNAs"). Duplex gRNAs comprise two separate RNA molecules, "crispr RNA" (or "crRNA") and "tracr RNA," and are well known to those of skill in the art.

[0035] Generally, various gene editing technologies are known that can be applied in accordance with various embodiments of the present disclosure, including, but not limited to, zinc finger (ZF), transcription activator-like effector (TALE), etc. Fusion proteins comprising one or more of these DNA binding domains and the cleavage domain of a Fokl endonuclease can be used to generate double-stranded breaks in desired regions of DNA within a cell (see, e.g., U.S. Patent Application Publication No. 2012 / 0064620, U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. 2011 / 0301073, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the entire contents of which are incorporated herein by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system (e.g., CRISPR-Cas9) known in the art. See, for example, US8,697,359, US8,906,616, and US8,999,641, each of which is incorporated herein by reference in its entirety. In various embodiments, gene editing employs a type II Cas endonuclease (such as Cas9) or a type V Cas endonuclease (such as Cas12a). Type II and type V Cas endonucleases are guide RNA dependent. Design of gRNAs to guide desired gene editing (while limiting or avoiding off-target editing) is known in the art. See, for example, Mohr SE, et al., CRISPR guide RNA design for research applications, FEBSJ. 2016 Sep; 283(17):3232-3238.In still other embodiments, non-canonical Type II or Type V Cas endonucleases with homology (albeit low primary sequence homology) to S. pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) can be employed. Many such non-canonical Cas endonucleases are known in the art. Nidhi S, et al. Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives, Int J Mol Sci. 2021 Apr;22(7):3327. In still other embodiments, gene editing employs base editing or prime editing to incorporate mutations without creating double-strand breaks. See, for example, Antoniou P, et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA, Front. Genet., 9 June 2020. Various other gene editing processes are known, including the use of dead Cas (dCas) systems (e.g., Cas fusion proteins) to target DNA-modifying enzymes to desired targets using dCas as a guide RNA-dependent system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications, Int J Mol Sci. 2019 Dec;20(23):6041.

[0036] Base editors that can introduce precise genome modifications without generating double-stranded DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., designing gene therapy vectors). Base editors inherently cannot create DSBs and contain a catalytically inactive nuclease, such as Cas9 nickase (nCas9), fused to a nucleic acid base deaminase enzyme, possibly a DNA glycosylase inhibitor. Currently, there are two major categories of base editors: cytidine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G transversions. Base editors can be delivered, for example, via HDAd5 / 35++ vectors, to efficiently edit promoters and enhancers to activate or inactivate genes. Exemplary methods are described in U.S. Patent Nos. 9,840,699, 10,167,457, 10,113,163, 11,306,324, 11,268,082, 11,319,532, and 11,155,803. Also contemplated are prime editors comprising a reverse transcriptase conjugated to (e.g., fused with) a Cas endonuclease and a polynucleotide useful as a DNA synthesis template conjugated to (e.g., fused with) a guide RNA, as described in WO2020 / 191153.

[0037] Exemplary vectors that can be used for genome editing applications include plasmids, retroviral vectors, lentiviral vectors, adenoviral vectors (e.g., Ad5 / 35, Ad5, Ad26, Ad34, Ad35, Ad48), parvoviruses (e.g., adeno-associated virus (AAV) vectors), herpes simplex virus vectors, baculovirus vectors, negative-stranded RNA viruses such as coronaviruses, orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses. Examples of vectors that can be used include, but are not limited to, double-stranded DNA viruses, including canarypox, vaccinia, or modified vaccinia viruses. Vectors containing a nucleic acid molecule of interest can be delivered to cells (e.g., iPS cells, endothelial cells, hemogenic endothelial cells, HSCs (ST-HSCs or LT-HSCs)) via any method known in the art, including, but not limited to, transduction, transfection, infection, and electroporation. Any of these vectors may contain a transposable element (such as a piggyBac transposon or a Sleeping Beauty transposon). Transposons insert specific sequences of DNA into the vertebrate genome. A gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar excision site present in the cell's nuclear genome.

[0038] For increased efficiency, in some embodiments, Cas and gRNA can be combined before delivery into cells. The Cas-gRNA complex is known as a ribonucleoprotein (RNP). Several methods have been developed for direct delivery of RNP into cells. For example, RNP can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nucleofection protocol can be employed, which allows RNP to rapidly enter the cell's nucleus and immediately begin cleaving the genome. See, for example, Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics. 2021 Jan 1;11(2):614-648, incorporated herein by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNP.

[0039] Generally, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage and is typically found 3-4 nucleotides downstream of the cleavage site. The PAM is a short DNA sequence (usually 2-6 base pairs in length) following the DNA region targeted for cleavage by a CRISPR system such as CRISPR-Cas9. In some embodiments, the PAM sequence, sgRNA, or base editing tool targeting a haplotype or polymorphism at an HLA locus does not contain four Gs, four Cs, a GC repeat, or a combination thereof.

[0040] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed by using the gRNAs described herein to design a single gRNA that targets each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example). To perform gene knockout, the gRNA targets the Cas9 protein to the appropriate site for editing. The Cas9 protein can then perform a double-strand break (DSB), which repairs the DNA through a non-homologous end joining (NHEJ) mechanism, generating an indel that results in a frameshift mutation and terminates the function of the resulting protein. However, off-target gene recombination can occur, altering the function of an otherwise intact gene. For example, the Cas9 endonuclease can generate DSBs at undesired off-target locations, even in the presence of some degree of mismatch. This off-target activity can result in genomic instability events such as point mutations and genomic structural perturbations. In various embodiments, an sgRNA targeting HLA-A can target a region of chromosome 6 defined as 29942532-29942626. In various embodiments, an sgRNA targeting HLA-DQB1 can target a region of chromosome 6 defined as 32665067-32664798. In various embodiments, an sgRNA targeting HLA-DPB1 can target a region of chromosome 6 defined as 33080672-33080935.

[0041] gRNAs can be used to develop clonal iPSCs. Such iPSC lines can be assessed for (i) on-target editing, (ii) off-target editing, and (iii) translocation editing, for example, using sequencing as described herein. Specifically, such assays can be performed by multiplex PCR using primers designed to target and enrich for the region of interest, followed by next-generation sequencing (e.g., Amplicon sequencing, AMP-seq). On-target and translocation panels can amplify the intended edited region and allow for the selection of iPSC clones with the expected edits that do not contain chromosomal translocations resulting from unintended DSB cleavage site fusions. Off-target panels can enrich for any potential off-target regions identified through sequencing, allowing for the selection of iPSC clones with negligible off-target mutations. Together, these assays enable the screening of iPSC clones to select clones with the desired edits while ruling out potential CRISPR / Cas9-related genome integrity issues.

[0042] In some embodiments, to further ensure the genomic stability and integrity of the reprogrammed and edited iPSCs, genetic and genomic assays can be performed to select clones that have not undergone translocation and mutation events and have not integrated the episomal vector. For example, whole genome sequencing (WGS) can be performed on CD34+ cells and iPSC clones after reprogramming, and the genomes can be compared for differences resulting from editing. These analyses provide an assessment of which iPSC clone genomes differ from the CD34+ starting material, allowing for the informed selection of iPSC clones that have not undergone mutations during reprogramming.

[0043] In some embodiments, karyotyping using a system such as the KARYOSTAT assay is used to select iPSC clones that did not develop indels or translocations during reprogramming, as described, for example, in Ramme AP, et al., "Supporting dataset of two integration-free induced pluripotent stem cell lines from related human donors," Data Brief. 2021 May 15;37:107140, incorporated herein by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal abnormalities with a resolution similar to G-banding karyotyping. The size of structural abnormalities that can be detected is >2 Mb for chromosome gains and >1 Mb for chromosome losses. The KARYOSTAT array is functionalized for balanced whole-genome coverage with low-resolution DNA copy number analysis, and the array covers all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay allows for the detection of aneuploidy, submicroscopic abnormalities, and mosaic events.

[0044] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that have not developed copy number aberrations (CNAs) during reprogramming, as described, for example, in Wiesner et al. "Molecular Techniques," Editor(s): Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, "Pathology of Melanocytic Tumors," Elsevier, 2019, pp. 364-373, ISBN 9780323374576, and Hussein SM, et al. "Copy number variation and selection during reprogramming to pluripotency," Nature. 2011 Mar 3;471(7336):58-62, which are incorporated herein by reference in their entireties. aCGH is a technique that analyzes the entire genome for CNAs by comparing sample DNA with reference DNA.

[0045] In some embodiments, a targeted hematologic malignancy NGS panel analysis is used to select iPSC clones that did not develop hematologic malignancy mutations during reprogramming. For example, a targeted hematologic malignancy NGS panel focuses on genes associated with myeloid leukemia, lymphoma, and / or other hematologic malignancies, generating smaller, more manageable datasets than more broad methods. A targeted hematologic malignancy NGS panel analysis involves the use of highly multiplexed PCR to amplify regions associated with hematologic malignancies, followed by next-generation sequencing.

[0046] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that have not integrated the episomal vector and have been passaged sufficiently for episomal vector clearance. As discussed herein, iPSC reprogramming of CD34+ cells can be achieved by delivering episomal vectors encoding reprogramming factors. However, episomal vectors can randomly integrate into the cellular genome, although this is rare, which can disrupt developmental processes, homeostasis, and the like. Therefore, ddPCR can be used to detect residual episomal vectors in iPSC cultures, allowing for the selection of iPSC clones that have not integrated the episomal vector.

[0047] In some embodiments, after assessing that selected clones do not contain editing-related genomic abnormalities, the clones can be further tested for spontaneous mutations that may arise during expansion, such as mutations affecting hematologic malignancies, indels, translocations, and numerical abnormalities, as described for pre-edited reprogrammed clones. Analysis of spontaneous mutations can include whole genome sequencing (WGS), KARYOSTAT analysis, array comparative genomic hybridization (aCGH) analysis, targeted hematologic malignancies NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0048] Somatic cells can be reprogrammed by expression of reprogramming factors selected from Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, and klf4. Methods for preparing iPSCs are described, for example, in U.S. Pat. Nos. 10,676,165, 9,580,689, and 9,376,664, which are incorporated by reference in their entireties. In various embodiments, reprogramming factors are expressed using well-known viral vector systems, such as lentivirus, Sendai, or measles virus systems. Alternatively, reprogramming factors can be expressed by introducing mRNA(s) encoding the reprogramming factors into somatic cells. Furthermore, iPSCs can be generated by introducing non-integrating episomal plasmids expressing reprogramming factors, i.e., for the generation of transgene-free and virus-free iPSCs. Known episomal plasmids that have limited replicative capacity and are therefore lost over several cell generations can be employed.

[0049] In some embodiments, human pluripotent stem cells (e.g., iPSCs) 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 HLA class II genes), deletion of β2 microglobulin (β2M), deletion of CIITA, or deletion or addition of receptor genes. Alternatively, engineered iPSCs with one or more HLA knockouts and TCR knockouts can be placed in a bioreactor for feeder-free and serum-free differentiation under GMP-grade conditions to generate fully functional megakaryocytes and resulting platelets.

[0050] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be recovered from the culture to generate embryoid bodies (EBs). EBs generated by differentiation of iPSCs are three-dimensional aggregates of iPSCs and contain three (or alternatively, two or one) embryonic germ cell layers based on the differentiation method(s). EB preparation is described, for example, in US 2019 / 0177695, which is incorporated herein by reference in its entirety. In some embodiments, EBs prepared by differentiation of iPSCs are expanded in a bioreactor, as described, for example, in Abecasis B. et al., "Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches." J. of Biotechnol. 246 (2017) 81-93. EBs can be used to generate any desired cell type. Other methods involving 3D suspension culture for the growth or differentiation of EBs are described in WO2020 / 086889, which is incorporated herein by reference in its entirety.

[0051] In some embodiments, processes according to each aspect can include generating CD34+ cells from pluripotent stem cells (e.g., EBs) and inducing endothelial-hematopoietic transformation. HSCs, including relatively high frequencies of LT-HSCs, can be generated from cell populations using a variety of stimuli or factors, including mechanical, biochemical, metabolic, and / or local stimuli, as well as factors such as extracellular matrix, niche factors, cell-extrinsic factors, induction of cell-intrinsic properties, and including pharmacological and / or genetic means.

[0052] In some embodiments, the method includes preparing hematopoietic endothelial cells from pluripotent stem cells prior to induction of EHT. In some embodiments, combined overexpression of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can lead to the formation of hematopoietic endothelial cells from PSC sources. In some embodiments, the method includes overexpression of the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSCs. ETV2 can be expressed by introduction of an encoding non-integrating episomal plasmid for constitutive or inducible expression of ETV2 and for the production of transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. The mRNA can be introduced using any available method, including electroporation or lipofection. Differentiation of ETV2-expressing cells can include the addition of VEGF-A. See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA. Sci. Adv. Vol. 6 (2020). Cells generated in this manner can be used to produce CD34+ cells and induce EHT according to embodiments of the present disclosure.

[0053] After CD34+ enrichment, HSCs and / or HSPCs are then generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification.

[0054] In some embodiments, iPSC differentiation proceeds until the cells are at least about 10% CD34+, or at least about 20% CD34+, or at least about 25% CD34+, or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced on days 7-14 of iPSC differentiation, such as days 8, 9, 10, 11, 12, 13, or 14. Differentiation of iPSCs can be by known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, combinations of bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1. In some embodiments, hPSCs are differentiated using feeder-free, serum-free, and / or GMP-compatible materials. Serum-free cultures typically include a cytokine / growth factor / small molecule cocktail.

[0055] In some embodiments, hPSCs are co-cultured in serum-containing medium with mouse bone marrow-derived feeder cells, such as OP9, which are feeder layers of STO mouse fibroblasts, blood-derived peripheral blood mononuclear cells (PBMCs), or cord blood-derived mesenchymal stem cells or lymphocyte-derived cancer cell line cells. The cultures can contain growth factors and cytokines to support differentiation into embryoid bodies or monolayers. Feeder cell co-culture systems can be used to generate multipotent HSPCs that 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., Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells, Stem Cell Research & Therapy Vol. 11 Art. 481 (2020). Alternatively, a stepwise process using defined conditions in conjunction with specific signals can be used. For example, expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs favors direct differentiation into multipotent CD34+ / CD45+ progenitors. Furthermore, expression of factors such as HOXB4, CDX4, SCL / TAL1, or RUNX1a supports the hematopoietic program in human PSCs. See Doulatov S. et al., Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors, Cell Stem Cell. 2013 Oct 3;13(4).

[0056] Differentiation of iPSCs (e.g., into EBs) can employ a WNT agonist, such as CHIR99021. A WNT agonist is a molecule that mimics or increases WNT signaling. Non-limiting examples of WNT agonists include the small molecule CHIR-99021 (CAS 252917-06-9), 2-amino-4,6-disubstituted pyrimidines such as BML 284 (CAS 853220-52-7), SKL 2001 (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), Kenpaullone (CAS 142273-20-9), or (hetero)arylpyrimidines. In some embodiments, the WNT agonist is an agonist antibody or functional fragment thereof, or an antibody-like polypeptide.

[0057] Differentiation of iPSCs (e.g., into EBs) can employ ROCK inhibitors. Exemplary ROCK inhibitors used in establishing and differentiating iPSCs include, but are not limited to, thiazovivin, Y27632, fasudil, AR122-86, RevitaCell™ 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, as well as the ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated herein by reference in its entirety.

[0058] Induction of EHT can be by any known process. In some embodiments, induction of EHT generates a hematopoietic stem cell (HSC) population, including LT-HSC. In some embodiments, EHT generates HSCs through endothelial or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population, including one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), and hematopoietic stem progenitor cells. In various embodiments, EHT can be induced in culture for 2 to 12 days, such as 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 some embodiments, EHT is induced in culture for about 5 to about 7 days. In embodiments, EHT is performed using a medium comprising one or more growth factors and cytokines selected from TPO, SCF, Flt3L, IL3, IL-6, IL7, IL-11, IGF, bFGF, and IL15. The medium may optionally include one or more of VEGF, bFGF, a BMP activator, a Wnt pathway activator, or a ROCK inhibitor (e.g., thiazovivin or Y27632).

[0059] In some embodiments, HSC and / or HSPC populations or fractions thereof are differentiated independently of the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as Yoda1. In some embodiments, the use of an agonist of a mechanosensitive receptor or mechanosensitive channel (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. Endothelial-hematopoietic conversion of the CD34+ enriched cell population is induced over at least 2 days but not more than 12 days, optionally with the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as yoda1, jedi1, jedi2, or ssRNA40. In some embodiments, endothelial-to-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days, 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. Full EHT differentiation proceeds over 12 days or less.

[0060] In some embodiments, the method comprises increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34+ enriched cells, ECs, HECs, or HSCs, which may be by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the method comprises increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in the cells. See WO2019 / 236943 and WO2021 / 119061, which are incorporated by reference in their entireties. In some embodiments, inducing EHT comprises increasing the expression or activity of dnmt3b.

[0061] In some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. An exemplary Piezol agonist is yoda1. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. 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, Chemical activation of the mechanotransduction channel Piezol. eLife (2015).

[0062] Derivatives of Yodal can be employed in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are employed in some embodiments. Exemplary agonists are disclosed in Evans EL, et al., "Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation," British Journal of Pharmacology 175(1744-1759):2018. Still other Piezo1 agonists include Jedi1, Jedi2, single-stranded (ss) RNA (e.g., ssRNA40), and derivatives and analogs thereof. See Wang Y., et al., "A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel," Nature Communications (2018) 9:1300; and Sugisawa, et al., "RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis," Cell, Volume 182, Issue 3, 2020, Pages 609-624, which are incorporated herein by reference in their entireties. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of Piezo1 agonist or derivative ranges from about 1 μM to about 500 μM, or from about 5 μM to about 200 μM, or from about 5 μM to about 100 μM, or in some embodiments, from about 25 μM to about 150 μM, or from about 25 μM to about 100 μM, or from about 25 μM to about 50 μM. Alternatively, single-stranded (ss) RNA (e.g., ssRNA) and derivatives and analogs thereof can be used for Piezo1 activation.

[0063] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34+ enriched cells). In certain embodiments, pharmacological Piezo1 activation may be further applied to hematopoietic lineage(s), including iPSCs, embryoid bodies, ECs, hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and the megakaryocytic lineage involved in the generation of platelets. 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 superior generation of megakaryocytes compared to other methods for inducing EHT.

[0064] Alternatively, or in addition, Dnmt3b activity or expression can be increased directly in cells, for example, in CD34+ enriched cells. For example, Dnmt3b mRNA expression can be increased by, but not limited to, delivering a transcript encoding Dnmt3b to cells, or by introducing a transgene encoding Dnmt3b, or by transgene-free methods such as introducing a non-integrating episome into cells. In some embodiments, gene editing is employed to introduce genetic modifications into Dnmt3b-expressing elements in cells, such as, but not limited to, increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0065] In some embodiments, the method includes increasing the activity or expression of Gimap6 in a cell, alone or in combination with Dnmt3b and / or other genes that are up- or down-regulated upon cyclic strain or Piezol activation. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cell; a transgene-free approach can be employed, including, but not limited to, introducing an episome into the cell; or, alternatively, a transgene encoding Gimap6 can be introduced. In some embodiments, gene editing is employed to introduce genetic modifications (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or to affect RNA splicing) into a Gimap6-expressing element in the cell.

[0066] In embodiments of the present disclosure employing mRNA delivery to cells, known chemical modifications can be used to circumvent the innate immune response in cells. For example, synthetic RNAs containing only canonical nucleotides can bind to pattern recognition receptors and induce a strong immune response in cells. This response can result in translation block, secretion of inflammatory cytokines, and cell death. RNAs containing certain non-canonical nucleotides can avoid detection by the innate immune system and can be translated into proteins with high efficiency. See US Pat. No. 9,181,319, incorporated herein by reference, particularly for nucleotide modifications to circumvent the innate immune response.

[0067] In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into cells, which can direct the desired level of overexpression (with varying promoter strength or other selection of expression control elements). Various viral vectors or transfection reagents (including lipid nanoparticles) known in the art can be used to introduce the transgene. In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by transgene-free methods (e.g., episomal delivery). In some embodiments, expression or activity of Dnmt3b and / or Gimap6, or other genes disclosed herein, is increased using gene editing techniques, for example, to introduce one or more modifications to increase promoter strength, ribosome binding, or RNA stability.

[0068] In some embodiments, the method includes applying cyclic 2D, 3D, or 4D stretch to cells. In various embodiments, the cells subjected to cyclic 2D, 3D, or 4D stretch are selected from one or more of CD34+ enriched cells, iPSCs, ECs, and HECs. For example, the cell population is introduced into a bioreactor that provides cyclic strain biomechanical stretch, as described in WO 2017 / 096215, which is incorporated herein by reference in its entirety. The cyclic strain biomechanical stretch can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, the mechanical means applies a stretching force to the cells or to a cell culture surface having cells (e.g., ECs or HECs) cultured thereon. For example, cyclic 2D, 3D, or 4D stretching can be applied to cells ex vivo under defined and controlled cyclic strain conditions using a computer-controlled vacuum pump system or other means for providing a stretching force (e.g., the FlexCell™ Tension System, CytoStretcher System) attached to a flexible, biocompatible and / or biomimetic surface. For example, the applied cyclic stretching can be about 1% to about 20% cyclic strain (e.g., about 6% cyclic strain) for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours.

[0069] Alternatively, or in addition, EHT is stimulated by Trpv4 activation, which can be by contacting cells (e.g., CD34+ enriched cells, ECs, or HECs) with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0070] When a cell population is described herein as having a particular phenotype, it is understood that the phenotype represents a substantial portion of the cell population, such as at least 25%, at least 40%, or at least about 50%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90% of the cell population. Furthermore, in various steps, the cell population can be enriched for cells of the desired phenotype and / or depleted of cells of an undesired phenotype, such that the cell population contains at least about 75%, or at least about 80%, or at least about 90% of the desired phenotype. Such positive and negative selection methods are known in the art. For example, cells can be sorted based on cell surface antigens (including those described herein) using a fluorescence-activated cell sorter or magnetic beads that bind to certain cell surface antigens. Negative selection columns can be used to remove cells expressing undesirable cell surface markers. In some embodiments, cells are enriched for CD34+ cells (before and / or after undergoing EHT). In some embodiments, the cell population is cultured under conditions that promote the expansion of CD34+ cells, thereby producing an expanded population of stem cells.

[0071] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition between days 10 and 20 of iPSC differentiation, such as between days 12 and 17. In some embodiments, the CD34+ cells include non-adherent cells.

[0072] In various embodiments, the HSCs or CD34+ enriched cells are further expanded. For example, the HSCs or CD34+ enriched cells can be expanded according to the methods disclosed in US8,168,428, US9,028,811, US10,272,110, and US10,278,990, which are incorporated by reference in their entireties. In some embodiments, the ex vivo expansion of the HSCs or CD34+ enriched cells employs prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSCs comprise at least about 0.01% LT-HSCs, or at least about 0.05% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 0.5% LT-HSCs, or at least about 1% LT-HSCs.

[0073] HSCs and / or HSPCs that give rise to megakaryocytes and / or platelets can be identified based on the expression of CD34 and the absence of lineage-specific markers (referred to as Lin-). In some embodiments, a population of stem cells containing HSCs and / or HSPCs is enriched, for example, as described in US 9,834,754, which is incorporated herein by reference in its entirety. For example, this process can include sorting the cell population based on the expression of one or more of CD34, CD90, CD38, and CD43. CD34 + , CD90 - , CD38 - , and CD43 - In some embodiments, a stem cell population for differentiation into hematopoietic lineages is selected for further differentiation from a fraction that is at least about 80% CD34 + , or at least about 90% CD34 + , or at least about 95% CD34 + is.

[0074] In some embodiments, stem cell populations, or CD34+ enriched cell or fraction thereof, or derivative populations, are expanded as described in US 2020 / 0308540, which is incorporated herein by reference in its entirety. For example, the cells are expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55, and Boitano A., et al., Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells. Science 2010 Sep 10;329(5997):1345-1348.

[0075] In some embodiments, CD34 + Compounds that promote cell proliferation include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, incorporated herein by reference).

[0076] In some embodiments, the stem cell population or CD34+ enriched cells are further enriched for or modified to express periostin and / or platelet-derived growth factor receptor alpha (pdgfra), as described in WO2020 / 205969 (incorporated herein by reference in its entirety). Such expression can be by transgene-free methods, including but not limited to, by delivering an encoding transcript to the cells, by introducing an encoding transgene, or by introducing a non-integrating episome into the cells. In some embodiments, gene editing is employed to introduce genetic modifications into expression elements in the cells, such as to increase promoter activity or strength, ribosome binding, RNA stability, or affect RNA splicing.

[0077] In yet another embodiment, the stem cell population or CD34+ enriched cells are cultured with an inhibitor of histone methyltransferase EZH1. Alternatively, EZH1 is partially or completely deleted or inactivated, or transiently silenced, in the stem cell population. Inhibition of EZH1 can direct myeloid progenitor cells (e.g., CD34+CD45+) to the lymphoid lineage. See WO2018 / 048828, which is incorporated herein by reference in its entirety. In yet another embodiment, EZH1 is overexpressed in the stem cell population.

[0078] In various embodiments, the HSC and / or HSPC population or a fraction thereof can be differentiated ex vivo into megakaryocytes, which can then generate platelets.

[0079] In some embodiments, populations containing HSCs and / or HSPCs or their progeny can be cultured ex vivo with a partial or complete Notch ligand, SHH, extracellular matrix component(s), and / or a combination thereof to differentiate the cells. Furthermore, according to known processes, a feeder layer of heterologous OP9-DL1 or cancer cell line cells derived from STO mouse fibroblasts, blood-derived peripheral blood mononuclear cells (PBMCs), or cord blood-derived mesenchymal stem cells or lymphocytes is often employed for hematopoietic cell differentiation. The OP9-DL1 coculture system uses a bone marrow stromal cell line (OP9) transduced with the Notch ligand Delta-like-1 (DLL1) to support T cell development from a stem cell source. The OP9-DL1 system limits the potential of the cells for clinical use. There is a need for a feeder-free system capable of generating hematopoietic cells from hiPSCs for clinical use, and in some embodiments, the present invention fulfills this goal. In a non-limiting example, to generate megakaryocytes, iPSC expansion is performed for 6 days, followed by embryoid body formation, which takes about 8 days. The cells are further cultured for about 5 days to allow the development of CD34+ hemogenic endothelial cells from which the HSCs are derived. The HSCs are then cultured in specific media for differentiation into megakaryocytes and / or further differentiation into platelets.

[0080] The term "Notch ligand" as used herein refers to a ligand capable of binding to a Notch receptor polypeptide present on 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-Delta, S-Serrate, and L-Lag2) containing 20-22 amino acids at the amino terminus and 3-8 EGF repeats on the extracellular surface. In various embodiments, Notch is a ligand of Delta-like-1 (DLL1), Delta-like-4 (DLL4), SFIP3, Delta-like-5 (DLL5), SFIP4, SFIP5, SFIP6, SFIP7, SFIP8, SFIP9, SFIP10, SFIP11, SFIP12, SFIP13, SFIP14, SFIP15, SFIP16, SFIP17, SFIP18, SFIP19, SFIP20, SFIP21, SFIP22, SFIP23, SFIP24, SFIP25, SFIP26, SFIP27, SFIP28, SFIP29, SFIP30, SFIP31, SFIP32, SFIP33, SFIP34, SFIP35, SFIP36, SFIP37, SFIP38, SFIP39 ... Max (disclosed in PCT / US2020 / 041765 and PCT / US2020 / 030977, which are incorporated by reference in their entireties), Jagged1 (JAG1), Jagged2 (JAG2), Delta-like ligand 3 (DLL3), and X-delta2, or at least one of their functional portions.

[0081] As used herein, "Notch ligand" also includes intact (full-length), partial (truncated), or modified (containing one or more mutations, such as conservative mutations) Notch ligands, as well as Notch ligands or fragments thereof from any species 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 typically characterized by an N-terminal (NT) domain followed by a Delta / Serrate / LAG-2 (DSL) domain and an extracellular domain containing multiple tandemly arranged epidermal growth factor (EGF)-like repeats. The DSL domain, along with the adjacent NT domain and the first two EGF repeats containing Delta and OSM-11-like (DOS) motifs, are typically required for canonical ligands to bind to Notch. The intracellular domains of several canonical ligands contain a carboxy-terminal PSD-95 / Dlg / ZO-1-ligand (PDZL) motif, which plays a role independent of Notch signaling.

[0082] 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 a human or humanized antibody), a single-chain antibody (scFv), a nanobody, or other antibody fragment or antigen-binding molecule that can activate the Notch signaling pathway.

[0083] In some embodiments, the Notch ligand is a Delta family Notch ligand. In some embodiments, the Delta family ligand is Delta-1 (Genbank Accession No. AF003522, Homo sapiens), Delta-like 1 (DLL1, Genbank Accession Nos. NM_005618 and NP_005609, Homo sapiens; Genbank Accession Nos. X80903, 148324, M. musculus), Delta-4 (Genbank Accession No. AF273454, BAB18580, Mus musculus; Genbank Accession Nos. AF279305, AAF81912, Homo sapiens), and / or Delta-like 4 (DLL4, Genbank Accession Nos. Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank Accession No. NM 019454, Mus musculus). Notch ligands are commercially available or can be produced, for example, by recombinant DNA techniques.

[0084] 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. A functional derivative of a Notch ligand (including a fragment or portion thereof) will be capable of binding to and activating a Notch receptor. Binding to a Notch receptor can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cell signaling assays.

[0085] In some embodiments, the Notch ligand is DLL4 with one or more affinity-enhancing mutations, such as one or more (or all) of the following: for hDLL4, G28S, F107L, I143F, H194Y, L206P, N257P, T271L, F280Y, S301R, and Q305P. See Gonzalez-Perez, et al., Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling, Nature Chemical Biology (2022).

[0086] In various embodiments, the Notch ligand is soluble and optionally immobilized on microparticles or nanoparticles that are optionally paramagnetic to enable magnetic enrichment or concentration processes. In yet other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally using other adhesion molecules such as VCAM-1. See US 2020 / 0399599, incorporated herein by reference in its entirety. In other embodiments, the beads or particles are constructed of biological materials, such as polymers (e.g., polystyrene or PLGA), gold, iron dextran, or particles formed from lipids and / or proteins. In various embodiments, the particles have a diameter or largest dimension of about 0.01 μm (10 nm) to about 500 μm (e.g., about 1 μm to about 7 μm). In still other embodiments, polymer scaffolds with conjugated ligands can be employed, as described in WO 2020 / 131582, incorporated herein by reference in its entirety. For example, the scaffold can be constructed from polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazine, 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(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold comprises pores having diameters of about 1 pm to 100 pm.

[0087] In some embodiments, the C-terminus of the Notch ligand is conjugated to a support of choice. In some embodiments, this can involve adding a sequence to the C-terminus of the Notch ligand that can be enzymatically conjugated to the support, for example, via a biotin molecule. In another embodiment, the Notch ligand-Fc fusion is prepared such that the Fc segment can be immobilized by binding to Protein A or Protein G that has been conjugated to a support. Of course, any of the known protein conjugation methods can be employed.

[0088] Thus, in various embodiments, the Notch ligand can be immobilized, functionalized, and / or embedded in a 2D or 3D culture system. The Notch ligand can be incorporated with components of an extracellular matrix, such as one or more selected from fibronectin, retronectin, and laminin. In some embodiments, the Notch ligand 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, the Notch ligand, components of the extracellular matrix, or combinations thereof, are in contact with culture conditions that provide cells with a topographical pattern and / or texture (e.g., roughness) that promotes differentiation and / or proliferation.

[0089] In some embodiments, differentiating the population comprising HSCs and / or HSPCs into megakaryocytes comprises culturing with thrombopoietin (TPO). The culture may further comprise one or more additional cytokines 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, including TPO, can be selected to further expand megakaryocytes. In some embodiments, such additional cytokines or growth factors for expanding megakaryocytes may be selected from stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L), IL-6, IL-9, and erythropoietin (EPO).

[0090] In some embodiments, immature megakaryocytes are generated and can be identified as CD34-CD41+CD61+CD42b-. Megakaryocyte maturation can be promoted by culturing with one or more cytokines and growth factors selected from stem cell factor (SCF), IL-6, and IL-9. In some embodiments, erythropoietin (EPO) and / or IL-8 are excluded. Megakaryocyte maturation is accompanied by an increase in cytoplasmic volume, an increase in the number of alpha and dense granules, the formation of a dense tubular network, and / or the formation of an open tubule system for granule release. In some embodiments, mature megakaryocytes contain granules. In some embodiments, mature megakaryocytes do not contain granules.

[0091] 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 in accordance with 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.

[0092] Mature megakaryocytes can be cultured in the presence of fibroblast growth factor 4 (FGF4) and stromal cell-derived factor 1 (SDF1) to form proplatelets and platelets, as known in the art. In some embodiments, megakaryocytes or proplatelets are cultured in a bioreactor that subjects the cells to hydrodynamic shear stress. For example, platelets can be produced in static 2D, serum-free, cytokine-dependent conditions. Alternatively, platelets can be produced in a three-dimensional (3D) microenvironment. In various embodiments, the platelets will have a phenotype that is CD41+CD42b+. Recovered platelets can be activated by thrombin. Platelets can be collected and subjected to gamma irradiation prior to transfusion therapy.

[0093] Compositions of the present disclosure (e.g., including 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 acids or bases without a significant change in pH. Examples of buffers contemplated by the present invention include, but are not limited to, physiological saline (0.9% NaCl), 5% dextrose in water (D5W), Dulbecco's phosphate-buffered saline (PBS), and Ringer's solution. The compositions may include a vehicle suitable for intravenous infusion or other administration routes, and the compositions may include a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). Other carriers may include dimethoxyethane (DME), N,N-dimethylformamide (DMF), or dimethylacetamide, including mixtures or combinations thereof. The cell or platelet composition may be provided in an appropriate volume within an implantable device (eg, a scaffold), or within a bag, or within a vial, tube, or container, and stored frozen until use.

[0094] In various embodiments, the composition contains at least about 10 per 50 or 100 mL volume. 9 platelets, or at least about 10 10 platelets, or at least about 10 11platelets, or at least about 10 12 Contains platelets.

[0095] Pharmaceutical compositions for use in the disclosed methods may also contain additional therapeutic agents for the treatment of specific target disorders. For example, pharmaceutical compositions may also include cytokines and growth factors (e.g., interleukins, interferons, FGF, VEGF, PDGF, PIGF, STAT, etc.). Such additional factors and / or agents may be included in the pharmaceutical compositions to provide the benefits of the therapeutic approaches disclosed herein, i.e., improved therapeutic efficacy with reduced systemic toxicity.

[0096] In other aspects, platelets are used to enrich platelet-rich plasma (PRP) from a subject in need of PRP therapy. Here, a combination of intact platelets or lysates extracted therefrom can be used to enrich other platelet-enriched plasma sources, such as donor-derived PRP. Optionally, it can contain growth factors, cytokines, or other agents from other sources that complement the PRP-based therapeutic use in the patient.

[0097] In another aspect, the present invention provides methods for platelet therapy, 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 a subject with thrombocytopenia, such as ACTN1-associated thrombocytopenia, amegakaryocytic thrombocytopenia with radioulnar synostosis, ANKRD26-associated thrombocytopenia, autosomal dominant thrombocytopenia, congenital amegakaryocytic thrombocytopenia, CYCS-associated thrombocytopenia, FYB-associated thrombocytopenia, idiopathic thrombocytopenic purpura, or X-linked thrombocytopenia. In some embodiments, the platelets are administered to a subject experiencing bleeding.

[0098] As used herein, the term "about" means ±10% of the associated numerical value.

[0099] Certain aspects and embodiments of the present disclosure are further illustrated with reference to the following examples. [Example]

[0100] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances CD34+ cell formation during iPSC differentiation, but does not affect pluripotency. method iPSCs were developed from hCD34+ cells by episomal reprogramming, as known in the art and essentially as described in Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells, Science 318, 1917-1920, (2007), and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009). Embryoid bodies and hemogenic endothelial differentiation were performed essentially as described in R. Sugimura, et al., Hematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438, (2017); C. M. Sturgeon, et al., Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554-561, (2014); J. Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920, (2007); and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009).

[0101] Briefly, hiPSCs were dissociated and resuspended in medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holo-transferrin, monothioglycerol, BMP4, and Y-27632. The cells were then seeded onto 10 cm dishes (EZSPHERE or low-attachment plates) for EB formation. On day 1, bFGF and BMP4 were added to the medium. On day 2, the medium was replaced with medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. Cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, EBs were dissociated on day 8, cells were filtered through a 70 μm strainer, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0102] result Induced pluripotent stem cells (iPSCs) were transduced using an adenoviral vector containing both ETV2 and GFP sequences under the control of the EF1A promoter. After transduction, approximately 45% of the iPSC cultures were observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). It was further observed that ETV2-OE in iPSC cells retained the pluripotent properties of iPSCs, as indicated by the stemness marker expression TRA-1-60 (Figure 1). Figure 1 shows a FACS plot demonstrating the transduction efficiency of iPSCs using an adenoviral vector for overexpressing ETV2 and GFP sequences.

[0103] Next, ETV2-OE-iPSCs (along with control iPSCs transduced with a vector carrying the GFP sequence but without ETV2) were differentiated into embryoid bodies and then into hemogenic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 significantly increased the expression of CD235a. - CD34 in the population + and CD31 +These results suggest that ETV2-OE enhances the formation of hemogenic endothelial cells, as evidenced by the expression of markers (Figure 2). Specifically, Figure 2 shows representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (defined here as CD235a-CD34+CD31+), demonstrating that ETV2-OE enhances the formation of hemogenic endothelial cells compared to controls.

[0104] Furthermore, the results showed that ETV2-OE expresses CD34 + This suggests that ETV2-OE enhances CD34+ cell formation (Figure 3). Figure 3 shows representative flow cytometry analysis and relative quantification of CD34+ cells demonstrating that ETV2-OE enhances CD34+ cell formation.

[0105] Overall, these data indicate that ETV2 overexpression in iPSCs does not affect their pluripotent properties, but promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation.

[0106] Example 2 - iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs. method To analyze EHT, EB-derived CD34+ cells were suspended in medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. After the cells had adhered to the bottom of the well (by visual inspection) for approximately 4-18 hours, Yoda1 was added to the cultures for some experiments. After 4-7 days, cells were collected for analysis.

[0107] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells from the iPSC-derived embryoid bodies were harvested and cultured for an additional 5–7 days to induce endothelial-hematopoietic (EHT) transition (with or without Yoda1). CD34+ cells were then harvested from EHT cultures on days 5–7 for further hematopoietic lineage differentiation.

[0108] result Figure 4 shows FACS analysis of megakaryocytic differentiation, demonstrating commitment of cells to the megakaryocytic lineage. HSC populations generated by inducing EHT in CD34+ cells differentiated from iPSCs (D8+7 iPSC-CD34+, with or without Yoda1 "Y") outperform differentiated iPSCs (D8 iPSC-CD34+) and CD34+ cells isolated from bone marrow (BM) CD34+ cells in terms of supporting megakaryocyte differentiation. Figure 5 shows that megakaryocytic cells derived from D8+7 iPSC-CD34+ (with or without Y) are capable of ex vivo expansion and outperform megakaryocytes differentiated from D8 iPSC-CD34+ cells, resembling megakaryocytes differentiated from BM CD34+ cells.

[0109] Figure 6 shows immunofluorescence analysis of megakaryocytes (with or without Y) derived from D8+7 iPSC-CD34+ cells and platelet differentiation of CD34+ cells from bone marrow, showing that D8+7 iPSC-CD34+-derived megakaryocytes are phenotypically similar to BM CD34+-derived megakaryocytes and are capable of releasing platelets.

[0110] FIG. 7 shows thrombus formation from D8+7 iPSC-CD34(+Y)-derived platelets, indicating that these platelets can coagulate and promote thrombus formation upon activation.

[0111] Example 3 - Evaluation of off-target editing in HLA knockout HSCs HLA typing of triple knockout (HLA edited) HSC clones was performed to check for unwanted editing and ensure that major editing events, such as deletion(s), did not occur within other regions of chromosome 6. Sequencing methods and analyses were performed to assess the extent of gRNA off-target activity and select gRNAs that represent a low risk of affecting non-target HLA genes.

[0112] Sequencing was performed using in situ cleavage labeling in fixed and permeabilized cells by ligating full-length P5 sequencing adapters to end-prepared DSBs. Genomic DNA was extracted, fragmented, end-prepared, and ligated using chemically modified semi-functional P7 adapters. The resulting DNA library contained a mixture of functional DSB-labeled fragments (P5:P7) and non-functional genomic DNA fragments (P7:P7). Subsequent DNA sequencing of the DNA library enriched for the DNA-labeled fragments, eliminating all irrelevant non-functional DNA. Because the library preparation was PCR-free, each resulting sequencing read was equivalent to a single labeled DSB end from the cell. This generated a DNA cleavage readout, enabling direct detection and quantification of genomic DSBs by sequencing without the need for error correction, and allowed for the unambiguous mapping of off-target mutations.

[0113] Table 1 below summarizes the results of the editing strategy in two representative clones relative to wild-type cells. [Table 1]

[0114] Table 2 provides non-limiting examples of gRNAs used in experiments that can be used to knock out expression of the indicated HLA genes. [Table 2]

[0115] The results show that the editing strategy was successful in selectively targeting the HLA-A, DPB1, and DQB1 genes without affecting other HLA genes or introducing major deletions elsewhere.

[0116] These results were confirmed by phenotypic analysis of the HLA-edited clones by FACS and immunofluorescence. As shown in Figures 8A and 8B, the HLA-edited cells tested positive for overall expression of HLA class I molecules, comparable to that of wild-type cells. Specific expression of HLA-A via immunofluorescence confirmed that HLA-A was not expressed in the HLA-edited cells, supporting the finding that the gene editing strategy successfully deleted only the HLA-A gene. Specifically, Figure 8A shows that all HLA-edited cells were positive for HLA-like class I to the same extent as wild-type (WT) (i.e., non-HLA-edited) cells. This result indicates that despite the deletion of HLA-A, other class I molecules, such as HLA-B and HLA-C, were expressed and were not affected by the gene editing strategy.

[0117] To confirm that the HLA-A gene had been deleted, the specific expression of HLA-A was analyzed by immunofluorescence. As can be seen in Figure 8B, HLA-A was not expressed in the HLA-edited clones, indicating that the gene editing strategy was effective in specifically deleting only the HLA-A gene. Such preservation of overall class I expression with deletion of HLA-A would facilitate patient matching while avoiding NK cell-mediated rejection.

[0118] Example 4 - Evaluation of pluripotency and immune compatibility of HLA-edited HSCs The ability of HLA-edited cells to preserve pluripotency was assessed. As shown in Figure 9, immunofluorescence evaluation of HLA-edited iPSC clones showed that they maintained tri-lineage differentiation, with ectoderm differentiation indicated by NESTIN-488 and PAX6-594 staining, mesoderm differentiation indicated by GATA-488 staining, and endoderm differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0119] HLA class I molecules are expressed on the surface of all nucleated cells, and if HLA class I molecules are mismatched between donor and recipient, the cells can be recognized and killed by CD8+ T cells. In addition, HLA mismatches can lead to cytokine release syndrome (CRS) and graft-versus-host disease (GVHD). Conversely, complete deletion of HLA-I molecules via B2M KO would render the cells targets for NK cell-mediated cytotoxicity. Preservation of overall class I expression with deletion of HLA-A could facilitate patient matching while avoiding NK cell-mediated rejection. Therefore, the immune compatibility of HLA-edited HSCs was tested by coculture with peripheral blood mononuclear cells (PBMCs) to assess whether immune cells would reject grafts of HLA-edited and wild-type HSCs (gHSCs).

[0120] Wild-type (gHSC) and HLA-edited HSCs were cocultured with PBMCs bearing HLA-B and HLA-C markers matched but mismatched HLA-A. B2M KO HSCs, which lack expression of HLA class I molecules, and CIITA KO HSCs, which lack expression of class II molecules, were used as controls to compare the degree of PBMC-mediated cytotoxicity for HLA-null and mismatched HLAs, respectively. Figure 10 shows the results of a PBMC-mediated cytotoxicity assay in the coculture, measured by Annexin V staining. The results show that deletion of HLA-A in HLA-edited HSCs protected the cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO HSCs were susceptible to PBMC-mediated cytotoxicity. HSCs cocultured with sorted CD8+ T cells from the same PBMC donor protected HLA-edited and B2M KO HSCs from CD8+ T cytotoxicity. Conversely, HSCs co-cultured with sorted NK cells protected only WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0121] In summary, the immunocompatibility results indicate that CD8+ T cells present in the PBMC samples were involved in killing cells bearing mismatched HLA molecules (WT) and CIITA KO, while NK cells present in the PBMCs were involved in killing HLA-null cells (B2M KO). However, HLA-edited HSCs were protected from CD8+ T cell-mediated cytotoxicity (because mismatched HLA-A had been knocked out) and from NK cell-mediated cytotoxicity (because HLA class I molecule expression was largely preserved).

[0122] Example 5 - Evaluation of in vivo engraftment potential of HLA-edited HSCs To evaluate the engraftment potential of HLA-edited HSCs, the cells' ability to engraft in vivo was assessed by competitive transplantation against wild-type HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed and transplanted into mice. Bone marrow (BM) and peripheral blood samples were collected and assessed by FACS to compare the relative amounts of each cell type present in the samples. As shown in Figure 11, both HLA-edited HSCs and wild-type HSCs contributed to approximately equal engraftment in BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to wild-type HSCs in their engraftment and reconstitution potential. Therefore, the properties of wild-type (unedited, parental) HSCs are expected to match those of the HLA-edited HSCs of the present disclosure for generating T cell lineages.

[0123] Example 6: Differentiation of HLA-edited HSCs into promegakaryocytes / proplatelets It was determined that HLA-edited HSCs can differentiate into megakaryocytes (MKs) and further differentiate into platelets. Differentiation was compared to bone marrow (BM)-derived CD34+ cells and iPSC-CD34+ cells. As shown in Figure 12, HLA-edited HSCs showed a statistically significant increase in platelet content compared to BM CD34+ and iPSC-CD34+ cell populations. Thus, HLA-edited HSCs can differentiate into megakaryocytes (MKs), which can further support differentiation into platelets.

[0124] References 1. Nianias, A. & Themeli, M. Induced Pluripotent Stem Cell (iPSC)-Derived Lymphocytes for Adoptive Cell Immunotherapy: Recent Advances and Challenges. Curr Hematol Malig Rep 14, 261-268 (2019). 2. Brauer, P. M., Singh, J., Xhiku, S. & Zuniga-Pfluecker, J. C. T Cell Genesis: In Vitro Veritas Est? Trends Immunol 37, 889-901 (2016). 3. Kennedy, M. et al. T Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem Cell Differentiation Cultures. Cell Reports 2, 1722-1735 (2012). 4. Sturgeon, C. M., Ditadi, A., Awong, G., Kennedy, M. & Keller, G. Wnt Signaling Controls the Specification of Definitive and Primitive Hematopoiesis From Human Pluripotent Stem Cells. Nat Biotechnol 32, 554-561 (2014). 5. Chang, C.-W., Lai, Y.-S., Lamb, L. S. & Townes, T. M. Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells. PLoS One 9, (2014). 6.Nishimura,T.et al.Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation.Cell Stem Cell 12,114-126(2013). 7.Themeli,M.et al.Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.Nat Biotechnol 31,928-933(2013). 8.Vizcardo,R.et al.Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+T Cells.Cell Stem Cell 12,31-36(2013). 9.Montel-Hagen,A.et al.Organoid-induced differentiation of conventional T cells from human pluripotent stem cells.Cell Stem Cell 24,376-389.e8(2019). 10.Guo,R.et al.Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors.Cell Research 30,21-33(2020). 11.Nagano,S.et al.High Frequency Production of T Cell-Derived iPSC Clones Capable of Generating Potent Cytotoxic T Cells.Molecular Therapy-Methods & Clinical Development 16,126-135(2020). 12.Iriguchi,S.et al.A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.Nature Communications 12,430(2021).

Claims

1. 1. A method for preparing a cell population comprising megakaryocytes, comprising: enriching a population of differentiated pluripotent stem cells (PSCs) for CD34+ cells to prepare a CD34+ enriched population; inducing endothelial-to-hematopoietic transition (EHT) of said CD34+ enriched population to produce a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs), and optionally harvesting the cells that undergo endothelial-to-hematopoietic transition; differentiating said HSC population into megakaryocytes and, optionally, to form platelets.

2. 10. The method of claim 1, wherein EHT is induced for at least 2 days and not 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. 4. The method of any one of claims 1 to 3, wherein the PSC population is a human iPSC population derived from lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or primary human tissue.

5. 5. 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 HLA class II genes.

7. 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. 9. The method of any one of claims 1 to 8, wherein the iPSCs have been gene-edited to delete one or more HLA class I genes, one or more class II genes, and / or one or more genes that govern HLA or MHC expression or presentation capacity.

10. 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 iPSCs comprise a deletion of HLA-DPB1 and / or HLA-DQB1.

12. The iPSCs are HLA-A ｎｅｇ , homozygous for both HLA-B and HLA-C, HLA-DPB1 ｎｅｇ , and HLA-DQB1 ｎｅｇ and optionally is homozygous for HLA-DRB1.

13. The method according to claim 9, wherein the one or more genes controlling the ability to express or present HLA or MHC are β2-microglobulin and / or CIITA.

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

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

16. 16. The method of claim 14 or 15, wherein the CD34+ cells are harvested from a culture undergoing endothelial-hematopoietic transformation, including harvesting of CD34+ floating 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 the induction of endothelial-hematopoietic 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-to-hematopoietic transition comprises applying cyclic stretch to the CD34-enriched cells.

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

21. The method of claim 18, wherein the induction of endothelial-hematopoietic transition comprises Piezo1 activation.

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

23. 23. The method of claim 22, wherein the effective amount of the Piezo1 agonist is in the range of 0.1 to 500 μM, or in the range of 0.1 to 100 μM.

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

25. 25. The method of claim 24, wherein said Trpv4 activation is by contacting said CD34+ enriched cells with one or more Trpv4 agonists optionally selected from GSK1016790A, 4α-PDD, or analogs or derivatives thereof.

26. 26. The method of 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. 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 IL-1, IL-3, IL-6, IL-9, IL-11, SCF, SDF-1, and PDGF-BB.

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

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

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

32. 32. The method of claim 31, wherein the megakaryocytes or proplatelets are cultured in a bioreactor that subjects the cells to hydrodynamic shear stress.

33. 33. The method of claim 31 or 32, wherein the platelets are activatable by thrombin.

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

35. At least about 10 per 50 mL volume ９ platelets, or at least about 10 １０ platelets, or at least about 10 １１ platelets, or at least about 10 １２ 25. The composition of claim 24, comprising platelets.

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

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

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

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

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