WO2021055985A1 - Ipsc-derived, hypoimmunogenic, myeloid progenitor cells - Google Patents

Abstract

Provided herein are iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells comprising genetic modifications to evade destruction of the cells by a competent immune system. Such cells are useful as a universal composition in the treatment of conditions in which cells developing from the myeloid line are therapeutic, such as, neutropenia, thrombopenia and monocytopenia. Where such cells are hypoimmunogenic they are not rejected by a subject having a functioning or partly functioning immune system.

Description

IPSC-DERIVED, HYPOIMMUNOGENIC, MYELOID PROGENITOR CELLS

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH [0001] None. REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the filing date of U.S. provisional application 62/903,911 , filed September 22, 2019, the contents of which are incorporated herein by reference in its entirety.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT [0003] None.

SEQUENCE LISTING

[0004] None.

BACKGROUND

[0005] Mixed, allogeneic myeloid progenitor cells are useful as a universal cell therapy to treat subjects, for example, suffering from neutropenia as a result of myeloablative chemotherapy for cancer or acute radiation syndrome. The product can be used in combination with G-CSF (e.g., filgrastim). Mixed, allogeneic myeloid progenitor cells preferentially proliferate in presence of G-CSF and provide a source of mature neutrophils to mitigate the risk of infections due to chemotherapy-induced neutropenia. For example, the product is indicated for prevention of serious bacterial and fungal infections in patients with de novo acute myeloid leukemia (AML) undergoing induction chemotherapy.

[0006] Mixed, allogeneic myeloid progenitor cells can be made by ex vivo culture expansion of CD34+ cells isolated from cord blood, bone marrow, or from leukapheresis obtained from granulocyte colony-stimulating factor (G-CSF) mobilized blood from screened and tested donors. It can be stored by cryopreservation and formulated for intravenous administration. [0007] The use of allogenic myeloid progenitor cells for treatment of neutropenia is described, for example, in U.S. Patent 9,545,427 (Brown), U.S. Patent 8,252,587 (Fong et al.), and U.S. Patent 8,383,095 (Christiansen et al.).

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate exemplary embodiments and, together with the description, further serve to enable a person skilled in the pertinent art to make and use these embodiments and others that will be apparent to those skilled in the art. The invention will be more particularly described in conjunction with the following drawings wherein:

[0009] FIGURE 1 shows differentiation of cells along the hematopoietic line, from stem cells through mature cells.

[0010] FIGURE 2 shows an exemplary pathway for the development of hypoimmunogenic myeloid progenitor cells, and differentiation into mature cells.

SUMMARY

[0011] In one aspect, provided herein is a cell population comprising hematopoietic stem cells (“HSCs”) derived from induced pluripotent stem cells (“iPSCs”) or from other human pluripotent stem cells. In one embodiment the HSCs are hypoimmunogenic HSCs comprising genetic modifications to evade destruction of the cells by a competent immune system. In another embodiment expression of one or more of CD47, PD-L1 , Tim3, CTLA4-lg and HLA-G are up-regulated. In another embodiment expression of one or both of Human Leukocyte Antigen Class I (“HLA I”) and Human Leukocyte Antigen Class II (“HLA I”) HLA genes is/are knocked out. In another embodiment the cell population comprises at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% hypoimmunogenic HSCs.

[0012] In another aspect, provided herein is a cell population comprising myeloid progenitor cells (“MPCs”) derived from induced pluripotent stem cells (“iPSCs”) or from other human pluripotent stem cells. In one embodiment the MPCs are hypoimmunogenic MPCs comprising genetic modifications to evade destruction of the cells by a competent immune system. In one embodiment expression CD47, PD-L1 , Tim3, CTLA4-lg and/or and HLA-G are up-regulated. In another embodiment wherein expression of one or both of Human Leukocyte Antigen Class I (“HLA I”) and Human Leukocyte Antigen Class II (“HLA I”) HLA genes is/are knocked out. In another embodiment the population comprises at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% hypoimmunogenic MPCs. In another embodiment the MPCs comprise one or more of: Common Myeloid Progenitor Cells (“CMPs”) (CD34+, CD90-, CD123+, CD45RA-), Megakaryocyte/Erythroid Progenitor Cells (“MEPs”) (CD34+, CD90-, CD123-, CD45RA-) and Granulocyte/Monocyte Progenitor Cells (“GMPs”) (CD34+, CD90-, CD123+, CD45RA+). In another embodiment the cell population is substantially free of cells of lymphoid lineage, e.g., Common Lymphoid Progenitor Cells (“CLPs”) (CD34+, CD7+, CD10+), T cells (CD2+, CD3+), and B cells (CD19+, CD20+, CD33-), e.g., less than 5%. In another embodiment the cell population comprises Multi-Potent Progenitor Cells (“MPPs”) (CD34+, CD90+). In another embodiment the cell population has fewer than 40%

MPPs.

[0013] In another aspect, provided herein is a method of making hypoimmunogenic hematopoietic stem cells comprising: a) providing induced pluripotent stem cells (iPSCs); b) knocking out expression of one or both of Human Leukocyte Antigen Class I (“HLA I”) and/or Human Leukocyte Antigen Class II (“HLA I”) HLA genes; and/or, up- regulating expression of any or all of CD47, CD47, PD-L1 , Tim3, CTLA4-lg and HLA-G in said iPSCs to produce hypoimmunogenic iPSCs; and c) culturing said hypoimmunogenic iPSCs under conditions to differentiate into hypoimmunogenic hematopoietic stem cells (“HSCs”); thereby producing hypoimmunogenic HSCs. In one embodiment culturing comprising culturing cells for at least 4 days with one or more of bone morphogenic protein-4, FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. In another embodiment the cell population knocking out expression and up-regulating expression comprises using CRIPSR/Cas9 technology.

[0014] In another aspect, provided herein is a method of making iPSC-derived myeloid progenitor cells (“MPCs”) comprising: a) providing iPSC-derived hematopoietic stem cells (“HSCs”); and b) culturing said cells under conditions to differentiate into hypoimmunogenic myeloid progenitor cells. In one embodiment expression of Major Histocompatibility Complex I (“MHC I”) and/or Major Histocompatibility Complex II (“MHC II”) genes are knocked out; and/or any or all of CD47, CD47, PD-L1 , Tim3, CTLA4-lg and HLA-G are up-regulated. In another embodiment expression or one or more of CD47, PD-L1 , Tim3, CTLA4-lg and HLA-G are up-regulated. In another embodiment expression of one or both of Human Leukocyte Antigen Class I (“HLA I”) and Human Leukocyte Antigen Class II (“HLA I”) HLA genes is/are knocked out. In another embodiment culturing comprises culturing the cells with one or more of (e.g., all of) Stem Cell Factor, lnterleukin-3, FLT3 ligand, thrombopoietin (“TPO”) (e.g., mimetic TPO), and human serum albumin (“HSA”). In another embodiment culturing comprises culturing the cells for at least 4 days and no more than 28 days.

[0015] In another aspect, provided herein is a pharmaceutical composition comprising: a) a cell population comprising myeloid progenitor cells (“MPCs”) derived from induced pluripotent stem cells (“iPSCs”); and b) a pharmaceutically acceptable carrier. In one embodiment the MPCs are hypoimmunogenic MPCs comprising genetic modifications to evade destruction of the cells by a competent immune system. In another embodiment the pharmaceutically acceptable carrier comprises about 5% cryopreservative (e.g., DMSO), and about 5% human serum albumin (“HSA”). In another embodiment the pharmaceutically acceptable carrier comprises BioLife® CryoStor®. In another embodiment the pharmaceutical composition is cryopreserved.

In another embodiment, the pharmaceutical carrier is selected for intravenous injection, e.g., comprises normal saline and/or a buffer, dextrose or DMSO.

[0016] In another aspect, provided herein is a kit comprising: a) a first container and, contained within the first container, pharmaceutical composition comprising: i) a cell population comprising hypoimmunogenic myeloid progenitor cells derived from induced pluripotent stem cells (“iPSCs”); and ii) a pharmaceutically acceptable carrier. In one embodiment the MPCs are hypoimmunogenic MPCs comprising genetic modifications to evade destruction of the cells by a competent immune system. In another embodiment the first container contains 15x106 to 50x106 cells per mL (e.g., about 20x106 cells per mL to about 30x106 cells per mL, e.g., about 25 x 106/mL MPC cells. In another embodiment the first container contains about 12 mL of the pharmaceutically acceptable carrier. In another embodiment the first container comprises a bag or a bottle. In another embodiment the kit further comprises: b) a second container containing one or more cytokines selected from G-CSF, GM-CSF, TPO, M-CSF, IL-1 beta, and EPO. In another embodiment the kit further comprises a syringe.

[0017] In another aspect, provided herein is a method comprising administering to a subject in need thereof an effective amount of myeloid progenitor cells, wherein the cells are derived from induced pluripotent stem cells. In one embodiment the MPCs are hypoimmunogenic MPCs comprising genetic modifications to evade destruction of the cells by a competent immune system. In another embodiment the cells are derived from induced pluripotent stem cells. In another embodiment the subject suffers from neutropenia, anemia, monocytopenia, and thrombocytopenia or infection. In another embodiment the subject is undergoing stem cell transplantation. In another embodiment the subject has undergone myeloablative therapy. In another embodiment the subject is immunocompromised. In another embodiment the subject suffers from chemically induced neutropenia (e.g., from chemotherapy) or acute radiation poisoning. In another embodiment the subject is immunocompetent. In another embodiment the subject suffers from an infection. In another embodiment the subject is at increased risk for infection. In another embodiment the method comprises inducing in the subject production of cells selected from monocytes, megakaryocytes, platelets, erythrocytes, neutrophils, eosinophils, and basophils. In another embodiment administration comprises intravenous injection. In another embodiment the method further comprises co-administering to the subject a cytokine selected from one or more of G-CSF, GM- CSF, TPO, M-CSF, IL-1 beta, and EPO. In another embodiment the method comprises co-administering granulocyte colony growth factor (“G-CSF”) in an amount sufficient to induce differentiation of the MPCs into neutrophils. In another embodiment the method comprises co-administering thrombopoietin in an amount sufficient to induce differentiation of the MPCs into platelets. In another embodiment the method comprises co-administering macrophage colony stimulating factor (“M-CSF”) in an amount sufficient to induce differentiation of the MPCs into macrophages. In another embodiment the method comprises co-administering hemopoietic stem cells (“HSCs”).

In another embodiment the dose of HSCs is sub-optimal, that is, below an amount that would be effective to reconstitute an immune system if the HSCs were delivered without co-administration of the MPCs. In another embodiment the method comprises administering the cells up to 1.5 x 107 cells/kg body weight.

[0018] In another aspect, provided herein is a method comprising: a) providing a population comprising hypoimmunogenic hematopoietic stem cells or hypoimmunogenic myeloid progenitor cells; b) contacting the population with a test drug and, optionally, one or more cytokines or growth factors that promote differentiation of the cells along a path in the hematopoietic differentiation pathway; and c) determining whether the test drug promotes or inhibits differentiation along the pathway. [0019] In another aspect, provided herein is a composition as described herein for use in treating a disease, e.g., neutropenia, anemia, monocytopenia, and thrombocytopenia or infection.

[0020] In another aspect, provided herein is a use of a composition as described herein for treating a disease, e.g., neutropenia, anemia, monocytopenia, and thrombocytopenia or infection.

[0021] In another aspect, provided herein is a use of a composition as described herein in the preparation of a medicament for treating a disease, e.g., neutropenia, anemia, monocytopenia, and thrombocytopenia or infection.

DETAILED DESCRIPTION

I. Introduction

[0022] Disclosed herein are hematopoietic stem cells (“HSCs”) derived from induced pluripotent stem cells ("iPSCs") and myeloid progenitor cells (“MPCs”) derived from iPSCs and their progeny cells. As used herein a cell is “derived from an induced pluripotent stem cell” or is an ”iPSC-derived” cell, if it is differentiated beyond pluripotent stem cell stage and comprises genetic modifications consistent with those used to produce an induced pluripotent stem cell, e.g., presence of recombinant DNA comprising Oct4, Sox2, Klf4, and/or cMyc genes. Recombinant DNA is DNA in which two DNA sequences, not normally connected in nature, have been combined. For example, connection of a heterologous promoter to a gene to form an expression construct represents a recombinant DNA molecule.

[0023] Also provided herein are HSCs and MPCs derived from iPSCs, e.g., hypoimmunogenic HSCs and hypoimmunogenic MPCs. Hypoimmunogenic HSCs and MPCs comprise genetic modifications allowing the cells to evade destruction by a competent host immune system, e.g., after transplantation. Hypoimmunogenic HSCs and MPCs can be derived from hypoimmunogenic iPSCs. Also provided herein are methods of making and using such cells. Induced PSC-derived and hypoimmunogenic cells are animal cells, including mammalian cells and human cells.

[0024] Multiple studies have demonstrated the ability of induced pluripotent stem cells (“iPSCs”) to differentiate towards the hematopoietic lineage and produce functional neutrophils both in vivo and in vitro. While the process of differentiating towards neutrophils necessitates the creation of myeloid progenitor cells as an intermediate, no studies have shown the development of a highly pure myeloid progenitor population as an endpoint. Among the first steps of creating myeloid progenitor cells from iPSCs is to differentiate the iPSCs into CD34+ hematopoietic stem cells. This has been demonstrated by several studies, but the CD34+ purity and potency of the resultant cell population is low.

II. IPSC-derived, e.g., Hypoimmunogenic, Myeloid Progenitor Cells

[0025] Provided herein are compositions comprising myeloid progenitor cells derived from induced pluripotent stem cells. Further provided are hypoimmunogenic myeloid progenitor cells derived from iPSCs.

[0026] Myeloid progenitor cells, as well as terminally differentiated myeloid cells that develop from them, can be produced by a process that involves providing mature cells, reprogramming the mature cells to produce induced pluripotent stem cells, optionally rendering induced pluripotent stem cells hypoimmunogenic (e.g., by knocking out HLA Class I/ll genes and up-regulating CD47), differentiating the pluripotent stem cells into hematopoietic stem cells, differentiating hematopoietic stem cells into myeloid progenitor cells and differentiating myeloid progenitor cells into mature cells of the myeloid line, such as granulocytes (precursors to neutrophils, eosinophils, basophils), monocytes (precursors to macrophages), megakaryocytes (precursors to platelets) and erythroid progenitors (precursors to erythrocytes). At each stage, the cells also may be hypoimmunogenic.

A. Induced Pluripotent Stem Cells

[0027] Provided herein are compositions comprising induced pluripotent stem cells (“iPSC”). Induced pluripotent stem cells are reprogrammed mature cells that have the capacity to differentiate into any mature cell type. They are described, for example, in US patent 8,058,065 (Yamanaka et al.). They have the ability to differentiate along various differentiation pathways to mature cells. In particular, under proper culture conditions, they can differentiate into hematopoietic stem cells, into myeloid progenitor cells, and into finally differentiated mature cells in the myeloid line.

B. Hypoimmunogenic Induced Pluripotent Stem Cells

[0028] Provided herein are compositions comprising hypoimmunogenic iPSCs. Induced pluripotent stem cells have been made hypoimmunogenic by inactivating HLA class I and class II genes and/or overexpressing proteins such as CD47, PD-L1 , Tim3, CTLA4-lg and HLA-G.

C. IPSC-derived, e.g., Hypoimmunogenic, Hematopoietic Stem Cells

[0029] Provided herein are compositions comprising hematopoietic stem cells derived from induced pluripotent stem cells. Further provided are hypoimmunogenic hematopoietic stem cells derived from iPSCs. Hematopoietic stem cells are characterized by the surface expression of CD34 (“CD34+”).

[0030] Compositions comprising iPSC-derived, e.g., hypoimmunogenic, HSCs can comprise at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% HSCs. Compositions can be enriched for HSCs by, for example, specific capture of HSCs from a heterogenous population.

A. IPSC-derived, e.g., Hypoimmunogenic, Myeloid Progenitor Cells and Mature Myeloid Cells

[0031] Provided herein are compositions comprising myeloid progenitor cells derived from induced pluripotent stem cells. Further provided are hypoimmunogenic myeloid progenitor cells derived from iPSCs.

[0032] Myeloid progenitor cells (“MPCs”) are stem cells committed to developing into mature myeloid cells. Referring to Figure 1 , hematopoietic stem cells (“HSCs”) are the precursors of myeloid progenitor cells. Long-term self-renewing hematopoietic stem cells (“LT-HSC”) are CD34+ and CD38-. The cells differentiate into short-term hematopoietic stem cells (“ST-HSC”) (CD34+, CD38-). HSCs differentiate into multi- potent progenitor cells (“MPPs”) (CD34+, CD90+). MPPs can develop along the myeloid line or the lymphoid line.

[0033] Along the lymphoid line, MPPs can differentiate into common lymphoid progenitors (“CLP”), which are CD34+, CD7+ and CD10+. Common lymphoid progenitors, in turn, can differentiate into T cells (CD34-, CD2+, CD3+), B cells (CD34-, CD19+, CD20+), natural killer (NK) cells (CD34-, CD56+) and dendritic cells (CD34-, CD11c+).

[0034] Along the myeloid line, MPP’s can differentiate into common myeloid progenitors (“CMP”), which are CD34+, CD90-, CD123+, CD45RA-). Common myeloid progenitors can differentiate into granulocyte-macrophage progenitors (“GMP”) (CD34+,

CD90-, CD123+, CD45RA+ and megakaryocyte-erythrocyte progenitors (“MEP”) (CD34+, CD90-, CD123-, CD45RA-). Granulocyte-macrophage progenitors can differentiate into monocytes (CD34+, CD14+), which, in turn, differentiate into macrophages (CD34-, CD11b+, CD68+). They also can differentiate into granulocytes (CD34+, CD15+), which, in turn, differentiate into neutrophils (CD34-, CD15+, CD66b+, CD16+), basophils (CD34-, CD15+, CD123+), and eosinophils (CD34-, CD15+,

CD66b+, CD11b+). Megakaryocyte-erythrocyte progenitors can differentiate into megakaryocytes (CD34+, CD41+) which, in turn, differentiate into platelets (CD34-, CD41+, CD42+). They also can differentiate into erythroid precursor cells (CD34+, CD71+), which, in turn, can differentiate into erythrocytes (CD34-, CD45-, CD71-, CD235+).

[0035] Compositions comprising iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells include different mixtures of cells. In some embodiments, the predominant cell type in the population is myeloid progenitor cells (CD34+, CD90-). For example, the population can be at least any of 40%, 50%, 60%, 70%, 80%, 90% or 95% myeloid progenitor cells. This includes populations in which the myeloid progenitor cells comprise mixtures of CMPs, MEPs, and GMPs. It also includes populations in which CMPs predominate among the population of myeloid progenitor cells or MEPs and GMPs predominate among the population of myeloid progenitor cells. In other embodiments, certain cells may be not present or present in small quantities in the population. The population can be essentially free of certain cell types. As used herein, a population is “essentially free” of a cell type if the cell type comprises no more than 1% of the cells in the population. The population may comprise no more than 10%, no more than 5%, or no more than 1% of long-term hematopoietic stem cells. The population may comprise no more than 3% or no more than 1 % lymphoid progenitor cells. The population may comprise no more than 10%, no more than 5%, or no more than 1% mature myeloid cells (e.g., neutrophils, platelets, erythrocytes or macrophages.)

[0036] In another embodiment, the population comprises at least 50%, at least 60%, at least 70%, at least 80% or at least 90% myeloid progenitor cells and no more than 40%, preferably no more than 20%, multipotent progenitor cells. This population can include no more than 5% hematopoietic stem cells. This population can include no more than 5%, no more than 2% or no more than 1% lymphoid progenitor cells. [0037] In one embodiment, the population comprises -8.5% CD34+CD15+ (granulocytes), -8.5% CD34+CD41+ (megakaryocytes) -16% CD34+CD90+ (MPP) - most immature, -22% CD34+CD90-CD45RA-CD123+ (CMP), -12% CD34+CD90- CD45RA+CD123+ (GMP), -32% CD34+CD90-CD45RA-CD123- (MEP).

III. Methods of Making IPSC-derived, e.g., Hypoimmunogenic, Myeloid Progenitor Cells

A. Induced Pluripotent Stem Cells

[0038] Induced pluripotent stem cells can be obtained commercially or produced by genetic reprogramming or using recombinant proteins.

[0039] Commercially, induced pluripotent stem cells are available from, for example, Takara Bio USA (Mountain View, CA) (Cellartis Human iPSCs) and from NIH. Induced pluripotent stem cells also are available from the American Type Culture Collection (Manassas, VA). These include ATCC-HYR0103 Human Induced Pluripotent Stem (IPS) Cells ATCC® ACS-1007 developed from human fibroblasts, and ATCC-BXS0114 Human Induced Pluripotent Stem (IPS) Cells (ATCC® ACS-1028™), developed from human bone marrow CD34+ cells.

[0040] Induced pluripotent stem cells can be produced by methods well established in the art. Mature cells, such as fibroblasts, mesenchymal cells, and blood cells may be used as a starting point. These cells are genetically re-programmed to produce re establish a pluripotent state. In one embodiment, cells are re-programmed by introducing into them Oct4, Sox2, Klf4, and cMyc genes, e.g., using a retroviral system. (See, e.g., Takahashi K, Tanabe K, Ohnuki M, Narita M, lchisaka T, Tomoda K, Yamanaka S (November 2007). "Induction of pluripotent stem cells from adult human fibroblasts by defined factors". Cell. 131 (5): 861-72. doi: 10.1016/j.cell.2007.11.019. PMID 18035408.) In another embodiment, Oct4, Sox2, Nanog, and Lin28 are introduced, e.g., using a lentiviral system. (See, e.g., Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (December 2007). "Induced pluripotent stem cell lines derived from human somatic cells". Science. 318 (5858): 1917-20. doi: 10.1126/science.1151526. PMID 18029452.) See, e.g., US Patent 8,058,065, November 15, 2011 (Oct3/4, KLF4, C-myc and SOX2 produce induced pluripotent stem cells”) and WO 2009/157593, December 30, 2009 (“Method of efficiently establishing induced pluripotent stem cells”).

[0041] For propagating and banking iPSCs: Numerous methods exist in the art that demonstrate how to iPSCs can be maintained without loss of pluripotency. Nearly all methods utilize co-culturing the iPSCs on a layer of mouse feeder cells or, in a feeder- free system, using tissue culture vessels coated with an extracellular matrix component such as Matrigel, vitronectin, or collagen. The cells are also incubated with a nutrient- rich medium such as TeSR-E8. To maintain pluripotency, the medium is typically changed daily and cells are passaged 1 :5-1 :10 when confluency reaches 70%. Morphology of the cells is checked by microscopy and cells with atypical morphology can be removed from culture. To generate a bank of iPSCs, they should be maintained for at least 20 passages. While the period of time between passages is dependent on cell morphology, each passage is typically expected to occur every 6-7 days.

B. IPSC-derived, e.g., Hypoimmunogenic, Induced Pluripotent Stem Cells

[0042] Several methods exist to render cells able to avoid detection by a competent host immune system.

[0043] In one method, cells are made hypoimmunogenic when one or both of Major Histocompatibility Complex I (“MHC I”) and/or Major Histocompatibility Complex II (“MHC II”) genes are knocked out, and, optionally, when expression of CD47 is upregulated. In humans, the Major Histocompatibility Genes are referred to as the Human Leukocyte Antigen (“HLA”) genes. This includes HLA I and HLA II genes. It is understood that when “HLA” genes are referred to herein, cognate genes in cells of other species (e.g., “MHC” genes) may be similarly modified to produce similar results.

[0044] In another methods, independent of, or together with, knocking out HLA genes, cells can be made hypoimmunogenic by over-expressing proteins such as CD47, PD-L1 , Tim3, CTLA4-lg and HLA-G.

[0045] As used herein, a gene is “knocked out” when the gene rendered inoperative so that it no longer expresses a functional protein. Genes can be knocked out by any method known in the art. This includes, for example, homologous recombination in the use of site-specific nucleus such as zinc fingers, CRISPER/Cas9 and transcription activator-like effector nuclease (TALENs). It is understood that genes that have been knocked out may still possess residual levels of expression.

[0046] As used herein, expression of a gene is “up-regulated” when a cell expresses it in higher amounts than normal. This includes, for example, constitutive expression or induced expression. Constitutive expression can be achieved by introducing into the cell an expression construct comprising the gene operatively linked with a constitutive expression control sequence (e.g., promoter). Induced expression can be achieved by introducing into the cell an expression construct comprising the gene operatively linked with an inducible expression control sequence. Induction may be had by providing a drug that induces expression or by using an expression control sequence that induces by compounds normally present in the cell. Up-regulated expression can be expression increased over normal by at least 1.5-fold, at least 2-fold, at least 4-fold, at least 7-fold, or at least 10-fold over cells before modification.

[0047] An HLA gene or genes can be knocked out in iPSCs. By culturing knocked out iPSCs with appropriate cytokines and optionally on an extracellular matrix protein or cocultured with mouse feeder cells, they can be made to differentiate into hematopoietic stem cells and along the myeloid line into myeloid progenitor cells. Myeloid progenitor cells can further differentiate into any cell along the myeloid line.

[0048] Over-expression of CD47, PD-L1 , CTLA4-lg and HLA-G can be achieved by, for example, introducing into the cell an expression cassette comprising any of these genes. For example, the expression cassette can be introduced via lentivirus-mediated delivery of a CD47-expressing vector.

[0049] Such methods are described, for example, in T. Deuse et al., Nature Biotechnology, “Hypoimmunogenic derivatives of induced pluripotent stem cells invade immune rejection in fully immunocompetent allogeneic recipients”, https: //doi.org/10.1038/s41587-019-0016-3; Liu, X. et al. , “The Immunogenicity and Immune Tolerance of Pluripotent Stem Cell Derivatives,” Front. Immunol., 02 June 2017 | https:// doi.org/10.3389/fimmu.2017.00645; and Han, X. et al., “Generation of hypoimmunogenic human pluripotent stem cells”, PNAS May 21 , 2019 116 (21) 10441- 10446; first published April 30, 2019 https:// doi.org/10.1073/pnas.1902566116.

[0050] CRISPR technology can be used to target coding sequences of genes essential for HLA Class I and HLA Class II functionality. The linearized CRISPR sequences with a T7 promoter can be used to synthesize guide RNA using commercially available kits such as the MEGAshortscript T7 Transcription Kit (Thermo Fisher). The guide RNA is then delivered to iPSCs via electroporation. Following electroporation, viable single cells which express surface marker TRA-160 can be isolated using flow cytometry or magnetic bead-based selection columns to select for edited iPSCs. Single cells can then be expanded into colonies and tested for CRISPR editing by isolating the DNA, performing PCR, and performing sequencing using standard techniques. To overexpress CD47 in these cells, CD47 cDNA can be synthesized and cloned into a lentiviral plasmid with and EFS promoter and an antibiotic resistance cassette. Three days after transduction with lentiviral stocks and Polybrene, cells can be expanded and selected using the appropriate antibiotic. After 5 days of selection, antibiotic-resistant colonies should emerge and the cells can be further expanded to create a bank. Overexpression of CD47 can be confirmed by FACS and qPCR.

[0051] Another method of preventing rejection of iPSCs by the recipient’s immune system is to overexpress or upregulate PD-L1 (programmed death ligand 1). While overexpression of CD47 in iPSCs can prevent targeting by the recipient’s macrophages, overexpression of PD-L1 can prevent destruction of the iPSCs by the recipient’s T cells. Since the receptor for PD-L1 (i.e. , PD-1) is expressed in T cells, upregulation of PD-L1 on iPSCs may act as a barrier to protect tumor cells from T cell lysis. This has been demonstrated experimentally. (See, e.g., Han et al. “Generation of hypoimmunogenic human pluripotent stem cells.” PNAS 116 (2019): 10441-10446; Tanaka et al. “PDL1 is required for Peripheral Transplantation Tolerance and Protection from Chronic Allograft Rejection.” J Immunol 179 (2007): 5204-5210.) Methods to overexpress PD-L1 include, for example, use of a lentiviral vector containing the PD-L1 DNA along with an antibiotic cassette can be synthesized using standard techniques. The lentiviral particles can be used to transduce the iPSCs and the cells can be treated with antibiotic to induce PD-L1 expression. Other immune modulator proteins/receptors can be used to prevent rejection.

[0052] Similarly, CTLA4-lg and HLA-G also can be over-expressed to render cells able to avoid destruction by the immune system. C. IPSC-derived, e.g., Hypoimmunogenic, Hematopoietic Stem Cells

[0053] Induced pluripotent stem cells can be cultured to become hematopoietic stem cells. iPSCs, e.g., hypoimmunogenic iPSCs, can be cultured to differentiate into hematopoietic stem cells by culturing the cells with cytokines that promote differentiation along the hematopoietic line. See, for example, WO 2019/040448, February 28, 2019 (“Methods for inducing hematopoietic stem cell specificity”).

[0054] In one method, iPSCs can be re-specified into hematopoietic stem and progenitor cells through transient expression of a single transcription factor, MLL-AF4. (See, e.g., Tan, Y-T et al., “Respecifying human iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells with a single factor”, 2180-2185, PNAS, February 27, 2018, vol. 115, no. 9.)

[0055] In another method, iPSCs are sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. (Chicha L. et al.,

“Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics”, PLoS One. 2011 Feb 25;6(2):e14733. doi: 10.1371/journal. pone.0014733.)

[0056] Transient overexpression of HOXB4 also can promote differentiation of iPSCs into hematopoietic stem cells. (Kyba et al, “HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors”, Cell. 2002 Apr 5; 109(1 ):29-37.)

[0057] These methods typically produce a mixture of cell types. Accordingly, to produce populations enriched for iPSC-derived, e.g., hypoimmunogenic, HSCs, one can separate HSCs from other cells in the mixture. This can be performed, for example, by selective capture of HSCs. One such method involves the use of a magnetic selection column for hematopoietic stem cells, which are characterized as CD34+. Accordingly, isolation can include the use of solid supports, such as beads, bearing ligands for CD34. This could for example, be an anti-CD34 antibody. Compositions isolated in this manner can be at least any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% CD34+ cells.

[0058] For generating hematopoietic stem cells from iPSCs: To induce differentiation towards the hematopoietic lineage, the medium for the iPSCs would be changed to a different culture medium known to be more conducive to support hematopoietic cells. The options include, but are not limited to, X-VIVO, APEL, and Stemline cell culture media. During HSC differentiation cells may continue to be cultured on vessels coated with an extracellular matrix protein as well as supplemented with cytokines including, but not limited to, bFGF, VEG4, BMP4, and SCF. Cells may be cultured in this media for 3-14 days, with the possible addition or removal of other cytokines over time, and will be monitored for CD45 and CD34 expression via flow cytometry. These cells can then be isolated using magnetic bead-based technologies and their potency and ability to form other hematopoietic cells can be determined using cell-based assays such as hematopoietic colony assays and lineage-specific culture assays. Upon determining the culture duration that results in CD34+ cells of the best quality, the isolated CD34+ cells can then be placed into myeloid progenitor cell culture.

D. IPSC-derived, e.g., Hypoimmunogenic, Myeloid Progenitor Cells

[0059] Myeloid cells can be produced in vitro from human embryonic stem cells and from induced pluripotent stem cells. Hematopoietic stem cells, e.g., hypoimmunogenic, can be cultured to differentiate into myeloid progenitor cells by culturing the cells with appropriate cytokines. See, for example, M. Hansen et al. , “Efficient production of erythroid, megakaryocytic and myeloid cells, using single cell-derived iPSC colony differentiation,” Stem Cell Res. 29 (2018) 232-244. See also, N. Lachmann et al., “Large-scale hematopoietic differentiation of human induced pluripotent stem cells provided granulocytes or macrophages for cell replacement therapies,” Stem Cell Reports, Vol. 4, pp. 282-296, February 10, 2015. See also, A. Niwa et al., “A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors,” PLos One, July 2011 , Volume 6, Issue 7. See also, G. Salvagiotto et al., “A Defined, Feeder-Free, Serum-Free System to Generate In Vitro Hematopoietic Progenitors and Differentiated Blood Cells from hESCs and hiPSCs,” PLos One, March 2011 , Volume 6, Issue 3. See also, Y-T Tan et al., “Respecifying human iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells with a single factor,” PNAS, pp. 2180-2185, February 27, 2018, vol. 115, no. 9.

[0060] Depending on culture conditions and timing of culture, a population of myeloid progenitor cells can include various mixtures of cells from common myeloid progenitors, granulocyte-macrocyte progenitors, megakaryocyte-erythrocyte progenitors and immature monocytes, granulocytes, megakaryocytes or erythrocytes. Such a composition may also still include a proportion of multipotent progenitor cells. Cultured for around four days, a large proportion of the cells will be common myeloid progenitor cells. Cultured for around eight days the population will include a significantly greater proportion of lineage committed cells such as granulocytes and megakaryocytes. The number of days of culture to produce myeloid progenitors will depend on the primitiveness of HSCs, with more primitive HSCs requiring longer periods of culture.

[0061] In one method of making MPCs, HSCs are cultured with Stem Cell Factor (“SCF”), lnterleukin-3 (“IL-3”), FMS like tyrosine kinase 3 (“FLT3”) ligand, thrombopoietin (“TPO”) (e.g., mimetic TPO) and human serum albumin (“HSA”). Cells can be cultured for between about 4 days and about 20 days or about 28 days. During such period, the proportion of different cell types in the population changes. At around 4 days, MPPs and common myeloid progenitor cells predominate. By day 8, the myeloid population may have a preponderance of MEPs and GMPs. Culturing for longer periods results in the myeloid population maturing to have significant numbers of committed monocytes, granulocytes, megakaryocytes and erythrocyte precursors (depending on growth factors used).

[0062] A protocol for efficient generation of neutrophils, eosinophils, macrophages, osteoclasts, DCs in Langerhans cells from human embryonic stem cells has been developed. (K-D Choi et al, “Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin-CD34+CD43+CD45+ progenitors,” J. Clin. Invest. 2009 119:2818-2829.) As a first step, one can generate Lin- CD34+CD43+CD45+ hematopoietic cells highly enriched in myeloid progenitors through co-cultures of iPSCs and OP9 feeder cells. After expansion in the presence of GM-CSF, CD235a/41a-CD45+ cells can be separated, for example, by selective capture. These cells can be directly differentiated with specific cytokine combinations toward mature cells particular types. For example, culturing on a bed of OP9 cells with G-CSF for eight days produces neutrophils. Culturing on OP9 cells with IL-3, IL-5 for 12 to 14 days produces eosinophils. Culturing with GM-CSF, IL-4 and TNF-alpha for seven days produces DC’s. Culturing for seven days with GM-CSF, TGF-beta and TNF-alpha produces Langerhans cells. Culturing with M-CSF, IL-1 beta for 5 to 7 days produces macrophages. Culturing with GM-CSF, vitamin D3 and RANKL for, e.g., 14 days produces osteoclasts. [0063] For generating MPCs from HSCs: Purified CD34+ cells can be placed into a bioreactor or cell culture vessel with cell culture media, such as X-VIVO, supplemented with cytokines and growth factors including, but not limited to, SCF, IL-3, Flt3-ligand, mTpo, and human serum albumin. Cells will be expanded for at least 4 but not more than 20 days. During expansion, the cells will be monitored for granulocyte, MPP, CMP, GMP, and MEP content and potency will be assessed using colony assays and in vitro cell culture assays. Upon reaching the optimal potency and expressing the appropriate characteristics, the cells will be harvested, formulated in a media containing BioLife® CryoStor® buffer, human serum albumin, and DMSO and cryopreserved in either a vial or bag.

E. Differentiation of Cells

[0064] Myeloid progenitor cells, administered to a mammalian subject, will populate the bone marrow. There, they will differentiate into mature cells. The particular direction of differentiation can depend on growth factors that are co-administered to a subject.

So, for example, G-CSF promotes differentiation along the neutrophil line. GM-CSF and M-CSF promote differentiation along the macrophage line. TPO promotes differentiation toward megakaryocytes and platelets. Erythropoietin promotes differentiation toward erythrocytes.

[0065] Myeloid progenitor cells are committed to terminal differentiation and do not maintain self-renewal capacity. As they mature, the cells eventually perish without being replaced. Therefore, such cells will not initiate a graft-versus-host response. Furthermore, because they are temporary, they must eventually be replaced by the host immune system. Accordingly, such cells are particularly useful as a bridge between myeloablation, when the immune system of the subject is either destroyed or severely impaired, and reestablishment of a host immune system. An immune system can be reestablished either by replication in differentiation of remaining host cells or by replacement with homogeneity or allogeneic stem cells.

IV. Pharmaceutical Compositions

[0066] Also provided herein are pharmaceutical compositions comprising iPSC- derived, e.g., hypoimmunogenic, myeloid progenitor cells and a pharmaceutically acceptable carrier. [0067] The pharmaceutically acceptable carrier can include a cryopreservant, such as DMSO, or the commercially available cryopreservant BioLife® CryoStor®. Compositions can be cryopreserved (e.g., frozen) in this state.

[0068] As used herein, the term “pharmaceutical composition” refers to a composition comprising a pharmaceutical compound (e.g., a drug) and a pharmaceutically acceptable carrier.

[0069] As used herein, the term “pharmaceutically acceptable” refers to a carrier that is compatible with the other ingredients of a pharmaceutical composition and can be safely administered to a subject. The term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. Pharmaceutical compositions and techniques for their preparation and use are known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and techniques for their administration one may refer to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., “Goodman and Gilman’s The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.

[0070] Pharmaceutically acceptable carriers will generally be sterile, at least for human use. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration. Examples of pharmaceutically acceptable carriers include, without limitation, normal (0.9%) saline, phosphate-buffered saline (PBS), Hank’s balanced salt solution (HBSS), and multiple electrolyte solutions such as PlasmaLyte ATM (Baxter). An example of a pharmaceutically acceptable carrier for use with intravenous injection is a sterile solution of normal saline, having pH between about 4 to about 9, e.g., around 7, optionally buffered. The solution also may contain DMSO.

[0071] Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N- methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Pluronics, Triton-X, or polyethylene glycol (PEG).

V. Kits

[0072] Further provided herein are kits. Kits can include a first container containing pharmaceutical composition comprising iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells, and a second container containing a cell growth factor.

[0073] A container containing the pharmaceutical composition can include, for example, about 12 milliliters (ml_) of solution including between about myeloid progenitor cells 15*10⁶ to 50x10⁶ cells per mL (e.g., about 20x10⁶ cells per mL to about 30x10⁶ cells per mL, e.g., about 25x10⁶ cells per mL).

VI. Methods of Using

A. Therapeutic Methods

[0074] Compositions comprising iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells are useful to replace myeloid progenitor cells in any indication in which they are used. These include indications in which the end-point target cell is any cell along the myeloid line. These include, for example, neutrophils, megakaryocytes (platelets), eosinophils, basophils, monocytes (macrophages), and erythrocytes.

[0075] As used herein, the term terms “therapy,” “treatment,” “therapeutic intervention” and “amelioration” refer to any activity resulting in a reduction in the severity of symptoms. In the case of cancer, treatment can refer to, e.g., reducing tumor size, number of cancer cells, growth rate, metastatic activity, reducing cell death of non-cancer cells, reduced nausea and other chemotherapy or radiotherapy side effects, etc. The terms “treat” and “prevent” are not intended to be absolute terms. Treatment and prevention can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. Treatment and prevention can be complete or partial. Treatment and prevention can be complete (undetectable levels of neoplastic cells) or partial, such that fewer neoplastic cells are found in a patient than would have occurred without the present intervention. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects, the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.

[0076] As used herein, the terms “effective amount,” “effective dose,” and “therapeutically effective amount,” refer to an amount of an agent that is sufficient to generate a desired response, such as reduce or eliminate a sign or symptom of a condition or ameliorate a disorder. In some examples, an “effective amount” is one that treats (including prophylaxis) one or more symptoms and/or underlying causes of any of a disorder or disease and/or prevents progression of a disease. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of therapeutic effect at least any of 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least any of a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0077] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable label (if present). One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration. For example, a dosage form can be in a liquid, e.g., a saline solution for injection.

[0078] As used herein, the term “subject” refers to an individual animal. The term “patient” as used herein refers to a subject under the care or supervision of a health care provider such as a doctor or nurse. Subjects include mammals, such as humans and non-human primates, such as monkeys, as well as dogs, cats, horses, bovines, rabbits, rats, mice, goats, pigs, and other mammalian species. Subjects can also include avians. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.

[0079] Administration can include co-administering more than one therapeutic composition. The term “co-administration” includes simultaneous administration. It further includes administration of compounds separate in time but allowing effective therapeutic effect together.

[0080] Treatments intended to produce cells of a certain type along the myeloid line can include administration of iSPC-derived cells at any point along the developmental pathway that leads to the desired cells, and co-administering one or more cytokines that promote differentiation toward the ultimate cell type. So, for example, in the development of neutrophils, the cells can include MPPs, CMPs, or GMPs, and GM-CSF can be co-administered. a) Neutropenia

[0081] Neutropenia is a condition characterized by a deficiency in neutrophils and other immune cells of the myeloid line. Subjects suffering from neutropenia are immunocompromised. Neutropenia can result from several different causes. These include, for example, chemically-induced neutropenia or radiation-induced neutropenia.

[0082] Chemically induced neutropenia is a side effect of certain chemotherapies which kill a subject’s immune cells. For example, chemotherapy for various cancers can kill cells of the myeloid line. For example, the treatment of myeloid leukemias such as acute myeloid leukemia and chronic myeloid leukemia, can involve myeloablation.

[0083] Acute radiation syndrome can result in radiation-induced neutropenia. Acute radiation syndrome can result from exposure to radiation which may be either environmental or therapeutic. For example, radiation therapy for conditions such as cancer can result in neutropenia, compromising the immune system on the subject. Environmental conditions that can result in radiation poisoning can include, for example, nuclear accident, exposure to radioactive waste and explosion of radioactive devices.

[0084] Methods of treating neutropenia can involve administering to a subject in need thereof an effective amount of iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells as described herein. Granulocyte colony-stimulating factor (G-CSF) can be co-administered with myeloid progenitor cells to induce differentiation along the neutrophil line. [0085] Compositions comprising cells other than MPCs can be administered, so long as the amount of MPCs in the composition is effective to treat the condition. b) Infection

[0086] In certain instances, an infection can be so massive that it overwhelms the host’s immune system. This can occur, for example, when the subject’s immune system is weakened or compromised or absent. In cases where the host immune system is present but insufficient or not active to fight off infection, iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells of this disclosure can be administered to the subject to boost immunity.

[0087] Methods of treating infection can involve administering to a subject in need thereof an effective amount of iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells as described herein. Granulocyte colony-stimulating factor (G-CSF) or G-CSF biosimilars can be co-administered with myeloid progenitor cells to induce differentiation along the neutrophil line.

[0088] In other circumstances, risk of infection can be reduced by administering to a person at increased risk of infection, an effective dose of MPCs as described herein.

For example, the subject may be very young, or elderly, or ill. c) Monocytopenia

[0089] Monocytopenia is a condition characterized by deficiency of monocytes. Monocytopenia also can result in a deficiency in macrophages, which developed from monocytes. MonoMAC syndrome is a rare form of monocytopenia resulting from a dominant autosomal mutation.

[0090] Methods of treating monocytopenia can involve administering to a subject in need thereof an effective amount of iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells as described herein. Macrophage colony-stimulating factor (M-CSF) can be co-administered with myeloid progenitor cells to promote differentiation along the monocyte line and toward macrophages. d) Thrombocytopenia

[0091] Thrombocytopenia is a condition associated with a deficiency in platelets. [0092] Thrombocytopenia can be divided according to three major causes: low production of platelets in the bone marrow, increased breakdown of platelets in the bloodstream, and increased breakdown of platelets in the spleen or liver. Disorders that involve low production in the bone marrow include aplastic anemia and cancer in the bone marrow. Disorders that involve the breakdown of platelets include: Immune thrombocytopenic purpura (ITP), drug-induced immune thrombocytopenia, drug-induced nonimmune thrombocytopenia, thrombotic thrombocytopenic purpura, primary thrombocythemia, disseminated intravascular coagulation (DIC), hypersplenism, etc.

[0093] Thrombocytopenia can also result from the impaired development of megakaryocytes, complications from infections, and in transplant situations, e.g., where a patient undergoing myeloablative treatment receives hematopoietic stem cell (HSC) transplant. In this case, thrombocytopenia can result from delayed or low engraftment of HSCs and from graft versus host disease (GVHD).

[0094] Methods of treating thrombocytopenia can involve administering to a subject in need thereof an effective amount of iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells as described herein. Thrombopoietin (TPO) and/or agonists of c-MPL can be co-administered with myeloid progenitor cells to induce differentiation along the megakaryocytic line. e) Hematopoietic Stem Cell Transplantation

[0095] Hematopoietic stem cell transplantation (sometimes referred to as “bone marrow transplant”) is a treatment used for subjects whose immune systems have been destroyed, for example, by chemotherapy or by radiation. It involves the transplantation of hematopoietic stem cells. The stem cells can be autologous or allogeneic to the subject. In some circumstances hematopoietic stem cells are co-administered with myeloid progenitor cells. Such cells can provide a bridge of immune function until engraftment of the hematopoietic stem cells in their development.

[0096] Accordingly, provided herein are methods of performing hematopoietic stem cell transportation comprising, administering iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells of this disclosure as an adjunct to administration of the hematopoietic stem cells. In certain embodiments, administration of iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells allow administration of a sub-optimal dose of hematopoietic stem cells. A suboptimal dose can be, for example, a single cord blood unit or two cord blood units. Granulocyte colony-stimulating factor (G-CSF) can be co- administered with myeloid progenitor cells to induce differentiation along the neutrophil line. f) Anemia

[0097] Anemia is a condition associated with a deficiency in red blood cells.

Methods of treating anemia can involve administering to a subject in need thereof an effective amount of iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells as described herein (e.g., MPPs, CMPs ort MEPs). Erythropoietin can be co-administered with myeloid progenitor cells to induce differentiation along the erythroid line. g) Amounts

[0098] Therapeutic administration of iPSC-derived, e.g., hypoimmunogenic, myeloid progenitor cells as disclosed are administered in amounts effective to treat the condition. An effective amount of myeloid progenitor cells can be at least about any of 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, or 1 x 10⁸ cells/kg body weight.

B. Drug Testing

[0099] Cells as provided herein also are useful in methods of testing drugs for their ability to promote or inhibit differentiation of cells along any pathway in the hematopoietic, myeloid or lymphoid lines. The methods can involve culturing cells with (1) factors known promote differentiation along a particular line and (2) a test drug; and determining whether the test drug promotes or inhibits differentiation along the particular line. Methods also can involve culturing cells with (1) a test drug; and determining whether the test drug promotes or inhibits differentiation along the particular line.

EXAMPLES

[00100] Induced PSCs are cultured either on a layer of mouse feeder cells or in a feeder-free system whereby tissue culture vessels would be coated with Matrigel, collagen, vitronectin, or another extracellular matrix component. iPSCs are maintained and expanded for many passages and cells that begin to differentiate may be physically removed from culture. The culture medium may include, but are not limited to, growth factors such as BMP4, VEGF, and bFGF. The iPSC culture is monitored for expression of CD34 and after the optimal culture duration, the CD34+ cells would be selected using the magnetic bead-based selection system Miltenyi CliniMACS, so that they are selected at a point where hematopoietic commitment has occurred but they have not become more differentiated progenitors. Following selection, the CD34+ cells are cryopreserved. To begin MPC expansion, the CD34+ cells would be thawed and expanded in bioreactors or large-scale spinner flasks in an expansion medium containing myeloid-promoting cytokines (similar to romyelocel-L). The expanded MPCs are then cryopreserved.

[00101] This method can also be used to culture iPSCs that are genetically engineered. For example, immunocompatibility of the resultant MPCs is much improved if the initial iPSCs are engineered to remove genes (such as critical components of human leukocyte antigen) that result in rejection of the MPCs. Following gene editing using CRISPR/Cas9, the iPSCs can undergo a similar maintenance and expansion process as described above.

[00102] The selection of the CD34+ cells prior to MPC expansion results in a much purer and potent product. Neutrophil differentiation can occur among a cell population with impurities and non-target cell types. However, populations in which such cells are eliminated or reduced have greater clinical value due to decreased risk of cells that produce graft v. host disease.

[0100] As used herein, the following meanings apply unless otherwise specified. The word “may” is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The words “include”, “including”, and “includes” and the like mean including, but not limited to. The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.” The phrase “at least one” includes “one or more”, “one or a plurality” and “a plurality”. The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of” between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1 , 2 or 3” means “at least 1 , at least 2 or at least 3”. The term "consisting essentially of" refers to the inclusion of recited elements and other elements that do not materially affect the basic and novel characteristics of a claimed combination.

[0101] It should be understood that the description and the drawings are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description and the drawings are to be construed as illustrative only and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. Headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description. [0102] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A cell population comprising hematopoietic stem cells (“HSCs”) derived from induced pluripotent stem cells (“iPSCs”) or from other human pluripotent stem cells.

2. The cell population of claim 1 , wherein the HSCs are hypoimmunogenic HSCs comprising genetic modifications to evade destruction of the cells by a competent immune system.

3. The cell population of claim 2, wherein expression of one or more of CD47, PD-L1 , Tim3, CTLA4-lg and HLA-G are up-regulated.

4. The cell population of claim 2 or 3, wherein expression of one or both of Human Leukocyte Antigen Class I (“HLA I”) and Human Leukocyte Antigen Class II (“HLA I”) HLA genes is/are knocked out.

5. The cell population of any of claims 1-4, comprising at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% hypoimmunogenic HSCs.

6. A cell population comprising myeloid progenitor cells (“MPCs”) derived from induced pluripotent stem cells (“iPSCs”) or from other human pluripotent stem cells.

7. The cell population of claim 6, wherein the MPCs are hypoimmunogenic MPCs comprising genetic modifications to evade destruction of the cells by a competent immune system.

8. The cell population of claim 7, wherein expression CD47, PD-L1 , Tim3, CTLA4-lg and/or and HLA-G are up-regulated.

9. The cell population of claim 7 or 8, wherein expression of one or both of Human Leukocyte Antigen Class I (“HLA I”) and Human Leukocyte Antigen Class II (“HLA I”) HLA genes is/are knocked out.

10. The cell population of any of claims 6-9, comprising at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% hypoimmunogenic MPCs.

11. The cell population of any of claims 6-9, wherein the MPCs comprise one or more of: Common Myeloid Progenitor Cells (“CMPs”) (CD34+, CD90-, CD123+, CD45RA-), Megakaryocyte/Erythroid Progenitor Cells (“MEPs”) (CD34+, CD90-, CD123-, CD45RA-) and Granulocyte/Monocyte Progenitor Cells (“GMPs”) (CD34+, CD90-, CD123+, CD45RA+).

12. The cell population of any of claims 6-9, which is substantially free of cells of lymphoid lineage, e.g., Common Lymphoid Progenitor Cells (“CLPs”) (CD34+, CD7+, CD10+), T cells (CD2+, CD3+), and B cells (CD19+, CD20+, CD33-), e.g., less than 5%.

13. The cell population of any of claims 6-9, comprising Multi-Potent Progenitor Cells (“MPPs”) (CD34+, CD90+)

14. The cell population of claim 13, which has fewer than 40% MPPs.

15. A method of making hypoimmunogenic hematopoietic stem cells comprising: a) providing induced pluripotent stem cells (iPSCs); b) knocking out expression of one or both of Human Leukocyte Antigen

Class I (“HLA I”) and/or Human Leukocyte Antigen Class II (“HLA I”) HLA genes; and/or, up-regulating expression of any or all of CD47, CD47, PD-L1 , Tim3, CTLA4-lg and HLA- G in said iPSCs to produce hypoimmunogenic iPSCs; and c) culturing said hypoimmunogenic iPSCs under conditions to differentiate into hypoimmunogenic hematopoietic stem cells (“HSCs”); thereby producing hypoimmunogenic HSCs.

16. The method of claim 15, wherein culturing comprising culturing cells for at least 4 days with one or more of bone morphogenic protein-4, FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor.

17. The method of claim 15, wherein the knocking out expression and up- regulating expression comprises using CRIPSR/Cas9 technology.

18. A method of making iPSC-derived myeloid progenitor cells (“MPCs”) comprising: a) providing iPSC-derived hematopoietic stem cells (“HSCs”); and b) culturing said cells under conditions to differentiate into hypoimmunogenic myeloid progenitor cells.

19. The method of claim 18, wherein expression of Major Histocompatibility Complex I (“MHC I”) and/or Major Histocompatibility Complex II (“MHC II”) genes are knocked out; and/or any or all of CD47, CD47, PD-L1 , Tim3, CTLA4-lg and HLA-G are up-regulated.

20. The cell population of claim 19, wherein expression of one or more of CD47, PD-L1 , Tim3, CTLA4-lg and HLA-G are up-regulated.

21. The cell population of claim 19 or 20, wherein expression of one or both of Human Leukocyte Antigen Class I (“HLA I”) and Human Leukocyte Antigen Class II (“HLA I”) HLA genes is/are knocked out.

22. The method of claim 18, wherein culturing comprises culturing the cells with one or more of (e.g., all of) Stem Cell Factor, lnterleukin-3, FLT3 ligand, thrombopoietin (“TPO”) (e.g., mimetic TPO), and human serum albumin (“HSA”).

23. The method of claim 18, wherein culturing comprises culturing the cells for at least 4 days and no more than 28 days.

24. A pharmaceutical composition comprising: a) a cell population comprising myeloid progenitor cells (“MPCs”) derived from induced pluripotent stem cells (“iPSCs”); and b) a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, wherein the MPCs are hypoimmunogenic MPCs comprising genetic modifications to evade destruction of the cells by a competent immune system.

26. The pharmaceutical composition of any of claims 24 or 25, wherein the pharmaceutically acceptable carrier comprises about 5% cryopreservative (e.g.,

DMSO), and about 5% human serum albumin (“HSA”).

27. The pharmaceutical composition of any of claims 24 or 25, wherein the pharmaceutically acceptable carrier comprises BioLife® CryoStor®.

28. The pharmaceutical composition of any of claims 24 or 25, which is cryopreserved.

29. A kit comprising: a) a first container and, contained within the first container, pharmaceutical composition comprising: i) a cell population comprising hypoimmunogenic myeloid progenitor cells derived from induced pluripotent stem cells (“iPSCs”); and ii) a pharmaceutically acceptable carrier.

30. The kit of claim 29, wherein the MPCs are hypoimmunogenic MPCs comprising genetic modifications to evade destruction of the cells by a competent immune system.

31. The kit of claim 29, wherein the first container contains 15* 10⁶ to 50* 10⁶ cells per ml_ (e.g., about 20*10⁶ cells per mL to about 30*10⁶ cells per mL, e.g., about

25 x 10⁶/ml_ MPC cells.

32. The kit of claim 29, wherein the first container contains about 12 mL of the pharmaceutically acceptable carrier.

33. The kit of claim 29, wherein the first container comprises a bag or a bottle.

34. The kit of claim 29, further comprising: b) a second container containing one or more cytokines selected from G- CSF, GM-CSF, TPO, M-CSF, IL-1 beta, and EPO.

35. The kit of claim 29, further comprising a syringe.

36. A method comprising administering to a subject in need thereof an effective amount of myeloid progenitor cells, wherein the cells are derived from induced pluripotent stem cells.

37. The method of claim 36, wherein the MPCs are hypoimmunogenic MPCs comprising genetic modifications to evade destruction of the cells by a competent immune system.

38. The method of claim 36, wherein the cells are derived from induced pluripotent stem cells.

39. The method of claim 36, wherein the subject suffers from neutropenia, anemia, monocytopenia, and thrombocytopenia or infection.

40. The method of claim 36, wherein the subject is undergoing stem cell transplantation.

41. The method of claim 36, wherein the subject has undergone myeloablative therapy.

42. The method of claim 36, wherein the subject is immunocompromised.

43. The method of claim 36, wherein the subject suffers from chemically induced neutropenia (e.g., from chemotherapy) or acute radiation poisoning.

44. The method of claim 29, wherein the subject is immunocompetent.

45. The method of claim 44, wherein the subject suffers from an infection.

46. The method of claim 29, wherein the subject is at increased risk for infection.

47. The method of claim 41 , comprising inducing in the subject production of cells selected from monocytes, megakaryocytes, platelets, erythrocytes, neutrophils, eosinophils, and basophils.

48. The method of claim 29, wherein administration comprises intravenous injection.

49. The method of claim 29, further comprising co-administering to the subject one or more cytokines selected from G-CSF, GM-CSF, TPO, M-CSF, IL-1 beta, and EPO.

50. The method of claim 49, comprising co-administering granulocyte colony growth factor (“G-CSF”) in an amount sufficient to induce differentiation of the MPCs into neutrophils.

51. The method of claim 49, comprising co-administering thrombopoietin in an amount sufficient to induce differentiation of the MPCs into platelets.

52. The method of claim 49, comprising co-administering macrophage colony stimulating factor (“M-CSF”) in an amount sufficient to induce differentiation of the MPCs into macrophages.

53. The method of claim 41 , further comprising co-administering hemopoietic stem cells (“HSCs”).

54. The method of claim 53, wherein the dose of HSCs is sub-optimal, that is, below an amount that would be effective to reconstitute an immune system if the HSCs were delivered without co-administration of the MPCs.

55. The method of claim 29, comprising administering the cells up to 1.5 x 10⁷ cells/kg body weight.

56. A method comprising: a) providing a population comprising hypoimmunogenic hematopoietic stem cells or hypoimmunogenic myeloid progenitor cells; b) contacting the population with a test drug and, optionally, one or more cytokines or growth factors that promote differentiation of the cells along a path in the hematopoietic differentiation pathway; and c) determining whether the test drug promotes or inhibits differentiation along the pathway.