Immune cells derived from induced pluripotent stem cells

Abstract

Methods for producing immune cells from myeloid progenitor cells, pluripotent stem cells, or multilineage hematopoietic progenitor cells are provided. [Solution] In one embodiment, the method includes the steps of: a) generating multi-lineage hematopoietic progenitor cells from myeloid progenitor cells; b) inhibiting histone methyltransferase in the resulting multi-lineage hematopoietic progenitor cell population; and c) differentiating the resulting multi-lineage hematopoietic progenitor cell population in the presence of a notch ligand or stromal cells or both to promote differentiation into lymphocyte lineages. TIFF2023011849000015.tif22167

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 383,984, filed September 6, 2016, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Government support This invention was made with government support under Grant Nos. U01 HL100001 and R24DK092760 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Field of Disclosure The present disclosure relates generally to the fields of medicine, cell biology, and molecular biology. The present disclosure relates to methods for producing immune cells, such as B or T lymphocytes, from restricted lineage myeloid progenitor cells, or pluripotent stem cells (PSCs), or multilineage hematopoietic progenitor cells (MHPCs). [Background technology]

[0004] background There is a scarce supply of functional immune cells for in vivo cell replacement therapy, treatment of numerous diseases, disorders, and conditions, as well as for disease modeling, drug screening, and in vitro studies of hematologic disorders. Bone marrow transplantation is the most established cell replacement therapy for a variety of hematologic disorders. The functional unit of bone marrow transplantation is the hematopoietic stem cell (HSC), which sits at the apex of a complex cellular hierarchy and replenishes blood development throughout life. However, the lack of HLA-matched or patient-specific HSCs severely limits the ability to perform transplantation, disease modeling, drug screening, and in vitro studies of hematologic disorders. In many cases, cell populations are not transplanted large enough to ensure satisfactory in vivo engraftment and reconstitution in the recipient subject.

[0005] Thus, many studies have been developed to generate HSCs from alternative sources. For example, reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has enabled the availability of a wide variety of patient-specific pluripotent cells, which are promising sources for disease modeling, drug screening, and cell therapy. Pluripotent cells are induced in human and mouse somatic cells by forced expression of OCT4 (Oct4) and SOX2 (Sox2), either in combination with KLF4 (Klf4) and, optionally, c-MYC (c-Myc), or in combination with NANOG (Nanog) and LIN28 (Lin28). Alternative transactivator combinations include OCT4, SOX2, NANOG, and LIN28. Mouse iPS cell lines derived from bone marrow hematopoietic progenitor cells (HPCs) have been reported. Derivation of human iPSCs from postnatal human blood cells, from peripheral blood CD34+ cells mobilized with granulocyte colony-stimulating factor (G-CSF), and from human umbilical cord blood and adult bone marrow CD34+ cells without any prior treatment such as G-CSF mobilization have also been reported. All of these methods employ HPCs, stem cells, as the source of iPSCs. Somatic cells such as T lymphocytes, B lymphocytes, fibroblasts, and keratinocytes are also used as alternative sources of iPSCs.

[0006] iPSCs have been shown to differentiate into various cells belonging to the three germ layers, as demonstrated by analysis of teratomas generated from human and mouse iPSCs. Additionally, the pluripotency of iPSCs has been confirmed by the contribution of iPSC-derived cells to various organs in chimeric mice developed from iPSC-transfected blastocysts.

[0007] However, in addition to issues of cell quantity and cell source, there are still significant obstacles to generating iPSC-derived hematopoietic stem and progenitor cells (iPSC-HSPCs) or to generating differentiated cells from them whose progeny will engraft in vivo. As mentioned above, various studies aimed at generating HSCs in vitro from alternative sources have produced hematopoietic progenitor or stem cells, but they do not engraft well in vivo. Summary of the Invention

[0008] overview Aspects of the present disclosure relate to methods for producing patient-specific histocompatible multipotent hematopoietic progenitor cells (MHPCs) that can be subjected to specific directed differentiation to provide functional immune cells in greater quantities than previously possible in in vitro culture conditions. Aspects of the present disclosure also relate to compositions comprising these MHPCs, and the progeny cells resulting from the specific directed differentiation process, and uses of these cells.

[0009] There is a shortage of HLA-matched functional immune cells for in vivo cell replacement therapy to treat diseases, disorders, and medical conditions, as well as for disease modeling, drug screening, and in vitro studies of hematologic disorders. Immune cells primarily differentiate from hematopoietic stem cells (HSCs), but HLA-matched HSCs are scarce. This method solves this problem by reversing the lineage potential of pre-committed non-lymphoid myeloid progenitor cells into MHPCs and subsequently specifically promoting the differentiation of hematopoietic progenitor cells (HPCs) toward the lymphoid lineage. Additionally, MHPCs with reversed lineage potential are engineered to have enhanced in vivo engraftment and reconstitution properties. This production method is useful, for example, as a cell preparation method for immunotherapy.

[0010] Abbreviations used herein: HPC = hematopoietic progenitor cells MHPC = multilineage hematopoietic progenitor cells or multipotent hematopoietic progenitor cells iPSC=induced pluripotent stem cells HSC=hematopoietic stem cells

[0011] We were able to reverse the lineage differentiation potential of non-lymphoid lineage-committed myeloid progenitor cells by introducing at least three exogenous transcription factors: ERG, HOXA9, and RORA. The resulting cells were MHPCs.

[0012] Blood cells produced during hematopoiesis are divided into three lineages: (1) erythroid cells, (2) lymphoid cells, and (3) myeloid cells. See Figure 15. Erythroid cells, including normoblasts, erythroblasts, and mature red blood cells (RBCs), are the most common type of blood cell and are the primary means of delivering oxygen from the lungs to the body's tissues. Lymphoid cells, including B cells and T cells, are a type of white blood cell that plays a critical role in the body's immune defense. Myeloid cells, including granulocytes, megakaryocytes, and macrophages, are a diverse group of cells that include other white blood cells (e.g., neutrophils, eosinophils, and basophils) and platelets.

[0013] Myeloid progenitor cells are committed to the myeloid lineage, a non-lymphoid lineage. Myeloid progenitor cells in the myeloid lineage undergo further cell division, differentiation, and maturation, and the myeloid lineage produces the following cell types: megakaryocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, and macrophages. See Figure 15. The myeloid lineage is distinct from the lymphoid lineage, which produces immune cells such as T and B lymphocytes. By further inhibiting the histone methyltransferase EZH1 in these reversed lineage MHPCs, we were able to direct the differentiation of these cells into immune cells by co-culturing with OP9-DL1 / 4 cells or by activating the Notch signaling pathway in these cells. Furthermore, we found that incorporating two additional exogenous transcription factors, DACH1 and NFIA, into these cells enhanced their lymphoid differentiation potential by co-culturing with OP9-DL1 / 4 cells or by activating the Notch signaling pathway. Furthermore, we found that incorporating two other exogenous transcription factors, SOX4 and MYB, into these cells enhanced the in vivo engraftment and reconstitution of these cells in recipient subjects.

[0014] An advantage of the disclosed protocol is that this method allows for the semi-persistent mass production of desired and specific immune cells from somatic cells, such as blood cells, immune cells, and skin cells, that can be easily harvested from a patient's body. The production of functional immune cells is not limited to the use of stem or progenitor cells obtained from the patient. The produced immune cells can then be used for immunotherapy.

[0015] Therefore, it is an object of the present disclosure to provide a method for producing immune cells, comprising the steps of reversing the lineage differentiation potential of previously non-lymphoid lineage-committed myeloid progenitor cells into MHPCs and specifically directing the reverted-lineage MHPCs to desired immune cells. Non-lymphoid lineage-committed myeloid progenitor cells can be produced from any cell in a patient's body, e.g., iPSCs generated from somatic cells. Such cells can be easily harvested from a patient's body, e.g., from a blood sample, skin sample, oral swab, etc. Non-lymphoid lineage-committed myeloid progenitor cells may be collected from a patient's bone marrow.

[0016] It is also an object of the present disclosure to provide methods for enhancing or improving the in vivo engraftment, or reconstitution, or both, of hematopoietic-related cells implanted in a subject.

[0017] It is also an object of the present disclosure to provide compositions of modified (also called engineered) cells for use in medical therapies such as in vivo cell replacement therapy, cancer immunotherapy, and for disease modeling, drug screening, and in vitro study of hematological disorders.

[0018] Thus, disclosed herein are: (1) methods for preparing engineered immune cells, such as T or B cells, comprising using exogenous copies of transcription factors to reverse the lineage potential of myeloid progenitors to HPCs and specifically committed differentiation of the reversed-lineage HPCs into immune cells; (2) engineered myeloid progenitors with a reversed lineage and increased lymphoid lineage potential; (3) compositions containing engineered myeloid progenitors with a reversed lineage including increased lymphoid lineage potential; (4) engineered myeloid progenitors and compositions thereof described herein for use in the manufacture / production of the described engineered immune cells; (5) engineered myeloid progenitors and compositions thereof described herein for use in cell replacement therapy or for the treatment of cancer, autoimmune disorders, hematological disorders or other genetic diseases and disorders; (6) pharmaceutical compositions containing engineered immune cells prepared by the methods described herein; and (7) methods for using treatments using immune cells produced using the above methods, such as bone marrow transplantation and cancer immunotherapy, autoimmune disorders, hematological disorders or other genetic diseases and disorders. The modified immune cells are mammalian cells, such as human cells.

[0019] In one aspect, the present disclosure provides modified or engineered myeloid progenitor cells with a reverted lineage, including increased lymphoid lineage differentiation potential. In one aspect, the present disclosure provides modified or engineered myeloid progenitor cells with a lineage reverted to include increased lymphoid lineage differentiation potential, produced by the methods described herein. In some aspects, the modified or engineered myeloid progenitor cells have exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA via ERA transfection. In one aspect, the modified or engineered myeloid progenitor cells further comprise exogenous gene-encoding copies of SOX4, or MYB, or both SOX4 and MYB. In another aspect, the modified or engineered myeloid progenitor cells further comprise exogenous gene-encoding copies of DACH1, or NFIA, or both DACH1 and NFIA. In some aspects, the modified myeloid progenitor cells are lineage-restricted CD34 + CD45 +Derived from myeloid progenitor cells.

[0020] In another aspect, the present disclosure provides a composition comprising the modified or engineered myeloid progenitor cells described herein.

[0021] In another aspect, the present disclosure provides modified myeloid progenitor cells and compositions thereof described herein for use in the manufacture / production of the described modified immune cells, wherein the modified myeloid progenitor cells comprise an exogenous gene-coding copy of each of the following transcription factors: ERG, HOXA9, and RORA. In one aspect, the modified or engineered modified myeloid progenitor cells further comprise an exogenous gene-coding copy of SOX4, or MYB, or both SOX4 and MYB. In another aspect, the modified or engineered modified myeloid progenitor cells further comprise an exogenous gene-coding copy of DACH1, or NFIA, or both DACH1 and NFIA.

[0022] In another aspect, the present disclosure provides modified myeloid progenitor cells and compositions thereof described herein for use in cell replacement therapy or for the treatment of cancer, autoimmune disorders, hematological disorders, or other genetic diseases and disorders, wherein the modified myeloid progenitor cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, and RORA. In one aspect, the modified or engineered modified myeloid progenitor cells further comprise an exogenous gene-encoding copy of SOX4, or MYB, or both SOX4 and MYB. In another aspect, the modified or engineered modified myeloid progenitor cells further comprise an exogenous gene-encoding copy of DACH1, or NFIA, or both DACH1 and NFIA.

[0023] Thus, in one aspect, provided herein are methods comprising: (a) generating multi-lineage hematopoietic progenitor cells (MHPCs) from myeloid progenitor cells in vitro or ex vivo; (b) inhibiting histone methyltransferase in the resulting MHPC population; and (c) differentiating the resulting MHPC population in the presence of a Notch ligand, or predetermined stromal cells, or both, to promote differentiation into lymphoid lineages. In some aspects, in vitro culturing of the cells occurs between steps (a) and (b). In some aspects, selection of the cells occurs between steps (a) and (b).

[0024] In another aspect, provided herein is a method comprising: (a) transfecting myeloid progenitor cells in vitro with exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, wherein the transcription factors are expressed in the transfected cells to produce a resulting multi-lineage hematopoietic progenitor cell (MHPC) population with myeloid and erythroid differentiation potential; (b) (i) inhibiting histone methyltransferases in the resulting MHPC population to expand lymphoid differentiation potential, or (ii) transfecting the resulting MHPC population in vitro with exogenous gene-encoding copies of DACH1 and NFIA to expand lymphoid differentiation potential, or (iii) both (i) and (ii); and (c) differentiating the resulting MHPC population in the presence of a Notch ligand or supportive stroma, or both, to promote differentiation into lymphoid lineages.

[0025] In another aspect, the disclosure provides a method for producing a myeloid progenitor cell, comprising: (a) transfecting myeloid progenitor cells in vitro with exogenous copies of each of the following transcription factors: ERG, HOXA9, and RORA, wherein the transfected transcription factors are expressed in the cells in vivo to produce a multi-lineage progenitor cell (MHPC) population with myeloid and erythroid differentiation potential; (b)(i) histone methyltransferases targeting histone proteins at H3K9 and / or H3K27 in the resulting MHPC population; and (c) differentiating the resulting MHPC population in the presence of a Notch ligand to promote differentiation into lymphoid lineages. These immune cells are genetically modified to carry exogenous copies of ERG, HOXA9, and RORA, as compared to the original myeloid progenitor cells.

[0026] In another aspect, provided herein is a method comprising the steps of: (a) contacting or introducing into a population of myeloid progenitor cells in vitro one or more vectors collectively carrying exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, for in vivo expression of the exogenous copies of the genes in the contacted cells, wherein the transfected transcription factors are expressed in vivo in the contacted cells to produce a population of multi-lineage hematopoietic progenitor cells (MHPCs) with myeloid and erythroid differentiation potential; (b) contacting the MHPCs with an inhibitor of a histone methyltransferase enzyme; and (c) contacting the MHPCs with a Notch ligand, predetermined stromal cells, or both. In some embodiments, in vitro culturing of the cells occurs between steps (a) and (b). In some embodiments, selection of the cells occurs between steps (a) and (b). In one embodiment of the method, step (c) consists of activating the Notch signaling pathway in the MHPCs by any method known in the art.

[0027] In another aspect, the present disclosure provides a method for improving in vivo engraftment (and also reconstitution) of hematopoietic stem cells in a recipient host, the method comprising: (a) generating multi-lineage hematopoietic progenitor cells (MHPCs) from myeloid progenitor cells in vitro or ex vivo; (b) inhibiting histone methyltransferases in the resulting MHPC population; and (c) transplanting the resulting MHPCs into the recipient host.

[0028] In another aspect, the present disclosure provides modified or engineered immune cells produced by the methods described herein.

[0029] In another aspect, the present disclosure provides compositions comprising modified or engineered immune cells produced by the methods described herein.

[0030] In another aspect, the present disclosure provides modified or engineered immune cells derived from a myeloid progenitor cell population, the immune cells comprising exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA. In one aspect, the modified or engineered immune cells further comprise exogenous gene-encoding copies of SOX4, or MYB, or both SOX4 and MYB. In another aspect, the modified or engineered immune cells further comprise exogenous gene-encoding copies of DACH1, or NFIA, or both DACH1 and NFIA.

[0031] In another aspect, the present disclosure provides modified or engineered immune cells derived from a myeloid progenitor cell population, the immune cells comprising an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, DACH1, and NFIA.

[0032] In another aspect, the disclosure provides modified or engineered immune cells derived from a myeloid progenitor cell population, the immune cells comprising exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, and exogenous gene-encoding copies of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or NANOG and LIN28, or four reprogramming factors: OCT4, SOX2, NANOG, and LIN28. In another aspect, the modified cells further comprise exogenous gene-encoding copies of two additional transcription factors: SOX4 and MYB. In another aspect, the modified cells further comprise exogenous gene-encoding copies of two additional transcription factors: DACH1 and NFIA.

[0033] In one aspect, the disclosure provides a composition of modified cells derived from a population of myeloid progenitor cells, wherein the modified cells comprise an exogenous copy of each of the following transcription factors: ERG, HOXA9, and RORA. In another aspect, the modified cells further comprise exogenous gene-encoding copies of two additional transcription factors: SOX4 and MYB.

[0034] In one aspect, the present disclosure provides a composition of modified cells derived from a myeloid progenitor cell population, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, and MYB.

[0035] In one aspect, the disclosure provides a composition of modified cells derived from a myeloid progenitor cell population, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, DACH1, NFIA, SOX4, and MYB.

[0036] In one aspect, the disclosure provides a composition of modified cells derived from a myeloid progenitor cell population, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, and RORA, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. An alternative combination of reprogramming factors includes these four factors: OCT4, SOX2, NANOG, and LIN28.

[0037] In one aspect, the disclosure provides a composition of modified cells derived from a myeloid progenitor cell population, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, and MYB, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. An alternative combination of reprogramming factors includes these four factors: OCT4, SOX2, NANOG, and LIN28.

[0038] In one embodiment of any of the methods, cells, or compositions described, the myeloid progenitor cells, HPCs, MHPCs, iPSCs, modified or engineered cells, or modified or engineered immune cells are mammalian cells, e.g., the immune cells are human, rat, mouse, rabbit, or hamster cells.

[0039] In one embodiment of any of the methods, cells, or compositions described, the myeloid progenitor cells, HPCs, MHPCs, iPSCs, modified or engineered mammalian cells are primate cells.

[0040] In one embodiment of any of the methods, cells, or compositions described, the myeloid progenitor cells, HPCs, MHPCs, iPSCs, modified or engineered primate cells, or immune cells are human cells.

[0041] In one embodiment of any of the described methods, cells or compositions, MHPCs are generated by introducing each of the following transcription factors: ERG, HOXA9, and RORA into myeloid progenitor cells, such as common myeloid progenitor cells (CMPs), in vitro or ex vivo, e.g., by transfecting with one or more vectors, which collectively carry exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, for in vivo expression of the transcription factors in the transfected cells.

[0042] In one embodiment of any of the described methods, cells, or compositions, MHPCs are generated by contacting a population of myeloid progenitor cells with one or more vectors, wherein the vectors collectively carry exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, for in vivo expression of the factors in the contacted cells, and the transfected transcription factors are expressed in vivo in the contacted cells. For example, a first vector carries the nucleic acid sequence of an exogenous gene-encoding copy of ERG, a second vector carries the nucleic acid sequence of an exogenous gene-encoding copy of HOXA9, and a third vector carries the nucleic acid sequence of an exogenous gene-encoding copy of RORA. Alternatively, a single vector carries all three exogenous genes encoding the ERG, HOXA9, and RORA transcription factors.

[0043] In one embodiment of any of the described methods, cells, or compositions, the method further comprises transfecting an exogenous gene-encoding copy of the transcription factor SOX4 into myeloid progenitor cells in vitro, wherein the transfected transcription factor is expressed in vivo in the transfected cells.

[0044] In one embodiment of any of the methods, cells, or compositions described, the method further comprises transfecting an exogenous gene coding copy of the transcription factor MYB into myeloid progenitor cells in vitro, wherein the transfected transcription factor is expressed in vivo in the transfected cells.

[0045] In one embodiment of any of the methods, cells, or compositions described, the myeloid lineage progenitor cells are at least CD45 + In one embodiment of any of the methods, cells, or compositions described, the myeloid lineage progenitor cells are CD34 + CD45 + In one embodiment of any of the methods, cells, or compositions described, the myeloid lineage progenitor cells are at least CD45 + and CD11b +In some embodiments, the myeloid progenitor cells are negative for lymphoid lineage markers such as IL-7Ralpha / CD127, CD3, CD4, CD8 and CD19.

[0046] In one embodiment of any of the methods, cells, or compositions described, the myeloid progenitor cells are committed to a non-lymphoid lineage.

[0047] In one embodiment of any of the methods, cells, or compositions described, the resulting MHPCs are CD34+CD38 negative / low expressing.

[0048] In one embodiment of any of the methods, cells or compositions described, the resulting MHPCs have myeloid and erythroid differentiation potential, but no lymphoid differentiation potential or very limited lymphoid differentiation potential of less than 5%.

[0049] In one embodiment of any of the methods, cells, or compositions described, the myeloid lineage progenitor cells are embryoid body-derived progenitor cells obtained from a pluripotent stem cell population.

[0050] In one embodiment of any of the methods, cells or compositions described, the pluripotent stem cell population is iPSCs or embryonic stem cells (ESCs).

[0051] In one embodiment of any of the described methods, cells, or compositions, iPSCs are produced by introducing exogenous copies of only three reprogramming factors: OCT4, SOX2, and KLF4 into mature or somatic cells in vitro or ex vivo. Alternatively, iPSCs with exogenous copies of four reprogramming factors include OCT4, SOX2, NANOG, and LIN28.

[0052] In one embodiment of any of the methods, cells, or compositions described, iPSCs harboring exogenous copies of OCT4, SOX2, and KLF4 are further introduced into the cells in vitro or ex vivo with exogenous copies of c-MYC or nanog and LIN28.

[0053] In one embodiment of any of the methods, cells, or compositions described, iPSCs are produced by introducing exogenous copies of the reprogramming factors: OCT4, SOX2, and KLF4, optionally along with c-MYC or nanog and LIN28, into mature or somatic cells in vitro or ex vivo.

[0054] In one embodiment of any of the described methods, cells, or compositions, iPSCs are produced by contacting mature cells in vitro or ex vivo with one or more vectors, wherein the vectors carry exogenous copies of reprogramming factors: OCT4, SOX2, and KLF4, optionally together with c-MYC or nanog and LIN28, collectively within the mature cells, and the reprogramming factors are expressed in vivo in the contacted mature or somatic cells.

[0055] In one embodiment of any of the methods, cells, or compositions described, the cells from which iPSCs are generated can be from any cell type, any mature or somatic cell in the donor subject. For example, the cells can be from a blood sample, or a bone marrow sample, B lymphocytes (B cells), T lymphocytes (T cells), fibroblasts, keratinocytes, etc.

[0056] In one embodiment of any of the methods, cells, or compositions described, iPSCs are produced by introducing the disclosed reprogramming factors into mature or somatic cells two or more times in vitro or ex vivo.

[0057] In one embodiment of any of the methods, cells, or compositions described, iPSCs are produced by contacting mature cells with the disclosed vector factors in vitro or ex vivo two or more times within the mature or somatic cells.

[0058] In one embodiment of any of the methods, cells, or compositions described, the Notch ligand is Delta-like-1, Delta-like-4, and immobilized Delta 1 ext-IgG, which consists of the extracellular domain of human Delta-like-1 fused to the Fc domain of human IgG1.

[0059] In one embodiment of any of the described methods, cells, or compositions, Delta-like-1 or Delta-like-4 is delivered by co-culturing MHPCs with immobilized Delta 1 ext-IgG, OP9-DL1 cells, or OP9-DL4 cells, a bone marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (Dll1).

[0060] In one embodiment of any of the methods, cells or compositions described, the Notch signaling pathway of the inhibited MHPCs is stimulated in culture.

[0061] In one embodiment of any of the methods, cells, or compositions described, the histone methyltransferase catalyzes the addition of a methyl group to histone H3 lysine residue 9 (H3K9) and / or histone H3 lysine residue 27 (H3K27).

[0062] In one embodiment of any of the methods, cells, or compositions described, the histone methyltransferase inhibitor inhibits the G9a / GLP heteromeric complex.

[0063] In one embodiment of any of the methods, cells, or compositions described, the histone methyltransferase inhibitor inhibits EZH1 (Enhancer of Zeste 1 Polycomb Repressive Complex 2 Subunit).

[0064] In one embodiment of any of the methods, cells, or compositions described, H3K9 or H3K27 histone methyltransferase is inhibited by small molecule or nucleic acid or CRISPR-mediated targeted genetic interference.

[0065] In one embodiment of any of the methods, cells, or compositions described, the H3K27 histone methyltransferase is EZH1.

[0066] In one embodiment of any of the methods, cells, or compositions described, the H3K27 histone methyltransferase is not EZH2.

[0067] In one embodiment of any of the methods, cells, or compositions described, the histone methyltransferase small molecule inhibitor is specific for EZH1 and not EZH2.

[0068] In one embodiment of any of the described methods, cells, or compositions, histone methyltransferase small molecule inhibitors include AMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin, UNC0224, UNC0631, UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343, EPZ-6438, 3-deazaneplanocin A (DZNeP)HCl, UNC1999, MM-102, SGC 0946, entacapone, EPZ015666, UNC0379, EI1, MI-2 (menin-MLL inhibitor), MI-3 (menin-MLL inhibitor), PFI-2, GSK126, EPZ004777, BRD4770, and EPZ-6438.

[0069] In one embodiment of any of the methods, cells, or compositions described, the histone methyltransferase nucleic acid inhibitor is a nucleic acid that targets the expression of a histone methyltransferase.

[0070] In one embodiment of any of the methods, cells, or compositions described, the nucleic acid inhibitor is an RNA interference inhibitor.

[0071] In one embodiment of any of the methods, cells, or compositions described, the nucleic acid is TIFF0007635191000001.tif38164.

[0072] In one embodiment of any of the modified immune cells described, the immune cell further comprises an exogenous gene coding copy of SOX4 or MYB or both SOX4 and MYB.

[0073] In one embodiment of any of the modified immune cells described, the immune cell further comprises an exogenous gene coding copy of DACH1 or NFIA or both DACH1 and NFIA.

[0074] In one embodiment of any of the methods, cells, or compositions described, the specific directed differentiation of histone methyltransferase-inhibited MHPCs comprises contacting the cells with a cytokine selected from the group consisting of IL-7, IL-2, IL-15, and IL-4.

[0075] In one aspect, provided herein is a method of cell replacement therapy or a method for treating cancer, an autoimmune disorder, a blood disease, or other genetic diseases and disorders in a subject, the method comprising: (a) providing somatic cells from a donor subject; (b) generating multilineage hematopoietic progenitor cells from myeloid progenitor cells derived from the somatic cells as described in any preceding paragraph; (c) inhibiting histone methyltransferase in the resulting multilineage hematopoietic progenitor cell population as described in any preceding paragraph; (d) differentiating the resulting multilineage hematopoietic progenitor cell population in the presence of a notch ligand or stromal cells or both to promote differentiation into lymphoid lineages as described in any preceding paragraph, and transplanting the resulting differentiated lymphoid cells into a recipient subject.

[0076] In one embodiment of the above treatment method, the host subject and recipient subject are the same individual.

[0077] In one embodiment of the above treatment methods, the host subject and recipient subject are not the same individual, but are at least HLA-compatible.

[0078] definition In one aspect, the term "hematopoietic stem cell" or "HSC" as used herein refers to a stem cell that has the capacity for self-renewal and gives rise to all blood cell types of the three hematopoietic lineages: erythroid, lymphoid, and myeloid. These cell types include the myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineage (T cells, B cells, NK cells). Human HSCs express CD34 + , CD59 + , CD90 / Thy1 + , CD38 low / - , c-kit / CD117 - / low , and Lin - Mouse HSCs are CD34 low / - , SCA-1 + , CD90 / Thy1 + / low , CD38 + , c-Kit / CD117 + , and Lin - Detecting the expression of this panel of markers allows for the isolation of specific cell populations via techniques such as fluorescence-activated cell sorting (FACS). In one aspect, the term "hematopoietic stem cells" or "HSCs" refers to cells that have the capacity for self-renewal and express the following cell surface markers: CD34+, CD59+, Thy1 / CD90 + , CD38 lo / - , CD133+, c-Kit / CD117 - / lo , and Lin - In one embodiment, the term "hematopoietic stem cell" or "HSC" refers to a stem cell that is at least CD34+. In one embodiment, the term "hematopoietic stem cell" or "HSC" refers to a stem cell that has the capacity for self-renewal and has at least CD34 + and c-kit / CD117 lo / - In one aspect, the term "hematopoietic stem cell" or "HSC" refers to a stem cell that has the capacity for self-renewal and expresses at least CD38 low / - , c-kit / CD117 - / low represents a stem cell.

[0079] As used herein, the terms "iPS cells," "iPSCs," and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent cells artificially derived from differentiated cells, e.g., somatic cells, by transfection of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. Alternative combinations of reprogramming factors include OCT4, SOX2, NANOG, and LIN28.

[0080] As used herein, the term "lineage," when used in the context of stem cell and progenitor cell differentiation and development, refers to the cell differentiation and development pathway a cell can take to become a fully differentiated cell. For example, HSCs have three hematopoietic cell lineages: erythroid, lymphoid, and myeloid, and HSCs have differentiation potential, i.e., the ability, to differentiate and develop into known terminally differentiated cell types for all three lineages. When the term "multilineage" is used, this term means that the cell can eventually differentiate and develop into known terminally differentiated cell types for more than one lineage. For example, HSCs have multilineage potential. When the term "lineage-restricted" is used, this term means that the cell can differentiate and develop into known terminally differentiated cell types for one lineage. For example, common myeloid progenitor cells (CMPs) or megakaryocytic-erythroid progenitors (MEPs) (see Figure 15) are lineage-restricted because they can differentiate and develop only into terminally differentiated cell types of the myeloid lineage, but not the lymphoid lineage. Terminally differentiated cells of the myeloid lineage include erythrocytes, monocytes, macrophages, megakaryocytes, myeloblasts, dendritic cells, and granulocytes (basophils, neutrophils, eosinophils, and mast cells); terminally differentiated cells of the lymphoid lineage include T lymphocytes / T cells, B lymphocytes / B cells, dendritic cells, and natural killer cells.

[0081] The term "progenitor cell" as used herein refers to an immature or undifferentiated cell that has the ability to subsequently mature (differentiate) into a specific cell type (fully differentiated or terminally differentiated cell), such as a blood cell, a skin cell, a bone cell, or a hair cell. Progenitor cells have a primitive cell phenotype compared to cells that can be produced by differentiation (e.g., are earlier along a developmental pathway or progression than fully differentiated cells). In many cases, progenitor cells also have a significant or very high proliferation potential. Progenitor cells can give rise to multiple distinct differentiated cell types, or a single differentiated cell type, depending on the developmental pathway and the environment in which the cells develop and differentiate. Progenitor cells can also proliferate to generate more progenitor cells that are similarly immature or undifferentiated.

[0082] As used herein, the terms "multilineage hematopoietic progenitor cells," "multipotent hematopoietic progenitor cells," and "MHPCs" are used interchangeably and refer to hematopoietic cells (blood-forming cells) that have the ability or potency to generate or differentiate into multiple types of hematopoietic lineage cells. In one aspect, the terms include "reverted multilineage hematopoietic progenitor cells" and "reverted MHPCs" as described herein. Such cells are derived from myeloid progenitor cells following in vitro or ex vivo transfection to incorporate into the cells several exogenous copies of nucleic acids encoding the transcription factors ERG, HOXA9, and RORA. In one aspect, the terms include "embryoid body-derived progenitor cells" and "EB-derived progenitor cells."

[0083] As used herein, in one aspect, the term "myeloid progenitor cells" or "myeloid precursor cells" refers to immature or undifferentiated cells that are committed to the myeloid lineage and can differentiate and develop only into terminally differentiated cell types of the myeloid lineage. Examples are CMPs, MEPs, and GMPs of the myeloid lineage. In one aspect, the term "myeloid precursor cells" or "myeloid precursor cells" refers to CD34+CD45+ cells derived from pluripotent stem cells obtained from embryoid bodies. In one aspect, the term "myeloid precursor cells" or "myeloid precursor cells" refers to cells that differentiate and develop only into granulocytes and macrophages.

[0084] The term "differentiated cell" refers to any primary cell that is not pluripotent in its native form, as that term is defined herein. The term "differentiated cell" also encompasses partially differentiated cells, such as multipotent cells (e.g., adult somatic stem cells). In some embodiments, the term "differentiated cell" also refers to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., an undifferentiated cell or a reprogrammed cell), which has undergone a cell differentiation process.

[0085] The term "differentiate" or "differentiating" in the context of cellular ontogeny is a relative term, meaning that a "differentiated cell" is a cell that has progressed further down the developmental pathway than its progenitor cell. Thus, in some embodiments, a reprogrammed cell, as defined herein, can differentiate into a lineage-restricted progenitor cell (such as a mesodermal or endodermal stem cell), which can in turn progress further down the pathway to other types of progenitor cells (such as tissue-specific progenitor cells, e.g., cardiac progenitor cells, or pancreatic progenitor cells), and then to terminally differentiated cells that fulfill roles characteristic of a particular tissue type and may or may not retain the ability to proliferate further.

[0086] The term "multipotency" when used in relation to "multipotent cells" refers to cells that can differentiate into some, but not all, of the cells derived from all three germ layers. Thus, multipotent cells are partially differentiated cells. Multipotent cells are well known in the art, and examples of multipotent cells include adult somatic stem cells such as hematopoietic stem cells and neural stem cells, hair follicle stem cells, and liver stem cells. Multipotency means that stem cells can form many cell types of a given lineage, but not cells of other lineages. For example, multipotent blood stem cells can form many different blood cell types (red blood cells, white blood cells, platelets, etc.) but cannot form neurons; cardiovascular progenitor cells (MICPs) differentiate into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types; and pancreatic-derived multipotent progenitor cell (PMP) colonies produce cell types of the pancreatic lineage (cells that produce insulin, glucagon, amylase, or somatostatin) and neural lineage (cells that are morphologically neuron-, astrocyte-, or oligodendrocyte-like).

[0087] As used herein, the term "reprogramming gene" refers to a gene whose expression contributes to the reprogramming of differentiated cells, such as somatic cells, into undifferentiated cells (e.g., cells in a pluripotent state or a partially pluripotent state, a multipotent state). Reprogramming genes can be, for example, genes encoding master transcription factors: Sox2, Oct3 / 4, Klf4, Nanog, Lin-28, c-myc, etc. The term "reprogramming factor" refers to a protein encoded by a reprogramming gene.

[0088] The term "exogenous" refers to a substance present in a cell other than its native source. As used herein, the term "exogenous" refers to a nucleic acid (e.g., a nucleic acid encoding a reprogramming transcription factor, such as Sox2, Oct3 / 4, Klf4, Nanog, Lin-28, c-myc, etc.) or protein (e.g., a transcription factor polypeptide) that has been introduced by a process involving human intervention into a biological system, such as a cell or organism, in which the nucleic acid or protein is not normally found or is found in lower amounts. A substance (e.g., a nucleic acid encoding a sox2 transcription factor, or a protein, such as a SOX2 polypeptide) is considered exogenous if it was introduced into a cell or an ancestor of a cell that inherits the substance.

[0089] As used herein, the term "isolated" indicates that the cells are placed in a state other than their natural environment. The term "isolated" does not exclude the subsequent use of these cells in combination or admixture with other cells.

[0090] As used herein, the term "expanding" refers to increasing the number of similar cells via cell division (mitosis). The terms "multiplying" and "expanding" are used interchangeably.

[0091] As used herein, "cell surface marker" refers to any molecule expressed on the cell surface. Cell surface expression usually requires that the molecule possess a transmembrane domain. Some molecules not normally found on the cell surface can be engineered to be expressed on the cell surface using recombinant techniques. Many natural cell surface markers are termed "CD" or "cluster of differentiation" molecules. Cell surface markers often provide antigenic determinants to which antibodies can bind. A cell surface marker of particular relevance to the methods described herein is CD34. Useful hematopoietic progenitor cells according to the present disclosure preferably express CD34, or in other words, these cells are CD34 positive.

[0092] Cells can be referred to as "positive" or "negative" for any cell surface marker, and both such designations are useful for practicing the methods described herein. A cell is considered "positive" for a cell surface marker if it expresses the marker on its surface in an amount sufficient to be detected using methods known to those skilled in the art, such as contacting the cell with an antibody that specifically binds to the marker, followed by flow cytometric analysis of the cells after such contact to determine whether the antibody binds to the cell. It should be understood that, although a cell may express messenger RNA for the cell surface marker, the cell must express the marker on its surface to be considered positive for the methods described herein. Similarly, a cell is considered "negative" or "negative / low expressing" (abbreviated as "- / lo" or "lo / -") for a cell surface marker if it does not express the marker on its surface in an amount sufficient to be detected using methods known to those skilled in the art, such as contacting the cell with an antibody that specifically binds to the marker, followed by flow cytometric analysis of the cells after such contact to determine whether the antibody binds to the cell. In some embodiments in which agents specific for cell surface lineage markers are used, the agents can all contain the same label or tag, such as a fluorescent tag, thus eliminating or removing all cells positive for that label or tag, leaving untouched hematopoietic stem or progenitor cells for use in the methods described herein.

[0093] The term "histone methyltransferase inhibitor" or "inhibitor" used herein refers to any molecule that inhibits the expression of histone methyltransferase (e.g., G9a, GLP, EZH1) or inhibits the catalytic activity of the enzyme that methylates the lysine residue on substrate histone protein.For example, histone methyltransferase inhibitor can be siRNA or dsRNA that inhibits the expression of G9a, GLP, or EZH1 in inhibited cells, or gRNA that promotes the degradation of G9a, GLP, or EZH1 mRNA in inhibited cells.For example, histone methyltransferase inhibitor is a small molecule that antagonizes enzyme activity. Examples include, but are not limited to, the small molecules described herein: AMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin, UNC0224, UNC0631, UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343, EPZ-6438, 3-deazaneplanocin A (DZNeP)HCl, UNC1999, MM-102, SGC0946, entacapone, EPZ015666, UNC0379, EI1, MI-2 (menin-MLL inhibitor), MI-3 (menin-MLL inhibitor), PFI-2, GSK126, EPZ004777, BRD4770, and EPZ-6438.

[0094] As used herein, the term "small molecule" refers to chemical agents including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams / mole (i.e., including heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams / mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams / mole, organic or inorganic compounds having a molecular weight of less than about 500 grams / mole, as well as salts, esters, and other pharmaceutically acceptable forms of such compounds. In some embodiments, the small molecule is a heteroorganic or organometallic compound.

[0095] The term "inhibitory RNA" is meant to include nucleic acid molecules that contain a sequence complementary to a target nucleic acid (e.g., a target microRNA) that mediates a reduction in the level or activity of the target nucleic acid. Non-limiting examples of inhibitory RNA include interfering RNA, shRNA, siRNA, ribozyme, antagomir, and antisense oligonucleotide. Methods for producing inhibitory RNA are described herein. Additional methods for producing inhibitory RNA are known in the art. In one embodiment, the BCL11A microRNA described herein is an inhibitory RNA that causes a reduction in the activity of BCL11A mRNA.

[0096] As used herein, " interfering RNA " refers to any double-stranded or single-stranded RNA sequence that can directly or indirectly (i.e., by being converted) mediate RNA interference, thereby inhibiting or down-regulating gene expression.Interfering RNA includes, but is not limited to, small interfering RNA (" siRNA ") and short hairpin RNA (" shRNA ")." RNA interference " refers to the selective degradation of sequence-compatible messenger RNA transcripts.

[0097] As used herein, " shRNA " (short hairpin RNA) refers to an RNA molecule that comprises an antisense region, a loop portion, and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a double-stranded stem. Following post-transcriptional processing, short hairpin RNA is converted into small interfering RNA by a cleavage event mediated by the enzyme Dicer, a member of the RNase III family. As used herein, the phrase "post-transcriptional processing" refers to mRNA processing that occurs after transcription, for example, mediated by the enzyme Dicer and / or Drosha.

[0098] As used herein, "small interfering RNA" or "siRNA" refers to any small RNA molecule that can inhibit or downregulate gene expression by mediating RNA interference in a sequence-specific manner. Small RNAs can be, for example, about 18 to 21 nucleotides in length. Each siRNA duplex is formed by a guide strand and a passenger strand. The endonuclease, Argonaute 2 (Ago 2), catalyzes the unwinding of the siRNA duplex. After unwinding, the guide strand is incorporated into the RNA interference specificity complex (RISC), while the passenger strand is released. RISC uses the guide strand to find mRNA with a complementary sequence, resulting in endonucleolytic cleavage of the target mRNA.

[0099] Retroviruses are RNA viruses that utilize reverse transcriptase during their replication cycle. The term "retrovirus" refers to any known retrovirus (e.g., c-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), and spumaviruses.

[0100] Retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus can be integrated into the chromosomes of infected cells, where it is called a "provirus." The provirus serves as a template for RNA polymerase II, which directs the expression of RNA molecules encoding the structural proteins and enzymes necessary to produce new viral particles.

[0101] A structure called a "long terminal repeat" or "LTR" is present at each end of the provirus. The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA, which, in its natural sequence context, is a direct repeat and contains the U3, R, and U5 regions. LTRs generally provide functions fundamental to retroviral gene expression and viral replication (e.g., promotion, initiation, and polyadenylation of gene transcripts). LTRs contain multiple regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. Viral LTRs are divided into three regions, designated U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is a sequence between the primer binding site and the R region and contains a polyadenylation sequence. The R (repeat) region is adjacent to the U3 and U5 regions. LTRs, consisting of the U3, R, and U5 regions, appear at both the 5' and 3' ends of the viral genome. In one embodiment of the present invention, the promoter in the LTR, including the 5'LTR, is replaced with a heterologous promoter. Examples of heterologous promoters that can be used include, for example, the spleen focus-forming virus (SFFV) promoter, the tetracycline-inducible (TET) promoter, the β-globin locus control region and β-globin promoter (LCR), and the cytomegalovirus (CMV) promoter.

[0102] The term "lentivirus" refers to a group (or genus) of retroviruses that cause slow-onset diseases. Viruses in this group include HIV (human immunodeficiency virus; including HIV types 1 and 2), the etiological agent of human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep; caprine arthritis-encephalitis virus, which causes immunodeficiency, arthritis, and encephalitis in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia and encephalitis in horses; feline immunodeficiency virus (FIV), which causes immunodeficiency in cats; bovine immunodeficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalitis in subhuman primates. Diseases caused by these viruses are characterized by long incubation periods and a protracted course. Typically, viruses latently infect monocytes and macrophages, which then transmit the virus to other cells. HIV, FIV, and SIV also readily infect T lymphocytes, or T cells.

[0103] The term "R region" refers to the region within a retroviral LTR that begins at the beginning of the capping group (i.e., transcription start) and ends just before the beginning of the polyA tract. The R region is also defined as flanking the U3 and U5 regions. The R region plays an important role in allowing the movement of nascent DNA from one end of the genome to the other during reverse transcription.

[0104] The term "promoter / enhancer" refers to a DNA segment containing a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous," "exogenous," or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is placed proximal to a gene by genetic engineering (i.e., molecular biological techniques), thereby directing transcription of that gene by the linked enhancer / promoter.

[0105] The "nucleic acid" referred to herein can be RNA or DNA, can be single- or double-stranded, and can be selected from the group including, for example, nucleic acids encoding proteins of interest, oligonucleotides, nucleic acid analogs, such as peptide-nucleic acids (PNAs), pseudo-complementary PNAs (pc-PNAs), and locked nucleic acids (LNAs). Such nucleic acid sequences include, for example, nucleic acid sequences encoding proteins, such as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, including but not limited to, nucleic acid sequences acting as RNAi, shRNAi, siRNA, microRNAi (miRNA), and antisense oligonucleotides.

[0106] As used herein, the term "engraftment" in relation to a recipient host refers to the time when new blood-forming cells begin to develop that give rise to healthy blood stem cells that are derived from the implanted cells and appear in the recipient's blood at least 10 days after implantation. Engraftment can occur as early as 10 days after transplantation, but is more common between about 14 and 20 days.

[0107] As used herein, the term "reconstitution" in reference to the immune or hematologic systems in a recipient host refers to the restoration of a native reservoir or working system, or portion thereof, in the body of the recipient host to a natural or functional state, such as in bone marrow after chemotherapy, which has been depleted of bone marrow stem cells. [Brief explanation of the drawings]

[0108] [Figure 1]Figures 1A–1F summarize the in vitro screening for epigenetic modifiers that limit definitive lymphoid differentiation potential. Figure 1A shows a schematic diagram of embryoid body (EB) differentiation of human iPSCs into hematopoietic progenitors. EBs cultured in serum, BMP4, and hematopoietic cytokines were dissociated after 14 days. CD34+ progenitor cells were isolated by MACS sorting and transduced with HOXA9, ERG, RORA, SOX4, and MYB (5F) in a doxycycline (Dox)-inducible lentiviral vector. 5F cells were then transduced with individual shRNAs targeting each epigenetic modifier and then seeded on OP9-DL1 stromal cocultures in 96-well plates to induce T cell differentiation. Dox was added to the cultures for 20 days to sustain transgene expression and then removed. T cell differentiation potential was assessed by flow cytometry on day 35. Figure 1B shows a Venn diagram summarizing candidate hits from two independent experiments using two different iPSC lines, CD45-iPSCs and MSC-iPSCs. Screening was performed by transduction with 5F followed by shRNA co-infection, and then transduced cells were co-cultured with OP-DL1 stroma. The top candidates from the screen are listed. Each candidate was scored as a hit if at least two of the four shRNAs produced CD4+CD8+ T cells at higher frequencies and absolute cell numbers compared to a control shRNA targeting luciferase (shLUC). Figure 1C shows the relative expansion of 5F+shRNA cells after 14 days of respecification in +Dox cultures. Figure 1D shows a prospective analysis of CD4+CD8+ T cell frequencies from 5F+shRNAs targeting the indicated epigenetic modifiers. Figure 1E shows a prospective analysis of CD19+ B cell frequencies from the indicated 5F+shRNA cells. Figure 1F shows the expansion and differentiation potential of 5F+shEZH1 after long-term in vitro culture. 5F+shEZH1 cells were maintained in +Dox cultures for a typical 14-day respecification period (approximately 102-fold expansion) and for an additional 6 weeks (approximately 104-fold expansion), and then plated into OP9-DL1 stromal cocultures.Representative flow cytometry analysis of the T cell differentiation potential of 5F+shLUC and 5F+shEZH1 cells after long-term culture and differentiation (13 weeks) is shown. [Figure 2] Figures 2A-2F collectively demonstrate that suppression of EZH1 abrogated lymphoid bipotentiality and had minimal effect on myeloid-erythroid differentiation. Figure 2A shows flow cytometry analysis of CD4+CD8+ T cell development in 5F cells using two different shRNAs targeting luciferase (shLUC) or EZH1 (shEZH1). Cells were evaluated 35 days after coculture with OP9-DL1. Figure 2B shows that knockdown of EZH1 strongly promotes B cell (CD19+) differentiation potential in iPSC-derived 5F cells, as assessed by flow cytometry. Figure 2C shows that myeloid (CD11b) cell differentiation is intact in 5F+shEZH1 cells. Figure 2D shows that erythroid (CD71+GLYA+) cell differentiation is intact in 5F+shEZH1 cells. Figure 2E shows quantification of the T cell differentiation potential of 5F+shEZH1 compared to 5F+shLUC cells. Graphs are shown as the mean ± SEM of five independent replicates using CD34-iPS, CH45-iPS, and MSC-iPS lines. ***p<0.001. Figure 2F shows the colony-forming ability of 5F+shLUC or 5F+shEZH1 cells plated in the absence of Dox. (Top panel) Representative images of CFU-G, CFU-M, CFU-GM, CFU-GEMM, and CFU-E colonies on plates in the absence of Dox. (Bottom left panel) Representative images of 5F+shLUC or 5F+shEZH1 plates. (Bottom right panel) Quantification of the colony-forming ability of 5F+shLUC or 5F+shEZH1 cells in two independent experiments (n=2). [Figure 3]Figures 3A-3J collectively demonstrate that inhibition of canonical PRC2 subunits does not abrogate robust lymphoid differentiation potential. Figure 3A shows representative flow cytometry plots of 5F cells in which each of the indicated PRC2 subunits was knocked down using two different shRNAs. Figure 3B shows quantitative PCR analysis of mRNA knockdown efficiency of individual shRNAs. Figure 3C shows quantification of T cell frequencies from 5F cells treated with shRNAs targeting the indicated subunits, shown as the mean ± SEM of two independent experiments. Figure 3D shows a schematic diagram for rescue experiments. 5F cells are GFP+, and shRNAs are selectable with puromycin. 5F+shEZH1 cells were transduced with mouse EZH1 ORF (mEzh1) or catalytic SET domain-deleted mEzh1 (mEzh1ΔSet), both marked by mCherry fluorescence. GFP+, puro-resistant, mCherry+ cells were sorted and seeded into OP9-DL1 stromal cocultures for T cell differentiation. Figure 3E shows representative flow cytometry plots from the rescue experiment detailed in Figure 3D. All plots were gated on CD45+. Figure 3F shows quantification of the flow cytometry analysis in Figure 3E, with data presented as the mean ± SEM of two independent experiments. Figure 3G shows a dose-dependent decrease in EZH2 and EZH1 enzymatic activity with increasing GSK126 concentrations, as monitored by total H3K27me3 protein levels in 5F cells. At 3 μM, total H3K27me3 protein levels begin to decrease compared to DMSO controls, indicating an effective dose for EZH2 and EZH1 inhibition. Figure 3H shows flow analysis of T cell differentiation potential after treatment of 5F-free CD34+ d9 hemogenic endothelial (HE) cells with increasing doses of GSK126. Figure 3I shows representative images of colony assays in which 5F cells were plated after treatment with the indicated GSK126 concentrations. Figure 3J shows quantification of colonies in (G) as ±SEM of duplicates. [Figure 4-1]Figures 4A–4H summarize the gene expression and chromatin accessibility of definitively respecified progenitor cells. Figure 4A shows that 104 genes were significantly upregulated and 49 genes were significantly downregulated (>2-fold; t-test, p<0.1) when EZH1 was knocked down compared to control knockdown in 5F cells. Figure 4B shows GO analysis of the most significantly upregulated genes in Figure 4A. Figure 4C shows GSEA analysis of human HSC and progenitor signatures in 5F+shEZH1 cells compared to 5F+shLUC cells. HSC_MLP, MLP, and GMP signatures are significantly enriched in 5F+shEZH1 cells (FDR<0.25). Figure 4D shows a plot of all ATAC-seq peaks in 5F+shEZH1 and 5F+shLUC cells. Figure 4E shows GO analysis of enriched pathways in regions associated with upregulated ATAC-seq peaks. Figure 4F shows a comparison of genomic regions associated with upregulated ATAC-seq peaks with GRN and HSPC signatures for HSPCs, T, and B cells. *p<0.05. Figure 4G shows GO analysis of enriched pathways in regions associated with downregulated ATAC-seq peaks. [Figure 4-2] Figures 4A-4H summarize gene expression and chromatin accessibility in definitively respecified progenitors. Figure 4H shows a comparison of genomic regions associated with upregulated ATAC-seq peaks with GRN and HSPC signatures in HSPCs, T, and B cells. *p<0.05. [Figure 5]Figures 5A–5H collectively demonstrate that EZH1 directly binds to and regulates HSC and lymphoid gene networks. Figure 5A shows that V5 epitope-tagged EZH1 or EZH2 were overexpressed in 5F cells and subjected to ChIP-seq analysis. ChIP-seq peaks were defined within the proximal promoter region (-1 to +1 kb of the TSS). EZH1 and EZH2 ChIP-seq peaks were overlaid to identify unique EZH1-bound promoters. Figure 5B shows ChIP-seq density heat maps for H3K4me3, H3K27me3, EZH1, and EZH2. Figure 5C shows the ratio of histone marks associated with EZH1 and EZH2 promoters. Figure 5D shows mRNA expression heat maps of 152 unique EZH1-bound TFs out of a total of 1,069 genes and their regulated networks. Figure 5E shows that significantly upregulated networks of EZH1-binding TFs (FDR<0.25) are enriched in the GRNs of HSPCs, B, and T cells. Figure 5F shows that EZH1-binding TFs are specifically expressed in the HSC, MLP, and pro-B cell populations of the HSPC hierarchy. Figure 5G shows the enrichment of EZH1-binding genes into each population of the HSPC hierarchy (left) and the disruption of their associated histone marks (right). Figure 5H shows that EZH1-binding bivalent genes are highly expressed in the B, T, NK, granulocyte, and monocyte lineages. [Figure 6]Figures 6A-6N collectively demonstrate that Ezh1 deficiency enhances the lymphoid differentiation potential and engraftment of embryonic hematopoietic stem / progenitor cells. Figure 6A shows representative images of E9.5 embryo bodies (top) and yolk sacs (bottom). Figure 6B shows representative flow plots of T cell analysis from E9.5 WT or Ezh1- / - EP and YS. YS and EP were dissociated into single cells and plated in OP9-DL1 stromal cocultures supplemented with 5 ng / ml IL-7 and 5 ng / mL FLT3. After 12 days of stromal coculture, cells were harvested and analyzed for T cell development using the markers CD4 and CD8. All plots were gated on CD45. Figure 6C shows representative flow plots of TCRγδ and TCRβ from WT or Ezh1- / - EP and YS. Figure 6D shows quantification of the ratio of CD4+CD8+ T cells or TCRβ to TCRγδ from Ezh1- / - YS compared to WT from three independent experiments. Figure 6E shows representative images of E10.5 embryos. Figure 6F shows quantitative PCR of each PRC2 subunit in the YS and AGM from E10.5 WT embryos as the mean ± SEM of three replicates. Figure 6G shows sublethally irradiated adult NSG females intravenously transplanted with 3.5ee of whole E10.5 AGM. Mice were bled retro-orbitally every 4 weeks, and donor chimerism was monitored for up to 16 weeks after transplantation. Each dot represents one transplant recipient. Figure 6H shows the lineage distribution of the engrafted mice in Figure 6G. Figure 6I shows sublethally irradiated adult NSG females transplanted with 5ee of whole E10.5 YS via tail vein injection. Figure 6J shows the lineage distribution of the engrafted mice in (Figure 6I). Figure 6K shows whole marrow from the primary recipients in Figure 6G transplanted into secondary recipients 24 weeks after primary transplantation. Two to five primary recipients from each group were sacrificed and 4 x 106 whole marrow cells were transplanted into one to three secondary recipients. Figure 6L shows the lineage distribution of the secondary recipients in Figure 6K. Figure 6M shows the secondary transplant of the primary recipients in Figure 6I. Figure 6N shows the lineage distribution of the secondary recipients in Figure 6M. *p<0.05, **p<0.01, NE = no engraftment. [Figure 7]Figures 7A-7B summarize our screen for epigenetic modifiers that can limit T cell differentiation potential. Figure 7A shows candidate chromatin factors. Four shRNAs targeting each factor were used in the screen. Figure 7B shows representative flow plots demonstrating the T cell differentiation potential of 5F cells in which each of the top candidate factors was knocked down with shRNA. [Figure 8] Figure 1 shows significantly enriched GSEA networks. Pathways that were statistically significantly up- or downregulated 4, 14, or 28 days after knockdown of EZH1 in 5F cells, as assessed by RNA sequencing. [Figure 9A] Figures 9A-9C summarize the ATAC-sequencing analysis of 5F+shEZH1 cells compared to 5F+shLUC cells. Figure 9A: GO analysis of enriched pathways of nearest neighbor genes associated with upregulated ATAC-seq peaks. [Figure 9B] Figures 9A-9C summarize the ATAC-sequencing analysis of 5F+shEZH1 cells compared to 5F+shLUC cells. Figure 9B: GO analysis of nearest neighbor genes associated with upregulated ATAC peaks. [Figure 9C] Figures 9A-9C summarize the ATAC-sequencing analysis of 5F+shEZH1 cells compared to 5F+shLUC cells. Figure 9C: Comparison of up- and down-regulated ATAC-seq peaks in 5F+shEZH1 cells with GRN and HSPC hierarchical signatures of HSPC, B, and T cells. [Figure 10] Figures 10A-10F summarize the characterization of adult Ezh1-deficient mice. Figure 10A shows quantification of LSK SLAM HSCs in adult bone marrow. Figure 10B shows the lineage distribution of WT, Ezh1+ / -, and Ezh1- / - adult mice (8-12 weeks old). n=3 mice per genotype. Figure 10C shows WBC counts in peripheral blood. Figure 10D shows lymphocyte counts in peripheral blood. Figure 10E shows absolute cell counts in the thymus. Figure 10F shows representative images of two thymuses from WT, Ezh1+ / -, and Ezh1- / - mice. ****P<0.0001. [Figure 11] Figures 11A-11C summarize the lineage analysis of hematopoietic populations at E9.5 YS. Figure 11A shows the gating scheme for the (embryonic) EP and (yolk sac) YS. Figure 11B shows a representative flow plot (left) and quantification from two replicates (right) of B cells in the EP and YS from pooled embryos. Figure 11C shows a representative flow plot (left) and quantification from two replicates (right) of T cells in the EP and YS from pooled embryos. [Figure 12] Figures 12A-12C summarize the in vitro B cell differentiation potential of E9.5 EP and YS. Figure 12A shows representative flow plots of B1 and B2 progenitor cell frequencies after 9 days of differentiation on OP9-DL stroma. Figure 12B shows representative images of CD19+ B cells isolated from (Figure 13A) (left) and after 4 days (right) in conditions promoting class switch recombination. Figure 12C shows flow analysis of class switch recombination efficiency. [Figure 13] Figures 13A-13B collectively demonstrate that Ezh1-deficient embryonic HSPCs contribute to adult lymphopoiesis in vivo. Figure 13A shows a representative flow analysis of B1 and B2 progenitor cells in the peritoneal cavity of an engrafted primary recipient. Figure 13B shows a representative flow analysis of TCRβ and TCRγδ frequencies in donor-derived peripheral CD3+ T cells from an engrafted primary recipient. [Figure 14] Figures 14A-14D collectively show the EZH1 regulatory networks shared between mouse and human HSPCs. Figure 14A shows seven significantly up-regulated pathways that were shared between all mouse and human Ezh1-deficient HSPCs. Figure 14B shows three significantly down-regulated pathways that were shared between all mouse and human Ezh1-deficient HSPCs. Figure 14C shows the number of genes in each GSEA network that were shared between human 5F+shEZH1 and mouse HSPCs sorted from the indicated tissues / genotypes. Figure 14D shows the GO analysis of all shared genes in Figure 15C. [Figure 15]Schematic of hematopoiesis for multipotent hematopoietic stem cells. Hematopoiesis is the process of creating new blood cells in the body. The schematic shows the different lineages into which stem cells can differentiate. [Figure 16] FIG. 1 is a schematic diagram showing the production of various myeloid, erythroid, and immune cell types from pluripotent hematopoietic stem cells. [Figure 17] Figures 17A-17F show that NFIA and DACH1 are involved in lymphoid lineage development from hPSCs. Figure 17A shows a Venn diagram of 23 candidate transcription factors (TFs) specifically expressed in HSCs, downregulated in EB-derived CD34+ progenitor cells, and not induced in ERG, HOXA9, and RORA-transduced hematopoietic progenitor cells. Figure 17B shows that a new library of 23 TFs and 5 TFs (HOXA9, ERG, RORA, SOX4, and MYB) was cloned into a doxycycline-inducible lentiviral vector, transduced into EB-derived CD34+ hematopoietic progenitor cells, plated, and subjected to colony assays. Colonies were picked and analyzed for TF integration by genomic PCR using gene-specific primers. Figure 17C shows a schematic for assessing the lymphoid lineage differentiation potential of EB-CD34+ cells transduced with 28 or 13 TF subsets. Thirteen TFs (including 5 TFs) with the highest integration frequency were selected to evaluate their T and B cell differentiation potential in stromal coculture. Figure 17D shows flow cytometry analysis of the T cell differentiation potential of 28 TFs or 13 TFs in two different IPS lines (MSC-IPS1, CD34-IPS). Figure 17E shows flow cytometry analysis of the B cell differentiation potential of 28 TFs or 13 TFs in two IPS lines. All 13 TFs are sufficient to demonstrate T and B cell differentiation potential. Figure 17F shows a depletion strategy to determine the minimum TF requirements for multilymphoid lineage differentiation potential. Flow cytometry analysis of the lymphoid differentiation potential of EB-derived CD34+ cells transduced with 13 TFs or with one TF subtracted at a time. In addition to ERG and RORA, NFIA and DACH1 are required for both T and B cell differentiation potential. DETAILED DESCRIPTION OF THE INVENTION

[0109] Detailed Description Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by experts in the technical field to which this disclosure belongs.It should be understood that this disclosure is not limited to the specific methodology, protocols, and reagents described herein, and therefore may be modified.The terms used herein are only for describing specific embodiments and are not intended to limit the scope of the present invention, which is defined solely by the claims.

[0110] Definitions of common terms in molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3) or the 2015 digital online version at merkmanuals.com; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999–2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entirety. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0111] Unless otherwise noted, this disclosure is based on, for example, Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.), Current Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998), Methods in Molecular biology, Vol.180, Transgenesis Techniques by Alan R. Clark editor, second edition, 2002, Humana Press, and Methods in Meolcular Biology, Vo.203, 2003, Transgenic Mouse, edited by Marten H. Hofker and Jan van Deursen, all of which are incorporated herein by reference in their entireties.

[0112] Other than in the examples, or unless otherwise specified, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as being modified in each instance by the term "about." When used in connection with percentages, the term "about" means ±1%.

[0113] All patents and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, methodology described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0114] The disclosure herein, in preferred aspects, does not relate to processes for cloning humans, processes for altering the genetic identity of the human germ line, the use of human embryos for industrial or commercial purposes, or processes for altering the genetic identity of animals that may cause suffering to humans or animals without substantially providing any medical benefit to them, nor to the animals resulting from such processes.

[0115] The present disclosure relates to in vitro or ex vivo methods for producing functional immune cells from progenitor cells with little or no lymphoid differentiation potential. For example, myeloid progenitor cells lack lymphoid differentiation potential and do not proliferate and differentiate into lymphoid cells such as natural killer lymphocytes, dendritic cells, T lymphocytes, and B lymphocytes. Myeloid progenitor cells are committed to the myeloid lineage, and they undergo further cell division, differentiation, and maturation to produce the following cell types: megakaryocytes, platelets, granulocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, and macrophages. See Figure 15. Functional immune cells derived from non-lymphoid progenitor cells are modified to carry exogenous DNA copies encoding certain transcription factors. In one embodiment, patient-specific functional immune cells can be produced by this method. The cells are functional because they express T or B cell specific markers and also undergo T cell receptor (TCR) gene rearrangement.

[0116] Thus, in one aspect, provided herein are in vitro or ex vivo methods comprising: (a) generating multilineage hematopoietic progenitor cells (MHPCs) from myeloid progenitor cells; (b) inhibiting histone methyltransferase in the resulting MHPC population; and (c) differentiating the resulting MHPC population in the presence of a Notch ligand or selected stromal cells, or both, to promote differentiation into lymphoid lineages. In some embodiments, in vitro culturing of cells occurs between steps. In some embodiments, cell culturing serves to expand the number of cells of interest at each step before performing the next step of the method. In some embodiments, cell selection occurs between steps.

[0117] In another aspect, provided herein are methods comprising: (a) transfecting myeloid progenitor cells in vitro with exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, wherein the transcription factors are expressed in the transfected cells to produce a resulting MHPC population with both myeloid and erythroid differentiation potential; (b) inhibiting histone methyltransferase in the resulting MHPC population to expand its lymphoid differentiation potential; and (c) differentiating the resulting MHPC population in the presence of a Notch ligand or supporting stroma, or both, to promote differentiation into lymphoid lineages. In some embodiments, in vitro culture of the cells occurs between each step before performing the next step of the method. In some embodiments, cell selection occurs between steps. In some embodiments, culture serves to expand the number of cells of interest.

[0118] In another aspect, the present disclosure provides a method for generating modified immune cells or modified hematopoietic progenitor cells (HPCs) from a myeloid progenitor cell population, the method comprising: (a) transfecting myeloid progenitor cells in vitro with exogenous copies of each of the following transcription factors: ERG, HOXA9, and RORA, wherein the transfected transcription factors are expressed in the cells in vivo to produce a MHPC population with myeloid and erythroid differentiation potential; (b) inhibiting a histone methyltransferase that methylates histone 3 lysine 9 or lysine 27 residues (H3K9 or H3K27, or both) of histones in the resulting MHPC population; and (c) differentiating the resulting MHPC population in the presence of a Notch ligand to promote differentiation into lymphoid lineages. These immune cells are genetically modified. In some embodiments, in vitro culture of the cells occurs between steps. In some embodiments, selection of the cells occurs between steps. In some embodiments, culturing serves to expand the number of cells of interest at each step before performing the next step of the method.

[0119] In another aspect, provided herein are methods comprising the steps of: (a) introducing or contacting a population of myeloid progenitor cells with one or more vectors collectively carrying exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, for in vivo expression in the contacted cells, wherein the transfected transcription factors are expressed in vivo in the contacted cells to produce a population of MHPCs with myeloid and erythroid differentiation potential; (b) contacting the MHPCs with a histone methyltransferase inhibitor; and (c) contacting the MHPCs with a Notch ligand, predetermined stromal cells, or both. In some embodiments, in vitro culture of the cells occurs between steps. In some embodiments, selection of the cells occurs between steps. In some embodiments, culture serves to expand the number of cells of interest at each step before performing the next step of the method.

[0120] In another aspect, the present disclosure provides a method for improving in vivo engraftment of hematopoietic stem cells (HSCs) or HPCs in a recipient host, comprising: (a) generating MHPCs from myeloid progenitor cells; (b) inhibiting histone methyltransferases in the resulting MHPC population; and (c) transplanting the resulting MHPCs into the host. In some embodiments, in vitro culturing of the cells occurs between steps. In some embodiments, cell selection occurs between steps. In some embodiments, culturing serves to expand the number of cells of interest at each step before performing the next step of the method. Myeloid progenitor cells have no or limited lymphoid differentiation potential. In one aspect, co-culturing myeloid progenitor cells in OP9-DL1 / 4 cells enhances the proliferation and differentiation of HSCs and HPCs, thereby improving the survival and survival of HSCs and HPCs. + / CD8 + No cells are produced.

[0121] In another aspect, the present disclosure provides modified or engineered immune cells produced by the methods described herein, which immune cells are genetically modified to have exogenous copies of ERG, HOXA9, and RORA compared to the original myeloid progenitor cells.

[0122] In another aspect, the present disclosure provides a composition comprising an engineered immune cell produced by the methods described herein. In one aspect, the composition further comprises a pharmacologically acceptable carrier. In one aspect, the pharmacologically acceptable carrier is not a cell culture medium.

[0123] In one aspect, the present disclosure provides modified myeloid progenitor cells with reverted lineage to include increased lymphoid lineage differentiation potential.

[0124] In one aspect, the present disclosure provides compositions containing modified myeloid progenitor cells that have lineage reversion to include increased lymphoid lineage differentiation potential.

[0125] In one aspect, the present disclosure provides modified myeloid progenitor cells and compositions thereof described herein for use in the manufacture / production of the described modified immune cells.

[0126] In one aspect, the present disclosure provides modified myeloid progenitor cells and compositions thereof described herein for use in cell replacement therapy or for the treatment of cancer, autoimmune disorders, hematological disorders or other genetic diseases and disorders.

[0127] In one aspect, the disclosure provides engineered immune cells derived from a myeloid progenitor cell population, the engineered immune cells comprising an exogenous gene-coding copy of each of the following transcription factors: ERG, HOXA9, and RORA. In another aspect, the immune cells consist essentially of an exogenous gene-coding copy of each of the following transcription factors: ERG, HOXA9, and RORA. In a further aspect, the immune cells consist of an exogenous gene-coding copy of each of the following transcription factors: ERG, HOXA9, and RORA. In one aspect, the immune cells are further comprised of an exogenous gene-coding copy of the following transcription factors: SOX4 or MYB or both SOX4 and MYB. In one aspect, the immune cells are further comprised of an exogenous gene-coding copy of the following transcription factors: DACH1 or NFIA or both DACH1 and NFIA.

[0128] In another aspect, the disclosure provides engineered immune cells or modified myeloid progenitor cells derived from a population of myeloid progenitor cells, the immune cells or modified myeloid progenitor cells comprising exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, and exogenous gene-encoding copies of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. Alternatively, the reprogramming factors are OCT4, SOX2, NANOG, and LIN28. In another aspect, the immune cells or modified myeloid progenitor cells consist essentially of exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, and exogenous gene-encoding copies of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. In further embodiments, the immune cells or modified myeloid progenitor cells are comprised of exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, and exogenous gene-encoding copies of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. Alternatively, the reprogramming factors introduced into the modified cells are OCT4, SOX2, NANOG, and LIN28.

[0129] In one aspect, the disclosure provides compositions of modified or engineered immune cells or modified myeloid progenitor cells derived from a myeloid progenitor cell population, wherein the modified cells comprise an exogenous copy of each of the following transcription factors: ERG, HOXA9, and RORA. In another aspect, the modified cells or modified myeloid progenitor cells consist essentially of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, and RORA. In a further aspect, the modified cells or modified myeloid progenitor cells consist of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, and RORA.

[0130] In one embodiment, the modified cells described further comprise exogenous gene-encoding copies of one or both of two additional transcription factors: SOX4 and MYB. In another embodiment, the modified cells further consist essentially of exogenous gene-encoding copies of one or both of two additional transcription factors: SOX4 and MYB. In a further embodiment, the modified cells consist of exogenous gene-encoding copies of two additional transcription factors: SOX4 and MYB.

[0131] In one embodiment, the modified cells described further comprise exogenous gene-encoding copies of one or both of two additional transcription factors: DACH1 and NFIA. In another embodiment, the modified cells further consist essentially of exogenous gene-encoding copies of two additional transcription factors: DACH1 and NFIA. In a further embodiment, the modified cells consist of exogenous gene-encoding copies of one or both of two additional transcription factors: DACH1 and NFIA.

[0132] In another aspect, the present disclosure provides modified or engineered MHPCs produced by the methods described herein. In another aspect, the present disclosure provides compositions comprising engineered MHPCs produced by the methods described herein. The engineered MHPCs have exogenous gene-coding copies of one or more of the following transcription factors: ERG, HOXA9, RORA, SOX4, MYB, DACH1, NFIA, OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28. Combinations of exogenous transcription or reprogramming factors in engineered MHPCs include ERG, HOXA9, and RORA; ERG, HOXA9, RORA, SOX4 and MYB; ERG, HOXA9, RORA, DACH1, and NFIA; ERG, HOXA9, RORA, SOX4, MYB, DACH1, and NFIA; ERG, HOXA9, RORA, OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28; ERG, HOXA9, RORA, SOX4, MYB, OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28; ERG, HOXA9, RORA, SOX4, The composition includes MYB, DACH1, NFIA, OCT4, SOX2, KLF4, and optionally c-MYC or NANOG and LIN28; ERG, HOXA9, RORA, DACH1, NFIA, OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28; ERG, HOXA9, RORA, DACH1, NFIA, OCT4, SOX2, NANOG and LIN28; ERG, HOXA9, RORA, SOX4, MYB, OCT4, SOX2, NANOG and LIN28; and ERG, HOXA9, RORA, DACH1, NFIA, SOX4, MYB, OCT4, SOX2, NANOG and LIN28. In one embodiment, the composition further includes a pharmacologically acceptable carrier. In one embodiment, the pharmacologically acceptable carrier is not a cell culture medium.

[0133] In one aspect, the disclosure provides a composition of modified cells derived from a myeloid progenitor cell population, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, and MYB. In another aspect, the modified cells further consist essentially of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, and MYB. In a further aspect, the modified cells consist of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, and MYB.

[0134] In one aspect, the disclosure provides a composition of modified cells derived from a population of myeloid progenitor cells, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, DACH1, and NFIA. In another aspect, the modified cells further consist essentially of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, DACH1, and NFIA. In a further aspect, the modified cells consist of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, DACH1, and NFIA.

[0135] In one aspect, the disclosure provides a composition of modified cells derived from a myeloid progenitor cell population, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, and RORA, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. In another aspect, the modified cells consist essentially of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, and RORA, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, c-MYC or nanog, and LIN28. In a further aspect, the modified cells consist of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, and RORA, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. Alternatively, a combination of four reprogramming factors: OCT4, SOX2, NANOG and LIN28 are present in the modified cells.

[0136] In one aspect, the disclosure provides a composition of modified cells derived from a population of myeloid progenitor cells, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, and MYB, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. In another aspect, the modified cells consist essentially of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, and MYB, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. In a further embodiment, the modified cells are comprised of exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, and MYB, and exogenous gene-encoding copies of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. Alternatively, a combination of the four reprogramming factors: OCT4, SOX2, NANOG, and LIN28 is in the modified cells.

[0137] In one aspect, the disclosure provides a composition of modified cells derived from a myeloid progenitor cell population, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, DACH1, and NFIA, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. In another aspect, the modified cells consist essentially of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, DACH1, and NFIA, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. In a further embodiment, the modified cells are comprised of exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, RORA, DACH1, and NFIA, and exogenous gene-encoding copies of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. Alternatively, a combination of the four reprogramming factors: OCT4, SOX2, NANOG, and LIN28 is in the modified cells.

[0138] In one aspect, the disclosure provides a composition of modified cells derived from a population of myeloid progenitor cells, wherein the modified cells comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, MYB, DACH1, and NFIA, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. In another aspect, the modified cells consist essentially of an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, MYB, DACH1, and NFIA, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. In a further embodiment, the modified cells are comprised of exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, RORA, SOX4, MYB, DACH1, and NFIA, and exogenous gene-encoding copies of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. Alternatively, a combination of the four reprogramming factors: OCT4, SOX2, NANOG, and LIN28 is in the modified cells.

[0139] In one aspect, the present disclosure provides a pharmacological composition comprising an engineered immune cell described herein and a pharmacologically acceptable carrier, wherein the engineered immune cell comprises an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, RORA, and optionally each of the following transcription factors: SOX4, MYB, DACH1, and NFIA. In one aspect, the pharmacologically acceptable carrier is not cell culture medium. In one aspect, the pharmacological composition is a cryopreserved composition comprising at least one cryopreservative known in the art.

[0140] Pluripotent stem cells (PSCs) have the potential to generate all somatic tissues. Directed differentiation of PSCs aims to generate patient-matched tissues by recapitulating embryonic development and specifying the three germ layers. A common theme in directed differentiation across all germ layers is the tendency of PSCs to generate embryonic and fetal-like cell types, raising questions about their integration and function in adult recipients. This feature is particularly striking in the hematopoietic system, which emerges in temporally and spatially distinct waves during ontogeny (Dzierzak and Speck, 2008). The earliest "primitive" progenitors emerge within the yolk sac at 8.5 days per century and give rise to a limited repertoire of macrophages, megakaryocytes, and nucleated erythrocytes (Baron et al., 2005, Tavian and Peault, 2005, Ferkowicz et al., 2005). These early embryonic-like progenitors are generally myeloid-based and are unable to functionally repopulate the bone marrow of adult recipients. In contrast, "committed" cells with hematopoietic stem cell (HSC) differentiation potential subsequently emerge in the arterial endothelium within the aorta-gonad-mesonephros (AGM) and other anatomical sites (Dzierzak and Speck, 2008). Directed differentiation of PSCs generates hematopoietic progenitors similar to those found in the yolk sac of the early embryo. These lack functional reconstitution potential, are biased toward the myeloid lineage, and express embryonic globins. Therefore, understanding the key fate-determining mechanisms that drive the development of either primitive or definitive lineages is crucial for specifying other adult-like cell types (e.g., erythrocytes) from HSCs and PSCs.

[0141] Activation of the Wnt pathway in early mesoderm and Notch in hemogenic endothelium is critical for promoting definitive differentiation potential (Kennedy et al. 2012, Sturgeon et al. 2013, Ditadi et al. 2015). Definitive differentiation potential is characterized by B and T lymphopoiesis. While lymphoid activity emerges before HSCs (Boeiers et al. 2013, Yoshimoto et al. 2012, Yoshimoto et al. 2011), strong B and T cell differentiation potential remains a useful marker of definitive fate in vitro. Global gene expression profiling has shed light on molecular differences among hematopoietic progenitors across ontogeny (Mckinney-Freeman et al. 2012, Miranda-Saavedra et al. 2009). Several classes of homeobox (Hox) A and B cluster genes are expressed in definitive cells but not in yolk sac cells (Sauvageau et al. 1994, McGrath and Palis 1997). Thus, overexpression of HoxB4 is sufficient to generate engraftment-competent cells from mouse PSCs. Moreover, HOXA cluster genes enhance hematopoietic lineage commitment from human PSCs (Doulatov et al. 2013, Ramos-Mejia et al. 2015, Dou et al. 2016). Despite these advances, the definitive hematopoietic differentiation potential of PSCs remains limited.

[0142] Epigenetic regulation maintains cell identity during development. Differentiation is characterized by the progressive silencing of alternative lineage programs through repressive mechanisms, including methylation of DNA and histone residues associated with heterochromatin (Dambacher et al., 2010). For example, trimethylation of histone H3 on lysine 27 (H3K27) by Polycomb Repressive Complex 2 (PRC2) is required for blood development (Majewski et al., 2008, Majewski et al., 2010, Mochizuki-Kashio et al., 2011, Hidalgo et al., 2012, Xie et al., 2014, Kinkel et al., 2015, Lee et al., 2015, Ikeda et al., 2016). We demonstrated that primitive and definitive hematopoietic programs, as well as alternative lineage fates, are co-excluded by epigenetic mechanisms. The primitive program that emerges in the yolk sac and during directed differentiation of PSCs is reinforced by a repressive mechanism that prevents master transcription factors SCL, RUNX1, GATA2, and HOXA from activating stem cell and lymphoid genes characteristic of definitive progenitors. We found that relieving this repression establishes definitive differentiation potential from PSCs in vitro and early embryonic progenitors in vivo. We report here that haploinsufficiency reduction of the Polycomb group protein EZH1 enables multily lymphoid output from PSCs in vivo and HSC emergence at sites of primitive hematopoiesis. Thus, EZH1 is a novel regulator of definitive hematopoietic differentiation potential in vitro and in vivo.

[0143] The objective of the present disclosure is to provide a solution to the lack of HLA-matched HSCs for in vivo cell replacement therapy, treatment of various medical diseases / conditions, and disease modeling, drug screening, and in vitro study of hematologic disorders, particularly HLA-matched HSCs that ultimately produce immune cells. Another objective is to enhance the engraftment and reconstitution of transplanted hematopoietic-related or hematopoietic-derived cells in a subject.

[0144] By introducing at least three transcription factors: ERG, HOXA9, and RORA, into lineage-restricted myeloid progenitor cells, we were able to reverse the lineage differentiation potential of these cells, so that the resulting cells now have the ability to proliferate and produce more progeny cells, self-renew into progenitor cells, and differentiate into cell types of more than one lineage. This step provides another source of cell types for generating lymphoid and also erythroid cells. Myeloid progenitor cells are committed to the myeloid lineage for further differentiation and maturation, which produces the following cell types: megakaryocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, and macrophages. The myeloid lineage is distinct from the lymphoid lineage, which produces immune cells such as T and B lymphocytes. See Figure 15.

[0145] We previously demonstrated the ability of lineage-restricted, abundant CD34 + CD45 +We demonstrated that myeloid progenitor cells can be generated from iPSCs. (See S. Doulatov, et al. 2013, Cell Stem Cell. 13: 459-470, which is incorporated herein in its entirety.) As previously described, human iPSCs were differentiated into embryoid bodies (EBs) in the presence of BMP4 and cytokines (Chadwick et al., 2003, Blood, 102:906-915). Briefly, iPSC colonies were scraped and placed into rotating non-adherent 10 cm plates. EB medium was KO-DMEM + 20% FBS (Stem Cell Technologies), 1 mM L-glutamine, 1 mM NEAA, penicillin / streptomycin, 0.1 mM β-mercaptoethanol, 200 μg / ml h-transferrin, and 50 μg / ml ascorbic acid. After 24 hours, the medium was changed by allowing the EBs to settle by gravity and replaced with EB medium supplemented with growth factors: 50 ng / ml BMP4 (R&D Systems), 300 ng / ml SCF, 300 ng / ml FLT3, 50 ng / ml G-CSF, 20 ng / ml IL-6, and 10 ng / ml IL-3 (all Peprotech). Medium was changed on days 5 and 10. EBs were dissociated on day 14 by digestion with collagenase B (Roche) for 2 hours, followed by treatment with enzyme-free dissociation buffer (Gibco) and filtration through an 80 μm filter. Dissociated EBs can be frozen in a 10% DMSO, 40% FBS freezing solution.

[0146] EBs are three-dimensional aggregates of pluripotent stem cells generated and cultured in vitro in the presence of serum. EBs then proceed to generate a mixture of primitive and definitive hematopoietic progenitor cell types. Primitive progenitors are considered equivalent to cells that arise naturally in vivo during the earliest stages of embryonic development, while the embryonic population gives rise to definitive progenitors that behave similarly to cells typical of adult hematopoiesis at later stages of maturation. The lineage-restricted CD34 + CD45 +Myeloid progenitors appear on day 10, expand until day 14, and then express CD34 after dissociation of aggregated cells in EBs. + CD45 + These myeloid progenitors were isolated by cell sorting using surface markers. They showed strong myeloid colony-forming activity: macrophage colonies (CFU-M) and granulocyte colonies (CFU-G), but only produced a few erythroid colonies (CFU-E and BFU-E) or mixed colonies: granulocyte / erythroid / macrophage / megakaryocyte colonies (multilineage myeloid progenitors: CFU-GEMM) and granulocyte / macrophage colonies (CFU-GM). These lineage-restricted CD34 + CD45 + Myeloid progenitor cells did not have the ability to proliferate or self-renew in culture in the absence of serum. + CD45 + Progenitor cells were CD34 after 7 days of culture. - The cells were fully differentiated, consistent with their lack of clonogenic potential.

[0147] Mass-produced lineage-restricted CD34 + CD45 +From myeloid progenitor cells, the inventors demonstrated that expressing three transcription factors: ERG, HOXA9, and RORA (abbreviated herein as EAR factors) in these cells could reverse the myeloid lineage of these cells and induce cell proliferation and self-renewal. (See S. Doulatov, et al. 2013, Cell Stem Cell. 13: 459-470, which is incorporated herein in its entirety.) Briefly, open reading frames encoding the three transcription factors: ERG, HOXA9, and RORA were cloned into lentiviral vectors using LR Clonase (INVITROGEN™). Two lentiviral vectors were used: pSMAL-GFP (constitutive) and pINDUCER-21 (doxycycline-regulated) (Meerbrey et al., 2011, 108:3665-3670, which is incorporated herein in its entirety). Lentiviral particles were produced by transfecting third-generation packaging plasmids into 293T-17 cells (ATCC). Virus was harvested 12 and 36 hours post-transfection and concentrated by ultracentrifugation at 23,000 rpm for 2 hours. Constructs were titered by serial dilution on 293T cells. Sorted CD34 + CD45 + Progenitor cells were cultured on a fibronectin-coated (10 μg / cm 2 ) Cell density of 2–5 × 10 cells on a 96-well plate 4Cells were seeded at 1000 cells / well. Infection medium was IMDM + 20% BIT (StemCell Technologies), 1 mM L-glutamine, and 0.1 mM p-mercaptoethanol with 300 ng / ml SCF, 300 ng / ml FLT3, 50 ng / ml G-CSF, 20 ng / ml IL-6, and 10 ng / ml IL-3 (all Peprotech). Lentiviral infection was performed in a total volume of 150 μl. After transduction, progenitor cells were cultured in suspension in infection medium supplemented with 50 ng / ml SCF, 50 ng / ml FLT3, 50 ng / ml TPO, 50 ng / ml IL-6, and 10 ng / ml IL-3 (all R&D Systems). In all experiments using inducible constructs (including all transplantation experiments), the infection medium was replaced with StemSpan SFEM (StemCell Technologies). Dox was added at 2 ng / ml (Sigma). The culture medium was the same as above. Cultures were grown at cell densities <1 x 10 6 The cells were maintained at 1000 / ml and the medium was changed every 3-4 days. A single lentiviral system can also be used to introduce open reading frames encoding three transcription factors: ERG, HOXA9, and RORA, into selected myeloid progenitor cells. Single lentiviral expression systems and vector cassettes are known in the art, for example, as taught in U.S. Patent No. 8,865,467, the contents of which are incorporated herein by reference in their entirety. Alternatively, gene transfer can be achieved using an episomal vector. Episomal expression vector systems are known in the art. For example, as taught in U.S. Patent Nos. 5,624,820; 5,674,703; 6,339,065; 6,410,314; 6,479,279; 6,797,494; 6,808,923; 7,294,505; 7,790,446; 8,703,481; 8,187,836; and 9,068,200, the contents of which are incorporated herein by reference in their entireties.

[0148] The resulting transfected lineage-restricted CD34 + CD45 + Myeloid progenitor cells express CD34 after 7 days of culture in serum-free medium. + This CD34 + The cell population continued to expand from days 7 to 14 of culture. The production of cell populations in serum-free medium indicates the restoration of the self-renewal capacity of multilineage hematopoietic progenitor cells. + After this expansion, some of the cell populations were CD38 negative or low expressing, but CD90 + and CD49 + Multipotent hematopoietic progenitor cells found in umbilical cord blood and hematopoietic stem cells are CD34 + / CD38 - Therefore, EAR is a lineage-restricted CD34 + CD45 + By transfecting myeloid progenitor cells, a new cell population capable of proliferation and self-renewal in serum-free medium is generated using the original lineage-restricted CD34 marker used for EAR transfection. + CD45 + They also display cell surface markers characteristic of multilineage multipotent hematopoietic progenitor cells instead of myeloid progenitor cells.

[0149] Therefore, we have demonstrated that EAR transfection induces lineage-restricted CD34 + CD45 + Multilineage multipotent CD34 from myeloid progenitor cells + / CD38 lo / - It was able to produce hematopoietic progenitor cells.

[0150] In one embodiment, lineage-restricted CD34 + CD45 +Provided herein are modified myeloid progenitor cells derived from myeloid progenitor cells, the modified myeloid progenitor cells having a reverted lineage that includes increased lymphoid lineage differentiation potential. In one embodiment, the increased lymphoid lineage differentiation potential is at least 5% compared to prior EAR transfection. In other embodiments, the increased lymphoid lineage differentiation potential is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more compared to before EAR transfection.

[0151] Reverse lineage hematopoietic progenitor cells have the ability to differentiate into myeloid and erythroid lineages and generate myeloid-erythroid colonies. Furthermore, we have demonstrated that expression of two additional transcription factors, SOX4 and MYB, in progenitor cells enhances in vivo engraftment of reverse lineage hematopoietic progenitor cells and enhances the expression of CD34 in the reverse lineage. + CD45 + We found that there was an increase in the number of mixed myeloid-erythroid colonies from hematopoietic progenitor cells. In one embodiment, the increased in vivo engraftment is at least 0.1% compared to the absence of any additional transcription factor selected from the group consisting of SOX4 and MYB. In other embodiments, the increased in vivo engraftment is at least 0.2%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more compared to the absence of any additional transcription factor selected from the group consisting of SOX4 and MYB.

[0152] Additionally, the present inventors have found that two other transcription factors, DACH1 and NFIA, enhance lymphoid differentiation potential. In one embodiment, the enhanced in vivo lymphoid differentiation potential is at least 0.1% compared to the absence of any additional transcription factor selected from the group consisting of DACH1 and NFIA. In other embodiments, the enhanced in vivo lymphoid differentiation potential is at least 0.2%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more compared to the absence of any additional transcription factor selected from the group consisting of DACH1 and NFIA.

[0153] These reverted lineage multipotent CD34 + / CD38 lo / - By further inhibiting histone methyltransferase in hematopoietic progenitor cells, we were able to differentiate these cells into immune cells by co-culturing the reverted lineage multipotent hematopoietic progenitor cells with OP9-DL1 / 4 cells. OP9-DL1 / 4 cells express and secrete Notch ligand, a factor known to promote HSC differentiation into T lymphocytes. Notch ligand binds to histone methyltransferase-inhibited CD34. + / CD38 lo / - Activates the Notch signaling pathway in hematopoietic progenitor cells. Normally, in the absence of histone methyltransferase inhibitors, reverses lineage multipotency of CD34 + / CD 38 lo / -When hematopoietic progenitor cells are co-cultured with OP9-DL1 / 4 cells, approximately 0-5% of them produce colonies or cells with T cell differentiation potential. In contrast, knocking down EZH1 (e.g., by using siRNA or histone methyltransferase inhibitors) increased the incidence of T cell differentiation potential to 25-30%, an at least five-fold increase. See Figures 2A and 2B. In some embodiments, the term "OP9" cells referred to herein refers to OP9-DL1 or OP9-DL4 cells that secrete Notch ligands that activate the Notch signaling pathway.

[0154] In one embodiment of any method, cell, or composition described herein, the MHPCs exhibit an increased incidence of T cell differentiation potential compared to the absence of a histone methyltransferase inhibitor. In one embodiment, the increased incidence of T cell differentiation potential is at least 5% compared to before in the absence of a histone methyltransferase inhibitor. In other embodiments, the increased incidence of T cell differentiation potential is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more compared to before in the absence of a histone methyltransferase inhibitor.

[0155] Furthermore, the in vivo engraftment and reconstitution of these cells is enhanced by further incorporating in vivo expression of two other transcription factors: SOX4 and MYB. We also found that the lymphoid differentiation potential of these cells is enhanced by further incorporating in vivo expression of two other transcription factors: DACH1 and NFIA. See Figure 17F.

[0156] To understand the gene regulatory network of definitive lymphoid lineage development from human pluripotent stem cells (hPSCs) in parallel, we screened additional transcription factors known to be highly expressed in hematopoietic stem cells (HSCs). We selected 23 additional transcription factors (TFs) in addition to HOXA9, ERG, RORA, SOX4, and MYB, which were HSC-specific and not induced by HOXA9, ERG, and RORA, but were induced by CD34, which differentiated from embryoid bodies (EBs). + A library of 28 transcription factors was analyzed to identify CD34 + These libraries were transduced into EBs, which were plated and used for colony formation assays. Analysis of colony integration sites by PCR revealed enrichment for 13 TFs. To prospectively test these libraries, we used CD34 + EBs were transduced with 13 TF subsets or a 28-TF library, and the cells were plated on stromal co-cultures to induce T or B cell differentiation. The 13 TF library was CD34 + The 13 TF cocktail was sufficient to induce multilineage differentiation from EBs. To identify TFs required for lymphoid differentiation, we removed one TF at a time from the cocktail and performed T and B cell differentiation on transduced EBs. We found that the addition of NFIA and DACH1, along with HOXA9, ERG, and RORA, was required for T and B cell development from hPSCs.

[0157] NFI genes function as both positive and negative regulators of gene transcription. NFIA has been shown to regulate the erythroid / granulocytic lineage switch (Fazi et al., 2005, Starnes et al., 2009). DACH1 regulates cell cycle progression of myeloid cells, maintains colonogenic activity, and blocks differentiation of myeloid progenitor cells (Lee et al., 2012, Lee et al., 2012). However, their roles in hematopoietic stem cell and definitive lymphoid development have not previously been investigated. Here, we demonstrate that these TFs are part of a regulatory network required for lymphoid development from hPSCs.

[0158] An advantage of the disclosed protocol is that the method allows for the semi-permanent mass production of desired immune cells or other types of hematopoietic cells (i.e., cells differentiated from multipotent HSCs; see FIG. 15 ) from a variety of cell sources, including stem cells, hematopoietic progenitor cells, and mature and differentiated somatic cells, all of which can be easily harvested from the patient's body.

[0159] Generated engineered immune cells or histone methyltransferase-inhibited engineered CD34 + / CD38 lo / - Hematopoietic progenitor cells can be transplanted into patients for various medical treatments, such as immune system reconstitution therapy (e.g., after bone marrow ablation) or immunotherapy (e.g., in cancer therapy or autoimmune diseases). An additional advantage is that if the donor of the source cells and the recipient of the engineered immune cells are the same person, the engineered immune cells produced will have the same HLA as the recipient, which avoids host-graft immune rejection after transplantation. For recipient patients whose HLA is allogeneic with the donor of the source cells, host-graft immune rejection is greatly reduced.

[0160] The generated engineered immune cells or histone methyltransferase-inhibited engineered CD34+ / CD38- hematopoietic progenitor cells can also be cryopreserved until future need.

[0161] Currently, bone marrow transplantation is the most established cell replacement therapy for various hematological disorders. The functional unit of bone marrow transplantation is the hematopoietic stem cell (HSC), which is at the apex of a complex cellular hierarchy and replenishes blood development throughout life. 1 The scarcity of HLA-matched HSCs significantly limits the ability to perform transplantation, disease modeling, and drug screening. Thus, many studies have aimed to generate HSCs from alternative sources. Advantages of reprogramming into induced pluripotent stem cells (iPSCs) 2 The development of hematopoietic stem cells (HSCs) has provided access to a wide variety of patient-specific pluripotent cells, which are a promising source for disease modeling, drug screening, and cell therapy. However, the inability to derive engraftable hematopoietic stem and progenitor cells from human pluripotent stem cells (hPSCs) has limited the characterization of hematologic diseases to in vitro assays. The generation of HSCs by directed differentiation remains elusive, necessitating novel approaches to this problem.

[0162] One approach to generating HSCs from hPSCs is to specify HSCs from their ontogenetic progenitors. HSCs now differentiate into hemogenic endothelial (HE) cells in the aorta-gonad-mesonephros (AGM). 3 It is widely accepted that HE originates from the arterial endothelium in the endothelium of hPSCs and other anatomical sites. Recent studies on directed differentiation of HE from hPSCs have provided valuable insights into some of the signaling pathways that control the emergence of primitive or definitive populations. 4,5 however, the endothelial-hematopoietic transition remains incompletely understood in human hematopoietic development, making rational intervention challenging.

[0163] An alternative to specifying HSCs from their precursor HE is to start with short-lived progenitor cells and convert them to a stem cell state, a strategy defined as "respecification." 6 Respecification combines directed differentiation with transcription-based reprogramming to reestablish HSC fate. The molecular differences between human primary HSCs and progenitor cells have been well characterized by gene expression profiling.7,8 This study provides a rational approach to introduce stem cell genes back into progenitor cells. We were able to obtain transplantable HSCs by restoring the HSC transcription factor network in hPSC-derived primitive progenitor cells. The proof of principle for this approach is a seminal experiment demonstrating that HoxB4 can restore HSC properties in mouse primitive progenitor cells. 9 .

[0164] About Human Systems 10,11 Due to species-specific differences, different sets of factors were required to restore hHSC characteristics in human primitive progenitor cells. We created a combination of transcription factors for hPSCs. We previously reported that five transcription factors: ERG, HOXA9, RORA, SOX4, and MYB (abbreviated as 5F) could reversibly convert hPSC-derived myeloid-committed progenitors into immortalized multilineage hematopoietic progenitors. 6 Doxycycline (Dox)-regulated conditional induction of 5F expresses immature CD34 + CD38 - Immature CD34 cells expand and maintain a self-renewal state, while differentiation begins upon withdrawal of Dox. + CD38 - Self-renewal is a property of hHSCs. These cells, abbreviated as CD34-5F cells, result in short-term engraftment after transplantation into immune-deficient mice, and erythroid progenitors undergo maturation and hemoglobin switching in vivo. This system presents a useful platform for modeling hematologic disorders due to its ability to generate large numbers of engraftable disease cells for in vitro and in vivo screening.

[0165] Generation of iPSCs by somatic cell reprogramming involves global epigenetic remodeling, and chromatin-modifying enzymes have been characterized as barriers or facilitators of reprogramming. 12,13,14Within the hematopoietic system, there are many epigenetic changes that mediate blood development during ontogeny and differentiation of HSCs into mature progeny. Progression from HSCs to differentiated progeny involves coordinated regulation of gene expression programs that result in the activation or repression of lineage-specific genes. See Figure 5. The events that lead to the formation of mature lymphocytes that express antigen receptors involve the regulation of both gene expression and DNA recombination, primarily through control of chromatin accessibility. 15 The state of HSCs is controlled by numerous transcription factors and epigenetic modifiers. We used a screening strategy to identify additional factors that regulate HSC fate. We screened for transcription factors and epigenetic modifiers using an shRNA library that suppresses potential negative regulators of HSC fate.

[0166] Thus, in one embodiment of any method, cell, or composition described herein, MHPCs are generated by introducing exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA into myeloid progenitor cells in vitro. In one embodiment, a vector is used as a delivery vehicle for introducing any of the exogenous gene-encoding copies described herein into myeloid progenitor cells. For example, by transfecting one or more vectors into myeloid progenitor cells, the vectors collectively carry exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA for in vivo expression of the transcription factors in the transfected cells. For example, by contacting myeloid progenitor cells with one or more vectors, the vectors collectively carry exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA for in vivo expression of the transcription factors in the contacted cells. For example, myeloid progenitor cells are contacted with one or more nucleic acids, whereby the nucleic acids collectively carry exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, for in vivo expression of the transcription factors in the contacted cells. In one embodiment, a single vector is used as a delivery vehicle for introducing exogenous gene-encoding copies of all three transcription factors, ERG, HOXA9, and RORA, into myeloid progenitor cells. In one embodiment, one or more episomal vectors are used as a delivery vehicle for introducing exogenous gene-encoding copies of three transcription factors: ERG, HOXA9, and RORA, into myeloid progenitor cells.

[0167] In one embodiment of any method, cell, or composition described herein, MHPCs are generated by contacting a population of myeloid progenitor cells with one or more vectors, wherein the vectors collectively carry exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, for in vivo expression of the factors in the contacted cells, and the transfected transcription factors are expressed in vivo in the contacted cells. The contacting is in vitro or ex vivo. In one embodiment, a single vector is used as a delivery vehicle for introducing exogenous gene-encoding copies of all three transcription factors: ERG, HOXA9, and RORA, into the myeloid progenitor cells. In one embodiment, one or more episomal vectors are used as delivery vehicles for introducing exogenous gene-encoding copies of the three transcription factors: ERG, HOXA9, and RORA, into the myeloid progenitor cells.

[0168] In one embodiment of any method, cell, or composition described herein, MHPCs are generated by contacting myeloid progenitor cells with one or more nucleic acids, wherein the nucleic acids comprise an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, and RORA, collectively for in vivo expression of the transcription factors in the contacted cells. The contacting is in vitro or ex vivo.

[0169] In one embodiment of any method, cell, or composition described herein, contacting the myeloid progenitor cells with any vector, nucleic acid, or composition comprising a vector or nucleic acid described herein occurs in vitro or ex vivo.

[0170] In one embodiment of any method, cell, or composition described herein, the contacting or introducing is repeated at least once.

[0171] In one embodiment of any of the methods, cells, or compositions described herein, the method further comprises transfecting an exogenous gene-encoding copy of SOX4 or MYB or both SOX4 and MYB into a myeloid progenitor cell, wherein the transfected transcription factor is expressed in the transfected cell in vivo. The transfecting is in vitro or ex vivo.

[0172] In one embodiment of any of the methods, cells, or compositions described herein, the method further comprises transfecting an exogenous gene-encoding copy of DACH1 or NFIA, or both DACH1 and NFIA, into a myeloid progenitor cell, wherein the transfected transcription factor is expressed in the transfected cell in vivo. The transfecting is in vitro or ex vivo.

[0173] transcription factors ERG (ETS-related gene) is an oncogene, meaning that it encodes a protein that is typically mutated in cancer. ERG is a member of the ETS (erythroid transformation-specific) family of transcription factors. The ERG gene encodes a protein, also called ERG, that functions as a transcriptional regulator. Genes in the ETS family regulate embryonic development, cell proliferation, differentiation, angiogenesis, inflammation, and apoptosis. External identifications for the ERG gene are as follows: HGNC:3446; Entrez Gene:2078; Ensembl:ENSG00000157554; OMIM:165080; UniProtKB:P11308; EMBL:AY204741 mRNA and corresponding mRNA translation:AAP41719.1; and GENBANK:AY204742 mRNA and corresponding mRNA translation:AAP41720.1.

[0174] The homeobox protein Hox-A9 is encoded by the HOXA9 gene in humans. In vertebrates, genes encoding homeobox gene class transcription factors are found in clusters named A, B, C, and D on four separate chromosomes. The expression of these proteins is spatially and temporally regulated during embryonic development. Hox-A9 is part of the A cluster on chromosome 7 and encodes a DNA-binding transcription factor that can regulate gene expression, morphogenesis, and differentiation. The external identification for the HOXA9 gene is as follows: HGNC:5109; Entrez Gene:3205; Ensembl:ENSG00000078399; OMIM:142956; UniProtKB:P31269; EMBL:BT006990 mRNA and corresponding mRNA translation:AAP35636.1; and GENBANK:AC004080 genomic DNA.

[0175] RAR-related orphan receptor alpha (RORα), also known as NR1F1 (nuclear receptor subfamily 1 group F member 1) or RORA, is a nuclear receptor encoded by the RORA gene in humans. RORα is involved in the transcriptional regulation of several genes involved in circadian rhythms. This nuclear receptor binds DNA as a monomer to the ROR response element (RORE), which contains a single core motif half-site, 5'-AGGTCA-3', preceded by a short AT-rich sequence. It is a major regulator of embryonic development, cell differentiation, immunity, circadian rhythms, as well as lipid, steroid, xenobiotic, and glucose metabolism. It is thought to have intrinsic transcriptional activity and possesses several natural ligands, such as oxysterols, which act as agonists (25-hydroxycholesterol) or inverse agonists (7-oxidized sterols), respectively, enhancing or repressing transcriptional activity. Depending on the tissue, time, and promoter context, it recruits distinct combinations of cofactors to target gene regulatory regions to modulate their transcriptional expression. It regulates genes involved in photoreceptor development, including OPN1SW, OPN1SM, and ARR3, and skeletal muscle development using MYOD1. It is required for proper cerebellar development and regulates SHH gene expression, inducing the expression of genes involved in granule cell proliferation and calcium-mediated signaling, among others. It regulates the circadian expression of several clock genes, including CLOCK, ARNTL / BMAL1, NPAS2, and CRY1. It competes with NR1D1 for binding to their common DNA response elements on several clock genes, such as ARNTL / BMAL1, CRY1, and NR1D1 itself, resulting in NR1D1-mediated repression or RORA-mediated activation of clock gene expression, leading to the circadian pattern of clock gene expression. This therefore affects the period and stability of the clock. It also regulates genes involved in lipid metabolism, such as the apolipoproteins APOA1, APOA5, APOC3, and PPARG.In the liver, it specifically functions as a positive or negative modulator of the expression of genes encoding phase I and phase II proteins involved in lipid, steroid, and xenobiotic metabolism, such as CYP7B1 and SULT2A1, with overlapping functions with RORC. It induces rhythmic expression of some of these genes. In addition, it functionally interacts with NR1H2 and NR1H3 to regulate genes involved in cholesterol metabolism. It is also involved in regulating hepatic glucose metabolism by modulating G6PC and PCK1. In adipose tissue, it acts as a negative regulator of adipocyte differentiation, likely acting via a dual mechanism. It may suppress CEBPB-dependent adipogenesis by direct interaction and PPARG-dependent adipogenesis by competing for DNA binding. It acts downstream of IL6 and TGFB and synergistically with RORC isoform 2, undetermined CD4. + RORA is involved in the lineage specification of T helper (T(H)) cells to T(H)17 cells and antagonizes the T(H)1 program. It regulates IL17 and IL17F expression on T(H) cells, likely by binding to conserved noncoding sequence 2 (CNS2), an essential enhancer in the IL17-IL17F locus. It is involved in hypoxic signaling by interacting with and activating the transcriptional activity of HIF1A, which can inhibit cell growth in response to cellular stress. RORA may exert an anti-inflammatory role by inducing CHUK expression and inhibiting NF-kappa-B signaling. External identifications for the RORA gene are as follows: HGNC: 10258; Entrez Gene: 6095; Ensembl: ENSG00000069667; OMIM: 600825; UniProtKB: P35398; EMBL: U04899 mRNA and corresponding mRNA: AAA62660.1; GENBANK: L14611 mRNA and corresponding mRNA translation: AAA02963.1.

[0176] HOX and ETS family transcription factors, HOXA9 and ERG, are inducers of self-renewal and multilineage differentiation in hematopoietic progenitor cells differentiated from hPSCs. RORA is a nuclear receptor that plays a role in maintaining the quiescent state of hematopoietic progenitor cells. The addition of SOX4 and MYB modulates this network to enable in vivo engraftment of myeloid and erythroid lineages.

[0177] OCT4, SOX2, KLF4, and c-MYC are the four original transcription factors identified to reprogram mouse fibroblasts into iPSCs. These same four factors were also sufficient to generate human iPSCs. OCT3 / 4 and SOX2 function as core transcription factors in the pluripotency network by regulating the expression of pluripotency-related genes. Krüppel-like factor 4 (KLF4) is a downstream target of LIF-STAT3 signaling in mouse ES cells and regulates self-renewal. Human iPSCs can also be generated using four alternative factors; OCT4 and SOX2 are required, but KLF4 and c-MYC can be replaced by NANOG, a homeobox protein important for maintaining pluripotency in both ES cells and early embryos, and LIN28, an RNA-binding protein. A combination of OCT4, SOX2, NANOG, and LIN28 reprogramming factors has also been reported to be sufficient to generate human iPSCs.

[0178] Transcription factor SOX-4 (SOX4). This intronless gene encodes a member of the SOX (SRY-related HMG box) family of transcription factors, which are involved in regulating embryonic development and determining cell fate. The encoded protein acts as a transcriptional regulator after forming a protein complex with other proteins, such as syndecan-binding protein (syntenin). This protein may function in apoptotic pathways leading to cell death and tumorigenesis, and may mediate the downstream effects of parathyroid hormone (PTH) and PTH-related protein (PTHrP) in bone development. External identifications for the Homo sapiens (human) SOX4 gene are as follows: HGNC: 11200; Entrez Gene: 6659; Ensembl: ENSG00000124766; OMIM: 184430; UniProtKB: Q06945; EMBL: BC072668 mRNA and corresponding mRNA translation: AAH72668.1; GENBANK: X65661 mRNA and corresponding mRNA translation: CAA46612.1.

[0179] MYB proto-oncogene, transcription factor (MYB). This gene encodes a protein with three HTH DNA-binding domains that functions as a transcriptional regulator. This protein plays an essential role in regulating hematopoiesis. This gene can be aberrantly expressed, rearranged, or translocated in leukemias and lymphomas and is considered an oncogene. External identification for the MYB gene is as follows: HGNC:7545; Entrez Gene:4602; Ensembl:ENSG00000118513; OMIM:189990; UniProtKB:P10242; EMBL:AJ606319 mRNA and corresponding mRNA translation:CAE55170.1; GENBANK:AJ606320 mRNA and corresponding mRNA translation:CAE55171.1.

[0180] The NFI gene functions as both a positive and negative regulator of gene transcription. Nuclear factor type 1A (NFIA) has been shown to regulate the erythroid / granulocytic lineage switch (Fazi et al., 2005, Starnes et al., 2009). NFIA has been shown to recognize and bind to the palindromic sequence 5'-TTGGCNNNNNGCCAA-3' present in viral and cellular promoters and the adenovirus type 2 replication origin. The NFIA protein can independently activate transcription and replication. The external identification for the NFIA gene is as follows: HGNC:7784; Entrez Gene:4774; Ensembl:ENSG00000162599; OMIM:600727; UniProtKB:Q12857; EMBL:AK299579 mRNA and corresponding mRNA translation:BAG61515.1; GENBANK:AC092784 genomic DNA.

[0181] Dachshund family transcription factor 1 (DACH1) encodes a chromatin-associated protein that associates with other DNA-binding transcription factors to regulate gene expression and cell fate decisions during development. The protein contains a Ski domain that is highly conserved from Drosophila to humans. DACH1 regulates cell cycle progression in myeloid cells, maintains colony-forming activity, and blocks differentiation of myeloid progenitor cells (Lee et al., 2012, Lee et al., 2012). This transcription factor is involved in regulating organogenesis and may be a regulator of SIX1, SIX6, and possibly SIX5. Co-repression of progenitor proliferation in myoblasts by SIX1 is switched to co-activation by recruitment of EYA3 to the SIX1-DACH1 complex. Transcriptional activation also appears to involve the association of CREBBP. DACH1 also acts as a co-repressor of SIX6 in proliferation regulation by directly suppressing the cyclin-dependent kinase inhibitor containing p27Kip1 promoter. Furthermore, DACH1 inhibits TGF-beta signaling through interactions with SMAD4 and NCOR1 and binds to chromatin DNA via its DACH box-N domain. However, its role in hematopoietic stem cell and definitive lymphoid lineage development has not been previously investigated. The external identification for the DACH1 gene is as follows: HGNC:266; Entrez Gene:1602; Ensembl:ENSG00000276644; OMIM:603803; UniProtKB:Q9UI36; EMBL:AF356492. The mRNA and corresponding mRNA translation are: AAL08487.1.

[0182] The cDNA encoding the described and desired transcription factor can be cloned into an expression vector by methods known in the art for in vivo expression in cells. The expression vector can be a constitutive or inducible vector. Protein and DNA information about transcription factors can be found in publicly available databases such as the GenBank™ database of the National Institutes of Health, the UniProt Protein knowledgebase, and the GeneCard database of the Weizmann Institute for Science. cDNA clones or plasmids carrying cDNA can be purchased from BioCat GmbH, and lentiviruses carrying cDNA for expression can also be purchased from Applied Biological Materials (ABM) Inc.

[0183] Thus, in one embodiment, provided herein is a population of modified myeloid progenitor cells having exogenous gene-coding copies of the transcription factors: ERG, HOXA9, and RORA. In one embodiment, the modified myeloid progenitor cells further comprise exogenous gene-coding copies of SOX4, or MYB, or both SOX4 and MYB. In another embodiment, the modified myeloid progenitor cells further comprise exogenous gene-coding copies of DACH1, or NFIA, or both DACH1 and NFIA. In another embodiment, the modified myeloid progenitor cells further comprise exogenous gene-coding copies of the reprogramming factors: OCT4, SOX2, and KLF4, optionally together with c-MYC or nanog and LIN28, or exogenous gene-coding copies for the four reprogramming factors consisting of OCT4, SOX2, NANOG, and LIN28. In another embodiment, the modified myeloid progenitor cells can be culture-expanded in serum-free medium, ie, the modified myeloid progenitor cells undergo mitosis and self-renewal in serum-free medium.

[0184] Thus, in one aspect, provided herein are populations of modified myeloid-lineage progenitor cells having exogenous gene-coding copies of the transcription factors ERG, HOXA9, and RORA for use in producing blood cells such as immune cells, for medical treatments such as transplantation therapy and cancer immunotherapy, or for in vitro research purposes as described herein. In one aspect, the modified myeloid-lineage progenitor cells further comprise exogenous gene-coding copies of SOX4, or MYB, or both SOX4 and MYB. In another aspect, the modified myeloid-lineage progenitor cells further comprise exogenous gene-coding copies of DACH1, or NFIA, or both DACH1 and NFIA. In another aspect, the modified myeloid-lineage progenitor cells further comprise exogenous gene-coding copies of the reprogramming factors OCT4, SOX2, and KLF4, optionally together with c-MYC or nanog and LIN28, or exogenous gene-coding copies for the four reprogramming factors consisting of OCT4, SOX2, NANOG, and LIN28. In another embodiment, the modified myeloid progenitor cells can be culture-expanded in serum-free medium, ie, the modified myeloid progenitor cells undergo mitosis and self-renewal in serum-free medium.

[0185] In one embodiment of any method, cell, or composition described herein, the myeloid progenitor cells are progenitor cells derived from embryoid bodies (EBs) obtained from a pluripotent stem cell population. In one embodiment, the pluripotent stem cells are iPSCs. In one embodiment, the iPSCs are derived from mature differentiated somatic cells.

[0186] Thus, in one embodiment of any of the methods, cells, or compositions described, the method further comprises providing a population of pluripotent stem cells (PSCs) for generating myeloid lineage progenitor cells. In one embodiment, the PSCs are human cells.

[0187] In one embodiment of any of the methods, cells, or compositions described, the method further comprises producing myeloid lineage progenitor cells from the population of pluripotent stem cells (PSCs). In one embodiment, the PSCs are human cells.

[0188] In one embodiment of any of the methods, cells, or compositions described herein, myeloid lineage progenitor cells are prepared by first culturing a population of pluripotent stem cells in vitro in bone morphogenetic protein 4 (BMP4), stem cell factor (SCF), Fms-like tyrosine kinase 3 (FLT3 / CD135), granulocyte colony-stimulating factor (G-CSF / CSF3), IL-6, and IL-3 for about 10-21 days to form EBs from the pluripotent stem cells, dissociating the EB aggregates of cells into single cells, and isolating the dissociated cells from CD34. + and CD45 + It is produced by positive selection of cells. + and CD45 +The cells are myeloid progenitor cells. In one embodiment, the PSCs are cultured for at least 10 days. In other embodiments, the PSCs are cultured for several days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, or at least 21 days. In one embodiment, the transfected or contacted myeloid progenitor cells are cultured for about 7-21 days. In other embodiments, the PSCs are cultured for about 10-20 days, about 10-18 days, about 10-16 days, about 10-14 days, about 10-12 days, about 11-21 days, 11-20 days, about 11-18 days, about 11-16 days, about 11-14 days, about 11-13 days, about 11-12 days, about 12-21 days, about 12-20 days, about 12-18 days days, about 12-16 days, about 12-14 days, about 13-21 days, 13-20 days, about 12-18 days, about 13-16 days, about 14-20 days, about 14-18 days, about 14-16 days, 10-19 days, about 10-17 days, about 10-15 days, about 10-13 days, about 10-11 days, about 11-19 days, 11-19 days , about 11 to 17 days, about 11 to 15 days, about 12 to 19 days, about 12 to 17 days, about 12 to 15 days, 12 to 13 days, about 14 to 21 days, about 13 to 18 days, about 13 to 17 days, about 13 to 15 days, about 13 to 14 days, about 15 to 21 days, about 15 to 20 days, about 15 to 19 days, about 15 to 17 days, about 15 to 16 days , about 16 to 21 days, about 16 to 20 days, about 16 to 19 days, about 16 to 18 days, about 16 to 17 days, about 17 to 21 days, about 17 to 20 days, about 17 to 19 days, about 17 to 18 days, about 18 to 21 days, about 18 to 20 days, about 18 to 19 days, about 19 to 21 days, about 19 to 20 days, and about 20 to 21 days.

[0189] In one embodiment of any of the methods, cells, or compositions described herein, the myeloid lineage progenitor cells are CD34 + and CD45 +In other embodiments of any of the methods, cells, or compositions described herein, the myeloid progenitor cells are further positive for CD14, or CD15, or CD11b, or a combination of two or three of these cell surface CD antigens.

[0190] In one embodiment of any method, cell, or composition described herein, the myeloid progenitor cells are committed to a non-lymphoid lineage. In one embodiment, the myeloid progenitor cells exhibit primarily the following culture colony-forming activity: CFU-M and CFU-G colonies (>80% CFUs of total CFUs in a colony-forming assay). In other embodiments, the myeloid progenitor cells produce greater than 82%, greater than 84%, greater than 86%, greater than 88%, greater than 90%, greater than 92%, greater than 95%, greater than 97%, or greater than 99% CFU-M and CFU-G colonies of total CFUs in a colony-forming assay. In one embodiment, the myeloid progenitor cells produce only a small number of CFU-E, BFU-E, CFU-GEMM, and CFU-GM colonies (<20% of total CFUs in a colony-forming assay). In one embodiment, the myeloid progenitor cells produce CFU-E, BFU-E, CFU-GEMM, and CFU-GM colonies at less than 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, or 2% of the total CFUs in a colony-forming assay. In vitro colony-forming assays can be performed by any method known in the art. For example, as taught in Tashiro K, et al. 2012, Stem Cell Res. 8:300-311, U.S. Patent Nos. 6,103,522, 6,419,918, 683,854, 7,883,861, 7,989,178, and 9,273,285. These references are incorporated herein in their entirety. For example, commercially available kits such as the hematopoietic CFC assay from Cell Biolabs Inc. and the human colony forming cell (CFC) assay using methylcellulose-based medium from R&D Systems are used.

[0191] In another embodiment of any of the methods, cells, or compositions described herein, the myeloid progenitor cells are collected or harvested from peripheral blood, umbilical cord blood, chorionic villi, amniotic fluid, placental blood, or bone marrow. + and CD45 + The cells, myeloid lineage progenitors, are positively selected from these sources.

[0192] Peripheral blood progenitor cells (PBPCs) have become a preferred source of hematopoietic progenitor cells for allogeneic and autologous transplantation due to their technical ease of collection and shorter engraftment times. Traditionally, granulocyte colony-stimulating factor (G-CSF) has been used to stimulate the release of hematopoietic progenitor cells from bone marrow and larger numbers of PBPCs. While G-CSF-based regimens are usually successful in collecting sufficient numbers of PBPCs from healthy donors, 5%–10% of cases may result in insufficient stem cell mobilization, necessitating multiple large-volume apheresis or bone marrow collection.

[0193] In one embodiment of any method, cell, or composition described herein, the pluripotent stem cell population is induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).iPSCs and ESCs can be produced by any method known in the art.Methods for producing iPS cells are known in the art, for example, in U.S. Patent No. 8,058,065, and U.S. Patent Application Publication Nos. 20110223669, 20120214243, 20130059386, and 20130183759, all of which are incorporated herein by reference in their entirety.

[0194] In one embodiment of any of the methods, cells, or compositions described herein, iPSCs are produced by introducing exogenous copies of just three reprogramming factors: OCT4, SOX2, and KLF4 into mature or somatic cells.

[0195] In one embodiment of any of the methods, cells, or compositions described herein, iPSCs harboring exogenous gene-coding copies of OCT4, SOX2, and KLF4 are further introduced into mature or somatic cells along with c-MYC or nanog and LIN28.

[0196] In one embodiment of any method, cell, or composition described herein, iPSCs are produced by introducing exogenous copies of the reprogramming factors OCT4, SOX2, and KLF4, optionally along with c-MYC or nanog and LIN28, into mature or somatic cells.

[0197] In one embodiment of any of the methods, cells, or compositions described herein, iPSCs are produced by contacting mature cells with one or more vectors, wherein the vectors collectively carry exogenous gene coding copies for reprogramming factors: OCT4, SOX2, and KLF4, optionally together with c-MYC or nanog and LIN28, within the mature or somatic cells, and the reprogramming factors are expressed in vivo in the mature or somatic cells after contacting. The contacting is in vitro or ex vivo.

[0198] In one embodiment of any of the disclosed methods, the iPS cells contain at least one exogenous copy of a nucleic acid sequence encoding a reprogramming factor selected from the group consisting of the genes Oct4 (Pou5f1), Sox2, cMyc, Klf4, Nanog, Lin28, and Glis1. In some embodiments, a combination of reprogramming factors is used. For example, a combination of four reprogramming factors consisting of Oct4, Sox2, cMyc, and Klf4, or a combination of four reprogramming factors consisting of Oct4, Sox2, Nanog, and Lin28.

[0199] In one embodiment of any of the methods, cells, or compositions described herein, the mature cells from which iPS cells are generated include any somatic cell, such as B lymphocytes (B cells), T lymphocytes (T cells), fibroblasts, and keratinocytes.

[0200] In one embodiment of any of the methods, cells, or compositions described herein, iPSCs are produced by introducing the disclosed reprogramming factors into mature or somatic cells two or more times.

[0201] In one embodiment of any method, cell, or composition described herein, iPSCs are produced by contacting mature cells with the disclosed vector factors two or more times to enter the mature / somatic cells.

[0202] In some embodiments, in vitro culturing of the cells occurs between step (a) of generating MHPCs and step (b) of inhibiting histone methyltransferases in the multi-lineage hematopoietic progenitor cells. In some embodiments, selection of the desired cells occurs between step (a) of generating MHPCs and step (b) of inhibiting histone methyltransferases in the MHPCs.

[0203] In one embodiment of any method, cell, or composition described herein, the transfected or contacted myeloid progenitor cells carrying an added exogenous gene-encoding copy of the described transcription factor prior to inhibiting histone methyltransferase are further cultured in vitro for a time sufficient to expand the cell population. In one embodiment of any method, cell, or composition described herein, the transfected or contacted myeloid progenitor cells are cultured for at least 7 days. In other embodiments, the transfected or contacted myeloid progenitor cells are cultured for at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. In one embodiment, the transfected or contacted myeloid progenitor cells are cultured for about 7-21 days. In other embodiments, the myeloid progenitor cells after transfection or contacting are incubated for about 7-20 days, about 7-18 days, about 7-16 days, about 7-14 days, about 7-12 days, about 7-10 days, 8-20 days, about 8-18 days, about 8-16 days, about 8-14 days, about 8-12 days, about 8-10 days, 9-20 days, about 9-18 days, about 9-16 days, about 9-14 days, about 9-12 days, about 9-10 days, 10-20 days, about 11-12 days, about 12-14 days, about 13-14 days, about 14-14 days, about 15-16 days, about 16-16 days, about 17-18 days, about 18-16 ... The cells are cultured for 0 to 18 days, about 10 to 16 days, about 10 to 14 days, about 10 to 12 days, 11 to 20 days, about 11 to 18 days, about 11 to 16 days, about 11 to 14 days, about 11 to 13 days, about 11 to 12 days, about 12 to 20 days, about 12 to 18 days, about 12 to 16 days, about 12 to 14 days, 13 to 20 days, about 12 to 18 days, about 13 to 16 days, about 14 to 20 days, about 14 to 18 days, and about 14 to 16 days.

[0204] In one embodiment of any of the methods, cells, or compositions described herein, culture-expanded myeloid progenitor cells carrying added exogenous gene-encoding copies of the described transcription factors are further selected for the presence of the cell surface marker CD34 (CD34 positive) and for the absence or low expression of the cell surface marker CD38 (CD38 low / negative). In other words, for the periods described herein, the cells obtained after culture expansion are positively selected for CD34 and negatively selected for CD38. The selected CD34 + CD38 lo / - The cells are reverse lineage MHPCs.Selection can be carried out by any known method, for example, by fluorescence activated cell sorting (FACs), as described in U.S. Patent Application Publication Nos. 20090239235, 20090061513, 20140075593, and U.S. Patent Nos. 5,985,216, 6,455,263, 6,461,813, and 6,897,031. These references are incorporated herein by reference in their entirety.

[0205] In one embodiment of any method, cell, or composition described herein, the MHPCs have myeloid and erythroid differentiation potential and low or undetectable lymphoid differentiation potential, as determined by any method known in the art, e.g., as taught in the Examples section, or as measured during an in vitro differentiation protocol or after engraftment in a recipient mouse host.

[0206] In one embodiment of any of the methods, cells, or compositions described herein, the MHPCs are CD34 +They are CD38 low / negative. In one embodiment of any method, cell, or composition described herein, the MHPCs are CD90 positive or CD49f positive, or both. In one embodiment of any method, cell, or composition described herein, the MHPCs exhibit increased expression of HSC-specific transcription factors: HLF, or NF1B, or HOPX, or HMGA2 or RBPMS, or a combination thereof, compared to before introduction of the EAR into the cells. In one embodiment, the increased expression is at least 5% compared to before introduction of the EAR into the cells. In other embodiments, the increased expression is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more, compared to before introduction of the EAR into the cells.

[0207] In some embodiments of any of the methods, cells, or compositions described herein, the MHPCs contain a cell surface marker characteristic of human hematopoietic progenitor cells: CD34 + , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 lo / - and Lin - Preferably, the multilineage hematopoietic progenitor cells have several of these markers.

[0208] In some embodiments of any of the methods, cells, or compositions described herein, the MHPCs have cell surface markers characteristic of the erythroid lineage: CD71 and Ter119.

[0209] In some embodiments of any of the methods, cells, or compositions described herein, the myeloid lineage progenitor cells or MHPCs are transfected with CD34 + Selected for surface markers.

[0210] Inhibition of histone H3 methylation to promote and enhance lymphoid differentiation potential During these experiments, we discovered that inhibition of specific histone-modifying enzymes targeting H3K9 and H3K27 promotes lymphoid differentiation of pluripotent stem cell-derived hematopoietic progenitor cells. Histone-modifying enzymes are histone lysine methyltransferases. Post-translational modifications of histone proteins regulate chromatin compaction, mediate epigenetic regulation of transcription, and control cellular differentiation in healthy and disease states. Methylation of histone tails is one of the fundamental events in epigenetic signaling. Trimethylation of lysine 9 (H3K9) of histone H3 mediates chromatin recruitment of HP1, heterochromatin condensation, and gene silencing. Similarly, methylation of H3K27 and H4K20 is associated with repressive chromatin, whereas expressed genes are methylated at H3K4, H3K36, and H3K79. Methylation of H3K9 in humans depends primarily on members of the Suv39 family, namely EHMT1 / GLP, EHMT2 / G9a, SUV39H1, SUV39H2, SETDB1 and SETDB2, and secondarily on the non-Suv39 enzymes PRDM2 and ASH1L (Hong Wu et al., Structural Biology of Human H3K9 Methyltransferases, 2010, PLoS ONE, 5(2):e8570). In contrast, methylation of H3K27 is carried out by Polycomb Repressive Complex 2 (PRC2).

[0211] Di- and trimethylation of H3K9 is primarily catalyzed by the conserved SUV39H1 / 2 histone methyltransferases, while polycomb repressive complex 2 (PRC2) ensures di- and trimethylation of H3K27 (Rea S, 2000. Nature 406:593-599; Margueron R, and Reinberg D. 2011. Nature 469:343-349). PRC2 contains the EZH1 / 2 catalytic subunits, SUZ12, EED, and RBBP7 / 4 (Margueron R, and Reinberg D, 2011).

[0212] Without wishing to be bound by theory, inhibiting histone lysine methyltransferases that target H3K9 and H3K27 relieves transcriptional repression caused by histone H3 methylation, thereby promoting gene expression that facilitates cell differentiation.

[0213] In one embodiment of any method, cell, or composition described herein, the histone methyltransferase catalyzes the addition of a methyl group to histone H3 lysine residue 9 (H3K9) and / or histone H3 lysine residue 27 (H3K27).

[0214] In one embodiment of any of the methods, cells, or compositions described, the histone methyltransferase inhibitor inhibits the G9a / GLP heteromeric complex.

[0215] G9a (EC 2.1.1.43) (UniProtKB: Q96KQ7) is also known as EHMT2, (euchromatin histone-lysine N-methyltransferase 2), G9A histone methyltransferase and protein G9a.

[0216] GLP (EC 2.1.1.43) (UniProtKB: Q9H9B1) is also known as EHMT1 (euchromatin histone-lysine N-methyltransferase 1), G9a-like protein 1 and GLP1.

[0217] In one embodiment of any of the methods, cells, or compositions described, the histone methyltransferase inhibitor inhibits EZH1 (Enhancer of Zeste 1 Polycomb Repressive Complex 2 Subunit).

[0218] In one embodiment of any of the methods, cells, or compositions described, the H3K27 histone methyltransferase is EZH1 (EC:2.1.1.43) (UniproKB Q92800-1).

[0219] In one embodiment of any of the methods, cells, or compositions described, the H3K27 histone methyltransferase is not EZH2 (EC:2.1.1.43) (Unipro Q15910-1).

[0220] In one embodiment of any method, cell, or composition described herein, the inhibitor of histone methyltransferase inhibits gene expression or protein catalytic activity of the histone methyltransferase.

[0221] In one embodiment of any method, cell, or composition described herein, histone methyltransferases H3K9 and / or H3K27 are inhibited by a small molecule or nucleic acid or CRISPR-mediated targeted gene interference.

[0222] In one embodiment of any of the described methods, cells, or compositions, the histone methyltransferase small molecule inhibitor is a chemical agent, including but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight of less than about 10,000 grams / mole, organic or inorganic compounds having a molecular weight of less than about 5,000 grams / mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams / mole, organic or inorganic compounds having a molecular weight of less than about 500 grams / mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. In some embodiments, the small molecule is a heteroorganic compound or an organometallic compound.

[0223] In one embodiment of any of the described methods, cells, or compositions, histone methyltransferase small molecule inhibitors include AMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin, UNC0224, UNC0631, UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343, EPZ-6 438, 3-deazaneplanocin A (DZNeP)HCl, UNC1999, MM-102, SGC0946, entacapone, EPZ015666, UNC0379, EI1, MI-2 (menin-MLL inhibitor), MI-3 (menin-MLL inhibitor), PFI-2, GSK126, EPZ004777, BRD4770, and EPZ-6438.

[0224] In one embodiment of any of the methods, cells, or compositions described, the histone methyltransferase small molecule inhibitor is selected from the group consisting of UNC0631, BRD4770, UNC1999, CPI-360, and BIX01294.

[0225] In one embodiment of any method, cell, or composition described herein, the nucleic acid inhibitor is a nucleic acid that targets the expression of histone methyltransferase. For example, it targets the mRNA or primary transcript of the histone methyltransferase EZH1, thereby inhibiting the protein expression of the enzyme. The histone-lysine N-methyltransferase, also known as enhancer of Zeste 1 polycomb repressive complex 2 subunit (EZH1) or EC 2.1.1.43, is a component of the non-classical polycomb repressive complex-2 (PRC2), which mediates the methylation of histone H3 (see MIM602812) lys27 (H3K27) and functions in maintaining the pluripotency and plasticity of embryonic stem cells. External identifications for the human EZH1 gene are as follows: HGNC: 3526; Entrez Gene: 2145; Ensembl: ENSG00000108799; OMIM: 601674; UniProtKB: Q92800; EMBL: AB002386 mRNA and corresponding mRNA translation: BAA20842.2; GENBANK: BT009782 mRNA and corresponding mRNA translation: AAP88784.1.

[0226] In one embodiment, the nucleic acid inhibitor targets human EZH1 mRNA.

[0227] In one embodiment of any method, cell or composition described herein, nucleic acid inhibitor is RNA interference inhibitor or CRISPR-mediated gene interference inhibitor.RNA interference inhibitor can be designed by using EZH1 mRNA as target, using the predictive RNAi software found on the sirna website of Whitehead Institute, MIT, Invitrogen / ThermoFisher's BLOCK-iT™ RNAi Designer and other online siRNA design tools on RNAiWeb.

[0228] Similarly, Crisper guide RNAs can be designed using the mRNA or genomic gene of EZH1 as a target using the Broad Institute (MIT) Crispr software (see the MIT website), dna20, Clontech, AddGene, e-crisp, and Innovative Genomic.

[0229] CRISPR (clustered regularly interspaced short palindromic repeats) Cas9-mediated gene disruption has been widely used to generate loss-of-function mutations in diverse organisms, including mammals (reviewed in Cong et al., 2013, Science, 339(6121):819-23; Hsu et al., 2014, Cell, 157(6):1262-78). Cas9-based knockout screens have been applied to identify essential genes and genes involved in drug resistance in various cell lines.For general information regarding CRISPR-Cas systems, their components, and delivery of such components, including with respect to amounts and formulations, all useful in practicing the present invention, including methods, materials, delivery vehicles, vectors, particles, AAVs, and their manufacture and use, see U.S. Patent Nos. 8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945, and 8,697,359; U.S. Patent Application Publication No. 2014 -0310830, 2014-0287938, 2014-0273234, 2014-0273232, 2014-0273231, 2014-0256046, 2014-0248702, 2014-0242700, 2014-0242699, 2014-0242664 Nos. 2014-0234972, 2014-0227787, 2014-0189896, 2014-0186958, 2014-0186919, 2014-0186843, 2014-0179770 and 2014-0179006, 2014-0170753; European Patent EP Reference is made to EP 2 784 162 B1 and EP 2 771 468 B1; European Patent Applications EP 2 771 468 (EP13818570.7), EP 2 764 103 (EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCT Publication No. WO 2014 / 093661, all of which are incorporated herein by reference in their entireties.

[0230] The CRISPR / Cas system contemplated for use in connection with the present invention can utilize any suitable CRISPR enzyme. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Cas9 from Streptococcus pneumoniae (S. pneumoniae), Streptococcus pyogenes (S. pyogenes), or S. thermophilus, and can include mutant Cas9s from these organisms. The enzyme can be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells.

[0231] The CRISPR / Cas system described herein is used to specifically target multiple sequences within a contiguous genomic region of interest. Targeting typically involves introducing into each cell of a cell population a vector system of one or more vectors comprising an engineered non-native CRISPR-Cas system comprising at least one Cas protein and one or more guide RNAs from a guide RNA library described herein.

[0232] In these methods, Cas protein and one or more guide RNAs can be on the same or different vectors of the system and are incorporated into each cell, whereby each guide sequence targets the sequence in the continuous genomic region in each cell in the cell population.Cas protein is operably linked to a regulatory element to ensure expression in the cell, more particularly to a promoter suitable for expression in the cells of the cell population.In certain embodiments, the promoter is an inducible promoter, such as a doxycycline-inducible promoter.When transcribed in the cells of the cell population, the guide RNA comprising the guide sequence directs the sequence-specific binding of the CRISPR-Cas system to the target sequence in the continuous genomic region.Typically, the binding of the CRISPR-Cas system induces the cleavage of the continuous genomic region by the Cas protein.

[0233] RNA interference (RNAi), mediated by small interfering RNA (siRNA) or microRNA (miRNA), is a powerful method for post-transcriptional regulation of gene expression. RNAi has been widely used to study biological processes in mammalian cells and can also constitute a therapeutic approach to human diseases in which selective modulation of gene expression is desirable. Depending on the degree of complementarity between the miRNA and target mRNA sequence, lack of gene expression occurs by inducing degradation of the cognate mRNA or by translational attenuation. Endogenous miRNAs are transcribed as primary transcripts and subsequently processed by the RNAse III enzyme Drosha (1), creating a stem-loop structure. Nuclear export and cleavage by Dicer generates mature short double-stranded molecules (siRNAs) that are separated into a guide strand and a passenger strand. The guide strand is loaded into the RNA-induced silencing complex (RISC), an effector complex that mediates cleavage of the target mRNA. The functional guide strand binds to RISC proteins, while the passenger strand is degraded. The loading of the guide strand relative to the passenger strand into RISC is largely dependent on the stability of the 5' end of the siRNA, with the less stable strand preferentially being incorporated into RISC, but the precise regulation in mammalian cells is incompletely understood. The 5' end of the guide strand contains a "seed region" that is important for target identification. Accurate cleavage by Drosha and Dicer is important for generating guide RNAs with a specific seed region that mediates efficient binding with the appropriate target mRNA. Inaccurate processing can lead to binding with off-target molecules, but the movement of the cleavage site also changes the nucleotide composition of the duplex end, which can have a significant impact on strand loading into RISC.

[0234] Inhibiting the expression of selected target polypeptides is achieved through the use of RNA interference agents. RNA interference (RNAi) uses small interfering RNA (siRNA) duplexes to target the messenger RNA encoding the target polypeptide for selective degradation. siRNA-dependent post-transcriptional silencing of gene expression involves cleaving the target messenger RNA molecule at the site guided by siRNA. RNAi is an evolutionarily conserved process in which the expression or introduction of RNA with the same or highly similar sequence as the target gene leads to sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of the messenger RNA (mRNA) transcribed from the targeted gene (see Coburn, G. and Cullen, B. (2002) J. Virology 76(18):9225), thereby inhibiting the expression of the target gene. In one embodiment, the RNA is double-stranded RNA (dsRNA). This process has been described in plants, vertebrates, and mammalian cells. In nature, RNAi is initiated by Dicer, a dsRNA-specific endonuclease that promotes the processive cleavage of long dsRNA into double-stranded fragments called siRNAs. The siRNA is incorporated into a protein complex (called "RNA-induced silencing complex" or "RISC") that recognizes and cleaves target mRNA. RNAi can also be initiated by introducing nucleic acid molecules, such as synthetic siRNA or RNA interference agents, to inhibit or silence the expression of target genes. As used herein, "inhibition of target gene expression" includes any reduction in the expression or protein activity or level of a target gene or a protein encoded by a target gene, compared to the situation in which RNA interference is not induced. The reduction is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more, compared to the expression or activity or level of a target gene or a protein encoded by a target gene that is not targeted by an RNA interference agent.

[0235] As used herein, the terms "RNA interfering agent" and "RNA interference" are intended to encompass double-stranded RNA-mediated gene silencing, regardless of whether the RNA interfering agent comprises siRNA, miRNA, shRNA, or other double-stranded RNA molecules. siRNA is defined as an RNA agent that functions to inhibit the expression of target genes, for example, by RNAi. siRNA may be chemically synthesized, produced by in vitro transcription, or produced within a host cell. In one embodiment, siRNA is a double-stranded RNA (dsRNA) molecule about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, more preferably about 19, 20, 21, 22, or 23 nucleotides in length, and may contain 3' and / or 5' overhangs on each strand, each having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand does not depend on the length of the overhang on the second strand. Preferably, the siRNA is capable of promoting RNA interference via degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).

[0236] siRNA also includes short hairpin (also called stem-loop) RNA (shRNA). In one embodiment, these shRNAs are composed of a short (for example, about 19 to about 25 nucleotides) antisense strand followed by a nucleotide loop of about 5 to about 9 nucleotides, and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop structure, and the antisense strand may follow. These shRNAs can be contained in plasmids, retroviruses, and lentiviruses, and can be expressed from, for example, pol III U6 promoters or other promoters (see, for example, Stewart, et al. (2003) RNA April; 9(4):493-501, which is incorporated herein by reference in its entirety). The target gene or sequence of RNA interference agent can be a cellular gene or genomic sequence, such as the BCL11A sequence. siRNA can be substantially homologous to the target gene or genomic sequence, or a fragment thereof. The term "homologous" used in this context is defined as being substantially identical, sufficiently complementary, or similar to the target mRNA or its fragment to cause target RNA interference. In addition to native RNA molecules, RNA suitable for inhibiting or interfering with the expression of target sequences includes RNA derivatives and analogs. Preferably, siRNA is identical to its target. siRNA preferably targets only one sequence. Each RNA interference agent, such as siRNA, can be screened for potential off-target effects, for example, by expression profiling. Such methods are known to those skilled in the art and are described, for example, in Jackson et al. Nature Biotechnology 6:635-637, 2003. In addition to expression profiling, potential target sequences may be screened for similar sequences in sequence databases to identify potential sequences that may have off-target effects. For example, 15 or perhaps as few as 11 consecutive nucleotides of sequence identity are sufficient to direct the silencing of non-target transcripts.Therefore, to avoid potential off-target silencing, proposed siRNAs may first be screened using sequence identity analysis by any known sequence comparison method, such as BLAST. siRNA sequences are selected to maximize the incorporation of the antisense (guide) of siRNA into RISC, thereby maximizing the ability of RISC to target G9a / GLP or EZH1 mRNA for degradation. This can be achieved by scanning for sequences with the lowest binding free energy at the 5' end of the antisense strand. Lower free energy results in enhanced unwinding of the 5' end of the antisense strand of the siRNA duplex, thereby ensuring that the antisense strand is incorporated by RISC and directs the sequence-specific cleavage of human G9a / GLP or EZH1 mRNA. siRNA molecules are not necessarily limited to molecules containing only RNA, but also include, for example, chemically modified nucleotides and non-nucleotides, including molecules in which ribose sugar molecules are replaced with other sugar molecules or molecules that perform similar functions. In addition, non-natural bonds, such as phosphorothioate bonds, can be used between nucleotide residues. The RNA strand can be derivatized with a reactive functional group for a reporter group, such as a fluorophore. Particularly useful derivatives are modified at one or more ends of the RNA strand, typically the 3' end of the sense strand. For example, the 2'-hydroxyl at the 3' end can be easily and selectively derivatized with a variety of groups. Other useful RNA derivatives incorporate 2'O-alkylated residues or nucleotides with modified sugar moieties, such as 2'-O-methylribosyl and 2'-O-fluororibosyl derivatives. The RNA bases can also be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence can be used. For example, halogenated bases such as 5-bromouracil and 5-iodouracil can be incorporated. The bases can also be alkylated; for example, 7-methylguanosine can be incorporated in place of guanosine residues. Unnatural bases that result in successful inhibition can also be incorporated.The most preferred siRNA modifications include RNA duplexes containing 2'-deoxy-2'-fluorouridine or locked nucleic acid (LAN) nucleotides and either phosphodiester or various numbers of phosphorothioate bonds.Such modifications are known to those skilled in the art and are described, for example, in Braasch et al., Biochemistry, 42: 7967-7975, 2003.Using the chemical reactions established for antisense oligonucleotide technology, most of the useful modifications can be introduced into siRNA molecules.Preferably, modifications include at least 2'-O-methyl modifications, and preferably exclude 2'-O-methyl modifications.Preferably, modifications also exclude the modification of the free 5'-hydroxyl group of siRNA.The examples herein provide specific examples of RNA interference agents, such as shRNA molecules, that effectively target mRNA.

[0237] In one embodiment, the nucleic acid is a G9a / GLP or EZH1 specific RNA interfering agent or a vector encoding the RNA interfering agent. TIFF0007635191000002.tif37159.

[0238] In one embodiment of any of the methods, cells, or compositions described herein, the nucleic acid is: TIFF0007635191000003.tif37164.

[0239] In one embodiment of any method, cell, or composition described herein, multilineage hematopoietic progenitor cells are contacted with a viral vector or vectors carrying a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 27-30.

[0240] In one embodiment of any method, cell, or composition described herein, the contacting with the histone methyltransferase inhibitor occurs more than once. For example, after contacting multilineage hematopoietic progenitor cells a first time with a virus or vector carrying a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 27-30, or after contacting with a small molecule inhibitor described herein, the contacted cells are washed, and then the washed cells are contacted a second time with the same histone methyltransferase inhibitor used in the first contacting.

[0241] It is contemplated herein that the Cas9 / CRISPR system of gene editing is employed in the methods, cells and compositions described herein.Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system is useful for RNA programmable genome editing (see, for example, Jinek, M. et al. Science (2012) 337(6096):816-821).

[0242] Transactivating crRNA (tracrRNA) is a small trans-encoded RNA. It was first discovered in the human pathogen Streptococcus pyogenes. (See Deltcheva E, et al. (2011). Nature 471 (7340): 602-7.) In bacteria and archaea, CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins) constitutes an RNA-mediated defense system that protects against viruses and plasmids. This defense pathway has three steps. First, a copy of the invading nucleic acid is integrated into the CRISPR locus. Next, CRISPR RNA (crRNA) is transcribed from this CRISPR locus. Next, the crRNA is incorporated into an effector complex, where the crRNA guides the complex to the invading nucleic acid, and the Cas protein degrades the nucleic acid. (See Terns MP and Terns RM (2011). Curr Opin Microbiol 14 (3): 321-7.) There are several pathways for CRISPR activation, one of which requires the tracrRNA, which plays a role in the maturation of the crRNA. TracrRNA is complementary to and base-pairs with the pre-crRNA, forming an RNA duplex, which is cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid. (See Deltcheva E, et al. supra; Jinek M, et al. (2012), Science 337 (6096): 816-21; and Brouns SJ (2012), Science 337 (6096): 808-9.)

[0243] In some embodiments, the guide RNA of the Cas9 / CRISPR system is designed to target exon 3 of the EZH1 gene, which is present in all known transcripts of EZH1. The sequence of exon 3 is: The file is TIFF0007635191000004.tif17163.

[0244] Non-limiting exemplary gRNAs targeting exon 3 include: The file is TIFF0007635191000005.tif51169.

[0245] In other embodiments, the guide RNA of the Cas9 / CRISPR system is designed to target exon 4 of the EZH1 gene, which is also present in all known transcripts of EZH1. The sequence of exon 4 is: The file is TIFF0007635191000006.tif17163.

[0246] Non-limiting exemplary gRNAs targeting exon 4 include: The file is TIFF0007635191000007.tif38163.

[0247] In one embodiment of any of the methods, cells, or compositions described herein, a vector is used as a delivery vehicle to introduce any of the exogenous gene-encoding copies of transcription factors or reprogramming factors or nucleic acid inhibitors described herein into a target cell selected from the disclosed myeloid progenitor cells or the disclosed reverse lineage multipotent hematopoietic progenitor cells.

[0248] In one embodiment of any of the methods, cells, or compositions described herein, a vector is used as a delivery vehicle to introduce any of the nucleic acids described herein, including the described exogenous gene-encoding copies of transcription factors or reprogramming factors or nucleic acid inhibitors, into a target cell selected from the disclosed myeloid progenitor cells or the disclosed reverse lineage multipotent hematopoietic progenitor cells.

[0249] In one aspect, the present disclosure provides one or more vectors, collectively comprising exogenous gene-encoding copies of each of the described transcription factors or reprogramming factors or nucleic acid inhibitors. The exogenous gene-encoding copies are for expression of the transcription factors or reprogramming factors in cells. In vivo expression of the nucleic acid inhibitors is for degrading the mRNA of the targeted histone methyltransferase, such as G9a / GLP or EZH1, to reduce and / or inhibit expression of the respective histone methyltransferase, with the goal of reducing histone H3 methylation in the transfected cells and relieving repression of gene expression therein. In one embodiment, each vector consists essentially of the transcription factors or reprogramming factors described herein. In one embodiment, each vector consists essentially of two or more of the described transcription factors or reprogramming factors.

[0250] In one aspect, the description provides one or more vectors that collectively contain a nucleic acid that includes a described exogenous gene coding copy of a transcription factor or reprogramming factor or nucleic acid inhibitor, wherein the nucleic acid is for expression of the transcription factor or reprogramming factor in a cell.

[0251] In one aspect, the description provides one or more vectors that collectively comprise an exogenous gene-encoding copy of each of the following transcription factors described herein: ERG, HOXA9, and RORA. For example, a single vector carrying coding copies for all three transcription factors: ERG, HOXA9, and RORA. In another aspect, the vector collectively further comprises exogenous gene-encoding copies of SOX4 and MYB. For example, a single vector carrying coding copies for both SOX4 and MYB. In another aspect, the vector collectively further comprises exogenous gene-encoding copies of DACH1 and NFIA. For example, a single vector carrying coding copies for both DACH1 and NFIA.

[0252] In another aspect, the present disclosure also provides host cells comprising one or more vectors described herein, or nucleic acids of transcription factors or reprogramming factors, or both, described herein.

[0253] In another aspect, the disclosure herein also provides a host cell comprising one or more of the vectors described herein or nucleic acids of the transcription factors ERG, HOXA9, and RORA described herein.

[0254] In another aspect, the host cell further comprises one or more vectors or nucleic acids of the transcription factors SOX4 and MYB described herein.

[0255] In another aspect, the host cell further comprises nucleic acids of one or more vectors described herein or the reprogramming factors described herein, or both, OCT4, SOX2, and KLF4, and optionally c-MYC or NANOG and LIN28, or the four reprogramming factors: OCT4, SOX2, NANOG and LIN28.

[0256] In one embodiment of any host cell described herein, the host cell is an embryonic stem cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic stem cell, a hematopoietic progenitor cell, an immune cell such as a T cell or a B cell, an erythrocyte, a fibroblast, a keratinocyte, or a myeloid progenitor cell. In one embodiment, the host cell is isolated from a subject. In one embodiment, the host cell is isolated from a subject diagnosed with a blood disorder.

[0257] In one embodiment of any method, cell, or composition described herein, the vector further comprises a spleen focus-forming virus promoter, a tetracycline-inducible promoter, a doxycycline (Dox)-inducible promoter, or aβ-globin locus control region and a β-globin promoter. In one embodiment, the promoter provides targeted expression of the nucleic acid molecule therein. Other examples of promoters include, but are not limited to, the CMV promoter and the EF1α promoter for various transgenes, and the U6 promoter for shRNAs targeting EZH1.

[0258] In one embodiment of any method, cell or composition described herein, vector is virus or non-virus vector.Non-limiting examples of the virus vector for gene delivery and expression in cells are retrovirus, adenovirus (type 2 and 5), adeno-associated virus (AAV), helper-dependent adenovirus vector (HdAd), hybrid adenovirus vector, herpesvirus, poxvirus, human foamy virus (HFV) and lentivirus.

[0259] In one embodiment of any of the methods, cells, or compositions described herein, the vector is an episomal vector.

[0260] In one embodiment of any method, cell, or composition described herein, the vector is an integrating vector.

[0261] In one embodiment of any method, cell, or composition described herein, the vector is a non-integrating vector.

[0262] In one embodiment of any of the methods, cells, or compositions described herein, the vector is an excisable vector.

[0263] In one embodiment of any of the methods, cells, or compositions described herein, the in vivo expression of the described transcription factor is regulatable, i.e., the promoter used in the vector to drive gene expression is inducible.

[0264] In one aspect of any method, cell, or composition described herein, the lentivirus is selected from the group consisting of human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0265] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which represents a circular double-stranded DNA loop into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector, into which additional nucleic acid segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors," or more simply, "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because plasmids are the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, the methods and compositions described herein can include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, lentiviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0266] "Operably linked" in an expression vector is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a target cell when the vector is introduced into the target cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Furthermore, a DNA-targeting endonuclease can be delivered at specific intervals or for a specific period of time by a vector containing a regulatory sequence to direct the synthesis of the DNA-targeting endonuclease. It will be appreciated by those skilled in the art that the design of the expression vector can depend on factors such as the selection of the target cell, the desired expression level, and the like.

[0267] Suitable viral vectors include, but are not limited to, RNA virus-based vectors, such as retrovirus-derived vectors (e.g., Moloney murine leukemia virus (MLV)-derived vectors), and more complex retrovirus-derived vectors (such as lentivirus-derived vectors); and DNA virus-based vectors, such as adenovirus-based vectors and adeno-associated virus (AAV)-based vectors. In some embodiments, the polynucleotide delivery system comprises a retroviral vector, more preferably a lentiviral vector. Non-limiting examples of viral vectors include lentiviral vectors derived from human immunodeficiency virus 1 (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, simian immunodeficiency virus (SIV), and Maedi / Visna virus.

[0268] Lymphoid differentiation potential and induction of lymphoid differentiation In another aspect, the present disclosure provides immune cells produced by the methods described herein. These immune cells are genetically modified to have exogenous copies of ERG, HOXA9, and RORA compared to the original myeloid progenitor cells. These immune cells can also have exogenous copies of SOX4 and MYB compared to the original myeloid progenitor cells. These immune cells can also have exogenous copies of DACH1 and NFIA compared to the original myeloid progenitor cells.

[0269] In another aspect, the present disclosure provides immune cells derived from a myeloid progenitor cell population, the immune cells comprising an exogenous copy of each of the following transcription factors: ERG, HOXA9, and RORA.

[0270] In another aspect, the disclosure provides immune cells derived from a myeloid progenitor cell population, the immune cells comprising an exogenous gene-encoding copy of each of the following transcription factors: ERG, HOXA9, and RORA, and an exogenous gene-encoding copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or NANOG and LIN28, or four reprogramming factors: OCT4, SOX2, NANOG, and LIN28.

[0271] In one embodiment of any of the described immune cells, the immune cell further comprises an exogenous gene coding copy of SOX4 or MYB or both SOX4 and MYB.

[0272] In one embodiment of any of the described immune cells, the immune cells further comprise exogenous gene coding copies of DACH1 or NFIA or both DACH1 and NFIA, which enhance lymphoid differentiation potential in the reverse lineage MHPCs described herein.

[0273] In one embodiment of any of the described immune cells, the immune cells are B cells or T cells. In one embodiment of any of the described immune cells, the T cells are regulatory T (T Reg ) cells. In one embodiment of any of the immune cells described, the T cells are natural killer cells.

[0274] Reverse lineage multipotent hematopoietic progenitor cells represent a useful platform that is immortalized and readily amenable to further genetic modifications, such as removal of native T cell receptor loci for enhanced targeting specificity, deletion of class I and class II major histocompatibility complexes, and expression of non-canonical HLA-G and HLA-E to prevent NK cell-mediated lysis (Riolobos L et al. 2013), which can provide a source of universal T cells for immunotherapy, e.g., cancer immunotherapy. In one embodiment of any of the immune cells described, the immune cells can undergo further genetic modifications to edit endogenous HLA (see Riolobos L et al. 2013), or removal of endogenous TCRs or knock-in of chimeric antigen receptors (CARs) for targeting specificity.

[0275] Reverse lineage MHPCs also retained their lymphoid differentiation potential after long-term in vitro culture, averaging approximately 10% after 13 weeks of expansion and differentiation. 4 Approximately 10 EB cells 8 Produces T cells. See Figure 1F.

[0276] In essence, hematopoietic stem cells (HSCs) in the bone marrow give rise to multipotent progenitors (MPPs) before differentiating into common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs). CLPs migrate from the bone marrow to the thymus, where thymic epithelial cells expressing Delta-like ligand 4 (DLL4) trigger classical Notch 1 signaling in early thymic progenitors (ETPs). This Notch 1 signaling is essential for T cell lineage commitment and is required further during early thymocyte differentiation, up to the double-negative 3 (DN3) stage. Active Notch signaling during these early T cell development inhibits other lineage differentiation potentials, such as B cell and myeloid cell (including dendritic cell (DC)) differentiation potential. During β-selection, Notch signaling is released as a result of pre-T cell receptor signaling. Therefore, subsequent T cell developmental stages exhibit very low levels of Notch signaling. Notch regulates regulatory T (T Reg ) cells (specifically, thymic T RegIt has also been suggested that Notch signaling affects the development of mitochondrial cells (cells). Notch signaling is mediated by the Notch 2 receptor. The Notch signaling pathway is highly conserved in both vertebrate and invertebrate species, and it regulates many different cell fate decisions. It is important for developmental patterning, such as neurogenesis, angiogenesis, or myogenesis, and regulates T cell development and stem cell maintenance. Notch signaling is also involved in cellular processes throughout adulthood. Signaling through Notch occurs between neighboring cells, and both the receptor and its ligand are transmembrane proteins. Schmitt TM, Zuniga-Pfluecker JC (2002) Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17:749-756; Mohtashami M. (2010) Direct Comparison of Dll1- and Dll4-Mediated Notch Activation Levels Shows Differential Lymphomyeloid Lineage Commitment Outcomes. J Immunol. 185(2):867-76; Ohishi K et al. Delta-1 enhances marrow and thymus repopulating ability of human CD34( + )CD38( - ) cord blood cells. J Clin Invest. 2002 Oct;110(8):1165-74; and Dallas MH et al. Density of the Notch ligand Delta1 determines generation of B and T cell precursors from hematopoietic stem cells J Exp Med. 2005 May 2; 201(9): 1361-1366.

[0277] Thus, to initiate lymphoid and T cell lineage-committed differentiation in histone methyltransferase-inhibited multipotent hematopoietic progenitor cells, these cells are exposed to Notch ligands to activate the Notch signaling pathway therein.

[0278] Notch ligands are single-pass transmembrane proteins with DSL (Delta, Serrate, LAG-2) domains and a variable number of EGF-like repeats. There are two classes of classical Notch ligands: Delta / Delta-like and Serrate / Jagged. The latter have an additional domain of cysteine-rich repeats near the transmembrane domain. There are five classical Notch ligands in mammals: Jagged-1, Jagged-2, DLL1, DLL3, and DLL4. These can bind to four Notch receptors, Notch 1 to 4. DLL1, also known as the Notch Delta ligand or Delta-like 1, is a protein that interacts with the NOTCH2 receptor. Shimizu K, et al., 2001, J. Biol. Chem. 276 (28): 25753-8; Blaumueller CM, et al., 1997, Cell 90 (2): 281-91; Shimizu K, et al., 2000, Mol. Cell. Biol. 20 (18): 6913-22. DLL1 is a protein encoded by the DLL1 gene in humans. DLL1 is a human homolog of the Notch Delta ligand.

[0279] There are several ways to provide a Notch ligand, including, but not limited to, co-culturing with stromal cells such as OP-9-DL1, or similar cells that extracellularly express and display or secrete such a Notch ligand, and providing purified recombinant Notch ligand or a Notch receptor-binding fragment sufficient to trigger a cell signaling event in vivo upon contact and binding to the extracellular Notch receptor on these cells.

[0280] In one embodiment of any method, cell, or composition described herein, the histone methyltransferase-inhibited multipotent hematopoietic progenitor cells of step (b) are co-cultured with stromal cells that express a Notch ligand.

[0281] In one embodiment of any method, cell, or composition described herein, the co-culturing of the cells occurs in medium comprising Flt-3L and IL-7.

[0282] In one embodiment of any of the methods, cells, or compositions described herein, the co-culturing of the cells is performed in serum-free culture conditions.

[0283] In one embodiment of any method, cell, or composition described herein, the cell expressing a Notch ligand is an OP-9 cell. In one embodiment, the OP-9 cell, otherwise referred to as OP9-DL1 cell, expresses DLL1. In another embodiment, the OP-9 cell, otherwise referred to as OP9-DL4 cell, expresses DLL4.

[0284] In one embodiment of any of the methods, cells, or compositions described herein, the Notch ligand is selected from Delta-like-1 (DLL1), Delta-like-4 (DLL4), and immobilized Delta1 (DLL2), which comprises the extracellular domain of human Delta-like-1 (DLL1) fused to the Fc domain of human IgG1. ext-IgG"Immobilized Delta 1ext-IgG" refers to a recombinant Notch ligand made by fusing the extracellular domain of Delta-like 1 with the Fc domain of human IgG1. This is a synthetic method that provides titratable doses of NOTCH ligand. Varnum-Finney B et al. Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. J Cell Sci. 2000, 23:4313-8. These references are incorporated herein by reference in their entirety. Recombinant Notch ligands and Fc fusions are commercially available under AdipoGen™.

[0285] In one embodiment of any of the methods, cells, or compositions described herein, DLL1 or DLL4 immobilize multilineage hematopoietic progenitor cells. ext-IgG , OP9-DL1 cells, a bone marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (DLL1).

[0286] In one embodiment of any method, cell, or composition described herein, the Notch ligand is DLL1 or DLL4.

[0287] Methods for differentiating progenitor cells into T cells using the Notch signaling pathway and OP9-Notch ligand-expressing cells are known in the art. Any method can be used herein to generate engineered immune cells from multilineage hematopoietic progenitor cells that have previously been inhibited with histone methyltransferase inhibitors. For example, as described in the Examples section, and also in U.S. Patent Nos. 7,575,925, 8,772,028, 8,871,510, and 9,206,394, and U.S. Patent Application Publication Nos. 20090217403, 20110123502, 20110052554, 20110027881, 20110236363, 20120149100, 20130281304, 20140322808, 20140248248, and 20140037599. These references are incorporated herein by reference in their entirety.

[0288] To induce lymphocyte- and B-cell lineage-committed differentiation in histone methyltransferase-inhibited multipotent hematopoietic progenitor cells, these cells are exposed to (1) a B-cell priming factor; (2) coculture with support cells expressing one or more B-lineage growth factors; (3) coculture with support cells expressing CD40L in the absence or presence of one or more B-cell activating factors; (4) exposure to one or more B-cell activating factors; or a combination of (1) to (4) over a culture period.

[0289] In some embodiments, the B cell priming factor can also be a B lineage growth factor. In some embodiments, the B lineage growth factor can also be a B cell priming factor.

[0290] B cell priming factors are known in the art. For example, IL-3, Flt3 ligand, thrombopoietin, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte colony-stimulating factor (GM-CSF), IL-7, and IL-11. As used herein, the term "B cell priming factor" refers to any compound that can support or promote the commitment of hematopoietic stem cells and / or lymphoid progenitor cells to B lineage development. The compound can be a small molecule, polypeptide, protein, or nucleic acid. Various B cell priming factors can be used in the methods and systems described herein. Examples of B cell priming factors include, but are not limited to, interleukin 3 (IL-3), Flt3 ligand, thrombopoietin, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte colony-stimulating factor (GM-CSF), interleukin 7 (IL-7), interleukin 11 (IL-11), antiphosphatase (Sbfl), and mechano-growth factor (MGF). Those skilled in the art will be able to select the amount of B cell priming factor to use based on the particular circumstances. Generally, about 1 to about 1000 ng / ml of B cell priming factor can be used in the methods or systems described herein, although in typical situations, about 1 to about 100 ng / ml of B cell priming factor can be used. However, in some situations, greater or lesser amounts of B cell priming factor can be used. In situations where more than one B cell priming factor is used, the amount of each B cell priming factor can be the same, or the amount of each B cell priming factor can differ from one another.

[0291] B cell activators are known in the art. Examples include CpG DNA, IL-2, IL-10, IL-15, IL-6, IFNα, and anti-CD40L. As used herein, the term "B cell activator" refers to any compound that can promote the activation of naive B cells, preferably the antigen-independent activation of naive B cells. B cell activators can be small molecules, polypeptides, proteins, or nucleic acids. Conventional methods can be used to determine whether a compound has the ability to stimulate the antigen-independent activation of naive B cells. For example, compounds can be tested for the activation of naive B cells isolated from human peripheral blood. Non-limiting examples of B cell activators include CpG DNA; cytokines such as IL-2, IL-3, IL-4, IL-6, IL-10, IL-15, and IFNα; anti-CD40L; and lactic acid. Those skilled in the art will be able to select the amount of B cell activator based on specific circumstances. Generally, about 1 to about 1000 ng / ml of cytokine B cell activator can be used in the methods or systems described herein, but in typical situations, about 1 to about 150 ng / ml, or about 1 to about 100 ng / ml of cytokine B cell activator can be used. However, in some situations, greater or lesser amounts can be used. Generally, about 0.1 to about 5 μM of CpG DNA can be used, but in typical situations, about 0.5 to about 4 μM, or about 1 to about 3.5 μM, or about 1.5 to about 3 μM, or about 2 to about 2.5 μM of CpG DNA can be used. In situations where more than one B cell activator is used, the amount of each B cell activator can be the same, or the amounts of each B cell activator can differ from each other.

[0292] B-lineage growth factors are known in the art. For example, prepro-B cell growth-stimulating factor (PPBSF), insulin-like growth factor-1 (IGF-1), interleukin-3 (IL-3), Flt3 ligand, thrombopoietin, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-11 (IL-11), anti-phosphatase (Sbfl), and mechano-growth factor (MGF). As used herein, the term "B-lineage growth factor" refers to any compound that can promote one or more stages of B cell differentiation during B-lineage development. B-lineage growth factors can be small molecules, polypeptides, proteins, or nucleic acids. Non-limiting examples of stages in B-lineage development include the precursor B cell to early pro-B cell stage, the early pro-B cell to late pro-B cell stage, the late pro-B cell to large pre-B cell stage, the large pre-B cell to small pre-B cell stage, the small pre-B cell to immature B cell stage, and the immature B cell to mature B cell stage. Examples of B-lineage growth factors include, but are not limited to, interleukin-7 (IL-7), pre-pro-B cell growth-stimulating factor (PPBSF), insulin-like growth factor-1 (IGF-1), interleukin-3 (IL-3), Flt3 ligand, thrombopoietin, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-11 (IL-11), antiphosphatase (Sbfl), and mechano-growth factor (MGF). Those skilled in the art can select the amount of B-lineage growth factor based on the particular circumstances. Generally, about 1 to about 1000 ng / ml of B-lineage growth factor can be used in the methods or systems described herein. However, in typical situations, about 1 to about 300 ng / ml, about 20 to about 200 ng / ml, about 50 to about 150 ng / ml, or about 80 to about 150 ng / ml of cytokine B-cell activating factor can be used. However, in some situations, greater or lesser amounts of B-lineage growth factor can be used. In situations where more than one B-lineage growth factor is used, the amount of each B-lineage growth factor can be the same, or the amounts of each B-lineage growth factor can differ from one another.

[0293] The feeder cells used in co-culture for cell differentiation purposes are typically stromal cells. Various stromal cells can be used in the methods described herein. Examples of stromal cell lines include, but are not limited to, the mouse MS5 stromal cell line; mouse bone marrow-derived stromal cell lines, such as S10, S17, OP9, and BMS2 cell lines; and human bone marrow stromal cell lines, such as those described in U.S. Patent No. 5,879,940. This reference is incorporated herein by reference in its entirety. The feeder cells or stromal cells express one or more B-lineage growth factors, such as the growth factor IL-7.

[0294] As used herein, the term "feeder or stromal cells," when used in the context of cell differentiation, refers to any cells that can create, promote, or support a microenvironment for the growth, proliferation, differentiation, or expansion of multipotent hematopoietic progenitor cells or T or B cells. Suitable feeder cells that can be used in the systems and methods disclosed herein include, but are not limited to, stromal cells and fibroblasts.

[0295] In some embodiments, histone methyltransferase-inhibited multipotent hematopoietic progenitor cells are co-cultured with a population of first support cells expressing one or more B-lineage growth factors. In some embodiments, the first support cells are capable of expressing IL-7. In other embodiments, the first support cells are capable of expressing IL-7 and at least one B-lineage growth factor selected from prepro-B cell growth-stimulating factor (PPBSF), insulin-like growth factor-1 (IGF-1), interleukin-3 (IL-3), Flt3 ligand, thrombopoietin, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-11 (IL-11), anti-phosphatase (Sbfl), and mechano-growth factor (MGF). In some embodiments, the one or more B-lineage growth factors are human. In some embodiments, all of the B-lineage growth factors are human. In some embodiments, one or more B-lineage growth factors are derived from a mammal other than a human. In some embodiments, all of the B-lineage growth factors are derived from a mammal other than a human.

[0296] Entry and commitment to the B cell lineage can be monitored by the appearance of B cell-specific markers. Many early B lineage markers are known in the art. For example, pro-B cells can be identified by the co-expression of CD19 and CD10 (CD19+CD10+) and the lack of expression of surrogate light chains.

[0297] The method of differentiating progenitor cells into B cells is known in the art.Any method can be used herein to produce engineered immune cells from the multi-lineage hematopoietic progenitor cells that have been inhibited beforehand by using histone methyltransferase inhibitor.For example, US Patent No. 8034613, US Patent No. 8133727 and US Patent No. 8206979, and US Patent Application Publication No. 20030152558, US Patent No. 20040029271, US Patent No. 20050153443, US Patent No. 20100047854, US Patent No. 2012004036, US Patent No. 20120040362 and US Patent No. 20140273211.These references are incorporated herein by reference in their entirety.

[0298] induced pluripotent stem cells In some embodiments, the pluripotent stem cells (PSCs) described herein are derived from isolated induced pluripotent stem cells (iPSCs). An advantage of using iPSCs is that the cells can be derived from the same subject into which the final immune cells will be reintroduced. That is, somatic cells can be obtained from a subject reprogrammed into induced pluripotent stem cells and then transfected and differentiated into modified immune cells (e.g., autologous cells) to be administered to the subject. Because the progenitor cells are essentially derived from an autologous source, the risk of engraftment rejection or allergic response is reduced compared to the use of cells from another subject or group of subjects. In some embodiments, the cells used to generate iPSCs are derived from a non-autologous source. Additionally, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one embodiment, the PSCs used in the disclosed methods are not embryonic stem cells.

[0299] Although differentiation is generally irreversible in physiological situations, several methods have recently been developed to reprogram somatic cells into induced pluripotent stem cells. Exemplary methods are known to those skilled in the art and are briefly described herein below.

[0300] As used herein, the term "reprogramming" refers to the process of changing or reversing the differentiation state of differentiated cells (e.g., somatic cells). In other words, reprogramming refers to the process of driving cell differentiation back toward a less differentiated or more primitive type of cell. It should be noted that placing large numbers of primary cells in culture can lead to some loss of fully differentiated characteristics. Thus, simply culturing such cells encompassed by the term differentiated cells does not result in these cells becoming non-differentiated (e.g., undifferentiated) or pluripotent cells. The transition from differentiated cells to pluripotency requires a reprogramming stimulus beyond the stimulus that leads to partial loss of differentiation characteristics in culture. Reprogrammed cells also generally have the characteristic of being able to be passaged for extended periods without loss of growth potential, compared to parental primary cells, which only have the ability to divide a limited number of times in culture.

[0301] Cells to be reprogrammed can be in either a partially differentiated or terminally differentiated state prior to reprogramming. In some embodiments, reprogramming involves the complete reversal of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent or multipotent state. In some embodiments, reprogramming involves the complete or partial reversal of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming can result in the expression of specific genes by the cell, whose expression further contributes to the reprogramming. In certain embodiments described herein, reprogramming of a differentiated cell (e.g., a somatic cell) results in a differentiated cell that assumes an undifferentiated state (e.g., is an undifferentiated cell). The resulting cell is referred to as a "reprogrammed cell" or "induced pluripotent stem cell (iPSC or iPS cell)."

[0302] Reprogramming can involve alteration, e.g., reversal, of at least some of the inherited patterns of nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cell differentiation. Reprogramming is distinct from simply maintaining the existing undifferentiated state of a cell that is already pluripotent, or maintaining the existing less-than-fully differentiated state of a cell that is already multipotent (e.g., a common myeloid stem cell). Reprogramming is also distinct from promoting self-renewal or proliferation of cells that are already pluripotent or multipotent, although the compositions and methods described herein can be useful for such purposes in some embodiments.

[0303] The particular approach or method used to generate pluripotent stem cells from somatic cells (broadly referred to as "reprogramming") is not critical to the claimed invention. Thus, any method of reprogramming somatic cells to a pluripotent phenotype is suitable for use in the methods described herein.

[0304] Reprogramming methodologies for generating pluripotent cells using defined combinations of transcription factors have been described to induce pluripotent stem cells from somatic cells. Yamanaka and Takahashi converted mouse somatic cells into ES cell-like cells with expanded developmental potential by direct transduction of Oct4, Sox2, Klf4, and optionally c-Myc. See U.S. Patent Nos. 8,058,065 and 9,045,738 to Yamanaka and Takahashi. iPSCs resemble ES cells because they restore much of the pluripotency-associated transcriptional circuitry and epigenetic landscape. In addition, mouse iPSCs satisfy all standard assays for pluripotency: specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, chimera contribution, germline transmission, and tetraploid complementation.

[0305] Subsequent studies have shown that similar transduction methods can be used to obtain human iPS cells, and a trio of transcription factors: OCT4, SOX2, and NANOG, have been established as the core set of transcription factors governing pluripotency. iPS cell production can be achieved by using viral vectors to introduce nucleic acid sequences encoding stem cell-associated genes into adult somatic cells.

[0306] iPS cells can be generated or derived from terminally differentiated somatic cells and adult or somatic stem cells. That is, non-pluripotent progenitor cells can be reprogrammed to pluripotency or multipotency. In such cases, it may not be necessary to include the same number of reprogramming factors as is required to reprogram terminally differentiated cells. Furthermore, reprogramming can be induced by non-viral introduction of reprogramming factors, for example, by introducing the protein itself, or by introducing a nucleic acid encoding the reprogramming factor, or by introducing a messenger RNA that produces the reprogramming factor when translated (see, e.g., Warren et al., Cell Stem Cell, 2010 Nov 5;7(5):618-30, which is incorporated herein by reference in its entirety). Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-related genes, including, for example, Oct-4 (also known as Oct-3 / 4 or Pouf51), Sox1, Sox2, Sox3, Sox15, Sox18, NANOG, Klf1, Klf2, Klf4, Klf5, NR5A2, c-Myc, l-Myc, n-Myc, Rem2, Tert, and LIN28. In one embodiment, reprogramming using the methods and compositions described herein can further include introducing one or more of Oct-3 / 4, a member of the Sox family, a member of the Klf family, and a member of the Myc family into somatic cells. In one embodiment, the methods and compositions described herein further include introducing one or more of Oct4, Sox2, Nanog, c-MYC, and Klf4 for reprogramming. As noted above, the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, if cells differentiated from the reprogrammed cells are to be used, for example, in human therapy, in one embodiment, the reprogramming is not affected by methods that alter the genome, and thus, in such an embodiment, the reprogramming is achieved without the use of, for example, viral or plasmid vectors.

[0307] The efficiency of reprogramming (i.e., the number of reprogrammed cells) obtained from the starting cell population can be enhanced by adding various small molecules, as shown by Shi, Y., et al. (2008) Cell-Stem Cell 2:525-528, Huangfu, D., et al. (2008) Nature Biotechnology 26(7):795-797, and Marson, A., et al. (2008) Cell-Stem Cell 3:132-135. This reference is incorporated herein by reference in its entirety. Therefore, agents or combinations of agents that increase the efficiency or speed of induced pluripotent stem cell production can be used to produce patient-specific or disease-specific iPSCs. Some non-limiting examples of agents that enhance reprogramming efficiency include, among others, soluble Wnt, Wnt-conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA).

[0308] Other non-limiting examples of reprogramming enhancers include suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (e.g., (-)-depudecin), HC toxin, Nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyrate (e.g., sodium phenylbutyrate) and valproic acid ((VPA) and other short-chain fatty acids), scriptaid, suramin sodium, trichostatin A (TSA), APHA Compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate (pivaloyloxymethyl butyrate), and valproic acid ((VPA) and other short-chain fatty acids). These include Pivanex, AN-9, trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnamic acid bishydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50. Other reprogramming enhancer includes, for example, dominant negative HDAC (for example, catalytically inactive), siRNA inhibitor of HDAC, and antibody that specifically binds to HDAC.Such inhibitor is available from, for example, BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene and Sigma Aldrich.

[0309] To confirm the induction of pluripotent stem cells for use in the methods described herein, isolated clones can be tested for the expression of stem cell markers. Such expression in cells derived from somatic cells identifies the cells as induced pluripotent stem cells. Stem cell markers can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, cells expressing Oct4 or Nanog are identified as pluripotent. Methods for identifying the expression of such markers can include immunological methods that detect the presence of the encoded polypeptide, such as RT-PCR and Western blot or flow cytometry analysis. In some embodiments, detection not only involves RT-PCR but also includes the detection of protein markers. Intracellular markers can best be identified via RT-PCR, whereas cell surface markers are easily identified by, for example, immunocytochemistry.

[0310] The pluripotent stem cell characteristics of isolated cells can be confirmed by testing the ability of iPSCs to differentiate into cells of each of the three germ layers. As an example, the pluripotent characteristics of isolated clones can be evaluated using the formation of teratomas in nude mice. The cells are introduced into nude mice, and tumors arising from the cells are subjected to histological and / or immunohistochemical examination. The growth of tumors containing cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.

[0311] Numerous U.S. patents and published U.S. patent applications teach and describe methods for generating iPSCs and related subject matter, for example, U.S. Patent Nos. 9,347,044, 9,347,042, 9,347,045, 9,340,775, 9,341,625, 9,340,772, 9,250,230, 9,132,152, 9,045,738, 9,005,975, 9,005,976, 8,927,277, 8,993,329, 8,900,871, 8,852,941, Nos. 8802438, 8691574, 8735150, 8765470, 8058065, and 8048675, as well as U.S. Patent Application Publication Nos. 20090227032, 20100210014, 20110250692, 20110201110, 20110200568, 20110306516, No. 20100021437, No. 20110256626, No. 20110044961, No. 20120276070, No. 20120263689, No. 20120128 No. 655, No. 20120100568, No. 20130295064, No. 20130029866, No. 20130189786, No. 20130295579, No. 20 Nos. 130130387, 20130157365, 20140234973, 20140227736, 20140093486, 20140301988, 20140170746, 20140178989, 20140349401, 20140065227, and 20150140662. These references are incorporated herein by reference in their entirety.

[0312] Somatic cells for reprogramming Somatic cells, as the term is used herein, refer to any cell that forms the body of an organism, excluding germline cells. With the exception of sperm and eggs, the cells from which they are made (gametocytes), and undifferentiated stem cells, all cell types in the mammalian body are differentiated somatic cells. For example, internal organs, skin, bones, blood, and connective tissue are all made up of differentiated somatic cells.

[0313] Additional somatic cell types for use in the compositions and methods described herein include fibroblasts (e.g., primary fibroblasts), muscle cells (e.g., myocytes), cumulus cells, neural cells, mammary cells, hepatocytes, and pancreatic islet cells. In some embodiments, the somatic cells are primary cell lines or the progeny of primary or secondary cell lines. In some embodiments, the somatic cells are obtained from a human sample, such as a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy, or a fat biopsy), a swab sample (e.g., a buccal swab sample), and are therefore human somatic cells.

[0314] Some non-limiting examples of differentiated somatic cells include, but are not limited to, epithelial cells, endothelial cells, neural cells, adipocytes, cardiac cells, skeletal muscle cells, skin cells, immune cells, liver cells, splenocytes, lung cells, peripheral circulating blood cells, gastrointestinal cells, kidney cells, bone marrow cells, and pancreatic cells. In some embodiments, somatic cells can be primary cells isolated from any somatic tissue, including, but not limited to, brain, liver, gastrointestinal tract, stomach, intestinal tract, fat, muscle, uterus, skin, spleen, endocrine organs, bone, etc. Furthermore, somatic cells can be from any mammalian species, non-limiting examples of which include murine, bovine, simian, porcine, equine, ovine, or human cells. In some embodiments, somatic cells are human somatic cells.

[0315] When reprogrammed cells are used to generate thyroid progenitor cells to be used in the therapeutic treatment of a disease, it is desirable, but not necessary, to use somatic cells isolated from the patient to be treated. For example, somatic cells involved in the disease, somatic cells related to the therapeutic treatment of the disease, etc., can be used. In some embodiments, methods for selecting reprogrammed cells from a heterogeneous population containing reprogrammed cells and the somatic cells from which they were derived or generated can be performed by any known means. For example, reprogrammed cells can be isolated using a selectable marker, such as a drug resistance gene, as an indicator.

[0316] The reprogrammed somatic cells disclosed herein may express alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; β-III-tubulin; α-smooth muscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, and Dax1. ; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (embryonic stem cell-associated transcript 1 (ECAT1); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14; blastocyst transcription factor (Utf1); Rex1; p53; G3PDH The induced pluripotent stem cells can express any number of pluripotent cell markers, including telomerase, including TERT; silent X-chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box-containing protein 15 (Fbx15); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency-associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3 / Stella; DPPA4; and other general markers for pluripotency. Other markers include Dnmt3L; Sox15; Stat3; Grb2; β-catenin, and Bmi1. Such cells can also be characterized by down-regulation of markers characteristic of the somatic cells from which the induced pluripotent stem cells are derived.

[0317] Use of engineered immune cells derived from pluripotent stem cells In one aspect, provided herein is a population of engineered immune cells produced by the methods described herein, wherein the cells contain an exogenous gene-encoding copy of each of the transcription factors ERG, HOXA9, and RORA, and optionally an exogenous gene-encoding copy of each of the transcription factors SOX4 and MYB, or further contain an exogenous gene-encoding copy of each of the transcription factors DACH1 and NFIA, or further contain an exogenous gene-encoding copy of each of the transcription factors SOX4, MYB, DACH1, and NFIA. In one aspect, the cell population further contains a pharmaceutically acceptable carrier. These engineered immune cells can be culture-expanded to increase the number of cells for use.

[0318] The engineered immune cells described herein are useful in laboratories for biological research. For example, these cells can be derived from individuals with a genetic disease or defect and can be used in laboratories to study the biological aspects of the disease or defect and to screen and test for potential therapeutic agents for the disease or defect.

[0319] Alternatively, the engineered immune cells described herein are useful for cell replacement therapy and other medical treatments in subjects in need thereof, such as patients who have undergone chemotherapy or radiation or both and have demonstrated defects in immune function and / or lymphocyte reconstitution, or who have demonstrated incomplete cancer immunotherapy.

[0320] In various aspects, the engineered immune cells described herein are administered (ie, implanted or transplanted) to a subject in need of cell replacement therapy.

[0321] In one aspect, provided herein is a method for cell replacement therapy or treatment of cancer, autoimmune disorders, hematological diseases, or other genetic diseases and disorders in a subject, the method comprising: (a) providing somatic cells from a donor subject; (b) generating multilineage hematopoietic progenitor cells from somatic cell-derived myeloid progenitor cells as described in any of the preceding paragraphs; (c) inhibiting histone methyltransferase in the resulting population of multilineage hematopoietic progenitor cells as described in any of the preceding paragraphs; (d) differentiating the resulting population of multilineage hematopoietic progenitor cells in the presence of a notch ligand or stromal cells, or both, to promote differentiation into lymphoid lineages as described in any of the preceding paragraphs; and (e) transplanting or administering the resulting differentiated lymphoid cells to a recipient subject.

[0322] In one embodiment of the above treatment method, the host subject and recipient subject are the same individual.

[0323] In one embodiment of the above treatment method, the host subject and recipient subject are not the same individual, but are at least HLA-compatible.

[0324] Hematological disease is the disorder that mainly affects blood.Non-limiting such disease or disorder includes hemoglobinopathy (congenital abnormality of hemoglobin molecule or hemoglobin synthesis rate), such as sickle cell disease, thalassemia and methemoglobinemia, myeloid-derived disorder; anemia (deficiency of red blood cells or hemoglobin), pernicious anemia; myelodysplastic syndrome, neutropenia (reduced neutrophil count) and thrombotic thrombocytopenic purpura (TTP), thrombocytosis, hematological malignancies, such as lymphoma, myeloma and leukemia, which causes the disorder that reduces cell count. Lymphomas such as Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, splenic marginal zone lymphoma, hepatosplenic T-cell lymphoma, and angioimmunoblastic T-cell lymphoma (AILT); myelomas such as multiple myeloma, Waldenstrom's macroglobulinemia, and plasmacytoma; and leukemias with increased defective WBCs such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic idiopathic myelofibrosis (MF), chronic myelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), chronic neutrophilic leukemia (CNL), hairy cell leukemia (HCL), T-cell large granular lymphocyte leukemia (T-LGL), and aggressive NK-cell leukemia.

[0325] Autoimmune diseases such as diabetes, rheumatoid arthritis and multiple sclerosis.

[0326] As used herein, the terms "administering," "introducing," and "implanting" are used interchangeably in the context of the placement of the described cells, e.g., hematopoietic progenitor cells, into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, so that a desired effect occurs. Cells, e.g., hematopoietic progenitor cells, or their differentiated progeny, can be administered by any suitable route that results in delivery to a desired location within a subject, where at least a portion of the implanted cells or components of the cells remain viable.

[0327] In various aspects, the engineered immune cells described herein are optionally expanded ex vivo before administration to a subject. In other aspects, the engineered immune cells are optionally cryopreserved for a period of time and then thawed before administration to a subject.

[0328] Engineered immune cells used for cell replacement therapy can be autologous / autogenous ("self") or non-autologous ("non-self", e.g., allogeneic, syngeneic, or xenogeneic) to the recipient of the cells. As used herein, "autologous" refers to cells from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cells being compared. As used herein, "syngeneic" refers to cells from a different subject that are genetically identical to the cells being compared. As used herein, "xenogeneic" refers to cells of a different species from the cells being compared. In preferred embodiments, the cells of the present invention are allogeneic.

[0329] In various aspects, the engineered immune cells described herein to be implanted into a subject in need thereof are autologous or allogeneic to the subject.

[0330] In various embodiments, the engineered immune cells described herein can be derived from one or more donors or can be obtained from an autologous source. In some embodiments of the aspects described herein, the engineered immune cells are expanded in culture before being administered to a subject in need thereof.

[0331] In various embodiments, the engineered immune cells described herein can be derived from one or more donors or can be obtained from an autologous source.

[0332] In various embodiments, prior to implantation, the recipient subject is treated with chemotherapy and / or radiation.

[0333] In one embodiment, chemotherapy and / or radiation is to deplete endogenous stem cells and promote cell engraftment after implantation.

[0334] In various embodiments, prior to implantation, the engineered immune cells or inhibited reverse lineage multilineage hematopoietic progenitor cells are treated ex vivo with prostaglandin E2 and / or the antioxidant N-acetyl-L-cysteine ​​(NAC) to promote subsequent engraftment in the recipient subject.

[0335] In various aspects, the recipient subject is a human.

[0336] In various embodiments, the subject has been diagnosed with HIV or other viral disease, a blood disorder, or is undergoing treatment for cancer.

[0337] In one aspect of any of the methods, cells, and compositions described herein, a subject is selected to donate the somatic cells used to produce the iPSCs and engineered immune cells described herein. In one embodiment, the selected subject has a genetic disease or defect.

[0338] In various aspects, the donor subject is a human.

[0339] In various embodiments, the donor or recipient subject is an animal, human or non-human, and rodent or non-rodent. For example, the subject can be any mammal, such as a human, other primate, pig, rodent such as a mouse or rat, rabbit, guinea pig, hamster, cow, horse, cat, dog, sheep or goat, or non-mammal, such as a bird.

[0340] In various embodiments, the donor or recipient subject has been diagnosed with HIV, a blood disorder, or cancer.

[0341] In one aspect of any of the methods, cells, and compositions described herein, a biological sample or population of embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells is obtained from a donor subject.

[0342] In various embodiments, the biological sample or population of embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells described herein can be derived from one or more donors or can be obtained from an autologous source.

[0343] In one embodiment, embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells are isolated from a donor subject, transfected, cultured (optionally), and transplanted back into the same subject, i.e., an autologous cell transplant. Here, the donor and recipient subject are the same individual. In another embodiment, embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells are isolated from a donor whose HLA type is matched with that of the subject (recipient). Donor-recipient antigen type matching is well known in the art. HLA types include HLA-A, HLA-B, HLA-C, and HLA-D. These represent the minimum number of cell surface antigen matches required for transplantation. That is, the transfected cells are transplanted into a different subject, i.e., allogeneic to the recipient host subject. Donor or subject embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells can be transfected with a vector or nucleic acid comprising a nucleic acid molecule described herein, and the transfected cells are cultured, inhibited, differentiated as disclosed, optionally expanded, and then transplanted into a recipient subject. In one aspect, the engineered immune cells after transplantation engraft the recipient subject. In one aspect, the engineered immune cells after transplantation reconstitute the immune system in the recipient subject. The transfected cells can also be cryopreserved and stored after transfection or cryopreserved and stored after cell expansion.

[0344] The engineered immune cells or histone methyltransferase-inhibited reverse-lineage multilineage hematopoietic progenitor cells may be administered as part of a bone marrow or umbilical cord blood graft in individuals who have or have not undergone myeloablative therapy. In one aspect, the genetically modified cells contemplated herein are administered in a bone marrow transplant to an individual who has undergone chemoablative or radioablative bone marrow therapy.

[0345] In one embodiment, a dose of cells is delivered to the subject intravenously. In one embodiment, the cells are administered to the subject intravenously.

[0346] In certain embodiments, patients receive a dose of modified cells described herein, e.g., engineered immune cells or histone methyltransferase-inhibited reverse lineage multilineage hematopoietic progenitor cells, in an amount of about 1 x 10 5 pieces / kg, approximately 5×10 5 pieces / kg, approximately 1×10 6 pieces / kg, approximately 2×10 6 pieces / kg, approximately 3×10 6 pieces / kg, approximately 4×10 6 pieces / kg, approximately 5×10 6 pieces / kg, approximately 6×10 6 pieces / kg, approximately 7×10 6 pieces / kg, approximately 8×10 6 pieces / kg, approximately 9×10 6 pieces / kg, approximately 1×10 7 pieces / kg, approximately 5×10 7 pieces / kg, approximately 1×10 8 Receive a single intravenous dose of 100 mg / kg or greater.

[0347] In certain embodiments, patients receive a dose of at least 1 x 10 modified cells described herein, e.g., engineered immune cells or histone methyltransferase-inhibited reverse lineage multilineage hematopoietic progenitor cells. 5 pieces / kg, at least 5 x 10 5 pieces / kg, at least 1 x 10 6 pieces / kg, at least 2 x 10 6 pieces / kg, at least 3 x 10 6pieces / kg, at least 4 x 10 6 pieces / kg, at least 5 x 10 6 pieces / kg, at least 6 x 10 6 pieces / kg, at least 7 x 10 6 pieces / kg, at least 8 x 10 6 pieces / kg, at least 9 x 10 6 pieces / kg, at least 1 x 10 7 pieces / kg, at least 5 x 10 7 pieces / kg, at least 1 x 10 8 Receive a single intravenous dose of 100 mg / kg or greater.

[0348] In additional embodiments, patients receive a dose of modified cells described herein, e.g., engineered immune cells or histone methyltransferase-inhibited reverse lineage multilineage hematopoietic progenitor cells, in an amount of about 1 x 10 5 pieces / kg to approximately 1 x 10 8 pieces / kg, approximately 1×10 6 pieces / kg to approximately 1 x 10 8 pieces / kg, approximately 1×10 6 pieces / kg to approximately 9 x 10 6 pieces / kg, approximately 2×10 6 pieces / kg to approximately 8 x 10 6 pieces / kg, approximately 2×10 6 pieces / kg to approximately 8 x 10 6 pieces / kg, approximately 2×10 6 pieces / kg to approximately 5 x 10 6 pieces / kg, approximately 3×10 6 pieces / kg to approximately 5 x 10 6 pieces / kg, approximately 3×10 6 pieces / kg to approximately 4 x 10 8 Receive a dose of 100 mg / kg, or any intervening dose of 100 mg / kg.

[0349] Generally, the engineered immune cells or histone methyltransferase-inhibited reverse-lineage multilineage hematopoietic progenitor cells described herein are administered as a suspension in a pharmaceutically acceptable carrier, e.g., as a therapeutic composition. Therapeutic compositions contain a physiologically tolerable carrier together with the cell composition and, optionally, at least one additional bioactive agent described herein dissolved or dispersed therein as an active ingredient. In preferred embodiments, the therapeutic composition is substantially non-immunogenic when administered to a mammalian or human patient for therapeutic purposes, unless otherwise desired. Those skilled in the art will recognize that pharmaceutically acceptable carriers to be used in cell compositions will not contain buffers, compounds, cryopreservatives, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to a subject. Cell-containing formulations can include, for example, an osmotic buffer to maintain cell membrane integrity, and optionally nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with the cells described herein using routine experimentation.

[0350] As used herein, the terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof when describing compositions, carriers, diluents, and reagents are used interchangeably to indicate that these substances can be administered to a mammal without causing undesired physiological effects, such as nausea, dizziness, or stomach upset. A pharmaceutically acceptable carrier does not promote the development of an immune response to the drug with which it is admixed, unless so desired. The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared for injection as either liquid solutions or suspensions, although solid dosage forms suitable for dissolution or suspension in liquid prior to use can also be prepared. Preparations can also be emulsified or presented as liposomal compositions. The active ingredient can be mixed with an excipient that is pharmaceutically acceptable, compatible with the active ingredient, and in an amount suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient. The therapeutic compositions of the present invention may contain pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art. An exemplary liquid carrier is a sterile aqueous solution containing no materials other than the active ingredient and water, or containing both a buffer such as sodium phosphate, saline, or phosphate-buffered saline at a physiological pH value.Furthermore, aqueous carriers can also contain one or more buffer salts, and salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to and excluding water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent used in the methods described herein that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of the active ingredient in 0.9% sodium chloride solution.

[0351] In one aspect, a "pharmaceutically acceptable" carrier does not include in vitro cell culture media.

[0352] In some aspects, the described compositions of engineered immune cells further comprise a pharmaceutically acceptable carrier. In one aspect, the pharmaceutically acceptable carrier does not comprise tissue or cell culture medium.

[0353] In various embodiments, the second or subsequent dose of cells is administered to recipient subject.For example, the second and subsequent administrations can be given between about 1 day and 30 weeks after previous administration.2, 3, 4 or more total administrations can be delivered to individual as needed.

[0354] The cell composition can be administered by any suitable route that results in an effective cell replacement treatment in the subject, i.e., administration results in delivery to a desired location in the subject, where at least a portion of the composition is delivered, i.e., at least 1×10 4The cells are delivered to the desired site over a period of time. Modes of administration include injection, infusion, or drip infusion, where "injection" includes, but is not limited to, intravenous, intraarterial, intracerebroventricular, and intracardiac injection and infusion. For cell delivery, administration by injection or infusion is generally preferred.

[0355] Efficacy testing can be performed over the course of treatment using the methods described herein: measurements of the severity of several symptoms associated with a particular disease are recorded before treatment begins, and then at specific time periods thereafter after treatment begins.

[0356] The present invention can be defined in any of the following numbered paragraphs: [1]. a. Generating multilineage hematopoietic progenitor cells from myeloid progenitor cells; b. inhibiting histone methyltransferases in the resulting multi-lineage hematopoietic progenitor cell population; and c. Differentiating the resulting population of multilineage hematopoietic progenitor cells in the presence of notch ligand or stromal cells or both to promote differentiation into lymphoid lineages. A method comprising: [2]. a. transfecting myeloid progenitor cells in vitro with exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA, wherein the transcription factors are expressed in the transfected cells to produce a multilineage hematopoietic progenitor cell population with myeloid and erythroid differentiation potential; b. inhibiting histone methyltransferases in the resulting multilineage hematopoietic progenitor cell population to expand lymphoid differentiation potential; and c. Differentiating the resulting population of multilineage hematopoietic progenitor cells in the presence of a notch ligand or supportive stroma, or both, to promote differentiation into lymphoid lineages. A method comprising: [3]. The method of item 1, wherein the multilineage hematopoietic progenitor cells are produced by introducing each of the following transcription factors: ERG, HOXA9, and RORA into myeloid progenitor cells in vitro. [4] The method of items 2 or 3, further comprising transfecting the myeloid progenitor cells with exogenous gene-encoding copies of the transcription factors: SOX4 and MYB. [5]. The method of items 2, 3, or 4, further comprising transfecting exogenous gene-encoding copies of the transcription factors: NFIA and DACH1 into the myeloid progenitor cells. [6] The method of any one of items 1 to 5, wherein the myeloid progenitor cells are CD34+ CD45+. [7] The method of any one of items 1 to 6, wherein the multilineage hematopoietic progenitor cells are CD34+ CD38-negative / low expressing. [8] The method of any one of items 1 to 7, wherein the myeloid-lineage progenitor cells are embryoid body progenitor cells derived from a pluripotent stem cell population. [9]. The method of item 8, wherein the pluripotent stem cell population is induced pluripotent stem cells (iPS cells) or embryonic stem cells (ESCs).

[10] . The method of item 9, wherein the induced pluripotent stem cells are produced by introducing reprogramming factors: OCT4, SOX2, KLF4 alone, and optionally c-MYC or nanog and LIN28 into mature cells.

[11] The method of item 10, wherein the mature cells are selected from the group consisting of B lymphocytes (B cells), T lymphocytes (T cells), fibroblasts, and keratinocytes.

[12] . The method of items 9, 10 or 11, wherein the induced pluripotent stem cells are produced by introducing reprogramming factors into mature cells two or more times.

[13] . Notch ligands include Delta-like-1, Delta-like-4, and immobilized Delta1, which consists of the extracellular domain of human Delta-like-1 fused to the Fc domain of human IgG1. ext-IgG 13. The method of any of items 1 to 12, wherein the method is selected from the group consisting of:

[14] . Delta1 fixed multilineage hematopoietic progenitor cells ext-IgGItem 14. The method of item 13, wherein Delta-like-1 or Delta-like-4 is supplied by co-culturing with OP9-DL1 cells or OP9-DL4 cells.

[15] The method of any of items 1 to 14, wherein the histone methyltransferase catalyzes the addition of a methyl group to histone 3 lysine residue 9 (H3K9) and / or histone 3 lysine residue 27 (H3K27).

[16] The method of item 15, wherein histone methyltransferases H3K9 and / or H3K27 are inhibited by a small molecule or a nucleic acid.

[17] The method of item 16, wherein the histone methyltransferase H3K9 and / or H3K27 small molecule inhibitor is an organic or inorganic compound having a molecular weight of less than about 10,000 grams / mole, or a salt or ester or other pharmaceutically acceptable form of said compound, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a nucleotide, or a nucleotide analog.

[18] The method of items 16 or 17, wherein the histone methyltransferase H3K9 and / or H3K27 small molecule inhibitor is a heteroorganic or organometallic compound.

[19] . The method of any of items 16 to 18, wherein the small molecule inhibitor is selected from the group consisting of BIX-01294, UNC0638, E72, BRD4770, A-366, chaetocin, UNC0224, UNC0631, UNC0646, EPZ005687, EPZ-6438 (E7438), 3-deazaneplanocin A (DZNep), EI1, GSK343, GSK126, and UNC1999.

[20] . The method of item 16, wherein the nucleic acid inhibitor is a nucleic acid that targets the expression of a histone methyltransferase.

[21] The method of items 16 or 17, wherein the nucleic acid inhibitor is an RNA interference inhibitor or an RNA interference agent.

[22] . The nucleic acid inhibitor is an EZH1-specific nucleic acid selected from the group consisting of an aptamer that binds to EZH1, an EZH1-specific RNA interference agent, or a vector encoding an EZH1-specific RNA interference agent; The RNA interference agent comprises one or more of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-5, 27-30; Item 21 method.

[23] . Immune cells produced by any of the methods described in items 1 to 22.

[24] . Immune cells derived from myeloid progenitor populations contain exogenous copies of each of the following transcription factors: ERG, HOXA9, and RORA.

[25] . The immune cells of item 24 further contain exogenous copies of each of the following reprogramming factors: SOX4 and MYB.

[26] . The immune cells of paragraphs 24 or 25, further comprising an exogenous copy of each of the following reprogramming factors: NFIA and DACH1.

[27] . The immune cells of paragraphs 24, 25, or 26, further comprising an exogenous copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC.

[28] . The immune cells of any of paragraphs 24 to 27, which have been further genetically modified to ablate the native T cell receptor (TCR) locus, to delete class I or class II major histocompatibility complexes or both, to express non-classical HLA-G or HLA-E or both, or to edit the endogenous HLA therein.

[29] A composition comprising a population of immune cells according to any one of items 23 to 28.

[30] . The composition of item 29, further comprising a pharmaceutically acceptable carrier.

[31] A pharmaceutical composition comprising a population of immune cells according to any one of items 23 to 28 and a pharmaceutically acceptable carrier.

[32] . The pharmaceutical composition of item 31 for use in cell replacement therapy in a subject.

[33] . a. generating multilineage hematopoietic progenitor cells from myeloid progenitor cells by the method of paragraphs 2-12; b. inhibiting histone methyltransferases in the resulting multilineage hematopoietic progenitor cell population by the method of items 15-22; c. Differentiating the resulting population of multilineage hematopoietic progenitor cells in the presence of Notch ligand or supporting stroma or both to promote differentiation into lymphoid lineages by the method of items 13-14; and d. Transplanting the resulting multilineage hematopoietic progenitor cells into a host. 10. An ex vivo or in vitro method for improving in vivo engraftment of hematopoietic cells in a host, comprising:

[34] A method for cell replacement therapy or immunotherapy in a subject in need thereof, comprising administering to the recipient subject the population of immune cells of items 23-28, or the composition of items 29-30, or the pharmaceutical composition of items 31-32.

[35] The method of cell replacement therapy of item 34, wherein the subject is a patient who has undergone chemotherapy or irradiation or both and has demonstrated defects in immune function or lymphocyte reconstitution, or defects in both immune function and lymphocyte reconstitution.

[36] The method of cell replacement therapy of items 34 or 35, wherein prior to implantation into the subject, the immune cells are treated ex vivo with prostaglandin E2 and / or the antioxidant N-acetyl-L-cysteine ​​(NAC) to promote subsequent engraftment in the recipient subject.

[37] The cell replacement therapy method of items 34 or 35, wherein the immune cells are autologous to the recipient subject or at least HLA-type matched to the recipient subject.

[38] . Modified or engineered myeloid progenitor cells with reverted lineages, including increased lymphoid lineage differentiation potential.

[39] . A composition comprising modified or engineered myeloid progenitor cells with reverted lineage, including increased lymphoid lineage differentiation potential.

[40] . A composition comprising modified myeloid progenitor cells or modified or engineered myeloid progenitor cells for use in the manufacture / production of the described modified immune cells, wherein the modified myeloid progenitor cells have a reverse lineage and have increased lymphoid lineage differentiation potential, and the modified myeloid progenitor cells comprise an exogenous gene-coding copy of each of the following transcription factors: ERG, HOXA9, and RORA.

[41] . A composition comprising modified myeloid progenitor cells or modified or engineered myeloid progenitor cells for use in cell replacement therapy or for the treatment of cancer, autoimmune disorders, blood disorders, or other genetic diseases and disorders, wherein the modified myeloid progenitor cells have reverse lineage and increased lymphoid lineage differentiation potential, and the modified myeloid progenitor cells contain exogenous gene-encoding copies of each of the following transcription factors: ERG, HOXA9, and RORA.

[42] . The modified myeloid progenitor cells of items 38-41 further contain exogenous gene coding copies of SOX4, or MYB, or both SOX4 and MYB.

[43] . The modified myeloid progenitor cells of items 38-42 further contain exogenous gene coding copies of DACH1, or NFIA, or both DACH1 and NFIA.

[44] . The engineered myeloid progenitor cells in items 38–43 express the lineage-restricted CD34 + CD45 + It is derived from myeloid progenitor cells.

[45] . The modified myeloid progenitor cells of paragraphs 38-44 further comprise an exogenous copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC.

[46] . A method of cell replacement therapy or a method for treating cancer, an autoimmune disorder, a blood disorder, or other genetic diseases and disorders in a subject, comprising: (a) providing somatic cells from a donor subject; (b) generating multilineage hematopoietic progenitor cells from myeloid progenitor cells derived from the somatic cells of any of the preceding items; (c) inhibiting histone methyltransferase in the resulting population of multilineage hematopoietic progenitor cells of any of the preceding items; (d) differentiating the resulting population of multilineage hematopoietic progenitor cells in the presence of a Notch ligand or stromal cells or both to promote differentiation into the lymphoid lineage of any of the preceding items, and transplanting the resulting differentiated lymphoid cells into a recipient subject.

[47] The method of item 46, wherein the host subject and recipient subject are the same individual.

[48] ​​The method of item 46, wherein the host subject and recipient subject are not the same individual, but are at least HLA-compatible.

[0357] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references as well as figures and tables cited throughout this application are hereby incorporated by reference.

[0358] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims. [Example]

[0359] Experimental procedure Culture of hIPSCs All experiments were performed using MSC-IPS1 (Park et al., 2008), CD34-IPS, and CD45-IPS. Human IPS cells were maintained on mouse embryonic fibroblast (GlobalStem) feeders in DMEM / F12 + 20% knockout serum substitute (Invitrogen™), 1 mM L-glutamine, 1 mM NEAA, 0.1 mM β-mercaptoethanol, and 10 ng / ml bFGF. Medium was changed daily, and cells were passaged 1:4 onto fresh feeders every 7 days using standard clump passaging with collagenase IV.

[0360] EB differentiation EB differentiation was performed as previously described (Chadwick et al., 2003). Briefly, hPSC colonies were scraped and plated at a 2:1 ratio onto rotating, non-adherent 10 cm plates. EB medium was KO-DMEM + 20% FBS (Stem Cell Technologies), 1 mM L-glutamine, 1 mM NEAA, penicillin / streptomycin, 0.1 mM β-mercaptoethanol, 200 μg / ml h-transferrin, and 50 μg / ml ascorbic acid. After 24 h, the medium was changed by allowing the EBs to settle by gravity and replaced with EB medium supplemented with growth factors: 50 ng / ml BMP4 (R&D Systems), 200 ng / ml SCF, 200 ng / ml FLT3, 50 ng / ml G-CSF, 20 ng / ml IL-6, and 10 ng / ml IL-3 (all Peprotech). The medium was changed on days 5 and 10. On day 14, EBs were dissociated by digestion with collagenase B (Roche) for 2 hours, followed by treatment with enzyme-free dissociation buffer (Gibco) and filtration through an 80 μm filter. Dissociated EBs were frozen in a 10% DMSO, 40% FBS freezing solution.

[0361] Progenitor cell sorting Dissociated EB cells were thawed according to the Lonza Poietics protocol, which can be found in the manual and instructions section of the Lonza website for the Poietics Cell Thawing Procedure. Thawed cells were diluted to 1 x 10 per 100 μl of staining buffer (PBS + 2% FBS). 6 CD34+ cells were sorted from bulk EB cultures using human CD34 microbeads (Miltenyi Biotec) and passed through a magnetic column separator (MACS) according to the manufacturer's instructions.

[0362] Lentiviral and shRNA library plasmids 5F lentiviral plasmids: HOXA9, ERG, RORA, SOX4, and MYB were cloned into the pInducer-21 Dox-inducible lentiviral vector. Four shRNAs against each epigenetic modifier and three shRNAs against luciferase were obtained from the Broad Institute RNAi Consortium in the pLKO.1 or pLKO.5 lentiviral vector. Lentiviral particles were produced by transfecting 293T-17 cells (ATCC) with the lentiviral plasmids and third-generation packaging plasmids. Virus was harvested 24 hours post-transfection and concentrated by ultracentrifugation at 23,000 rpm for 3 hours. All viruses were titered using 293T cells by serial dilution. All TRC numbers for the shRNAs used in this study are provided in Table 1.

[0363] Gene transfection of 5F and culturing of 5F MACS isolation CD34 + EB progenitor cells were cultured on a retronectin-coated (10 μg / cm 2 ) Cell density of 2–5 × 10 cells in a 96-well plate 4Cells were seeded at 1000 cells / well. Infection medium was SFEM (StemCell) containing 50 ng / ml SCF, 50 ng / ml FLT3, 50 ng / ml TPO, 50 ng / ml IL6, and 10 ng / ml IL3 (all from R&D Systems). Lentiviral infection was performed in a total volume of 150 μl. The multiplicity of infection (MOI) for factors was 5 for ERG and HOXA9, 3 for RORA, SOX4, and MYB, and 2 for all shRNAs. Virus was concentrated on the cells by centrifuging the plates at 2300 rpm for 30 minutes at room temperature. Infection was carried out for 24 hours. After transfection, 5F cells were cultured in SFEM containing 50 ng / ml SCF, 50 ng / ml FLT3, 50 ng / ml TPO, 50 ng / ml IL6, and 10 ng / ml IL3 (all from R&D Systems). Dox was added at 2 μg / ml (Sigma). Cultures were grown to a density <1 × 10 6 The cells were maintained at 0.1% / ml and the medium was changed every 3-4 days. After 14 days of culture, 5F cells were plated using the T cell differentiation protocol.

[0364] Co-culture with the Notch ligand delta-like 1-expressing OP9 (OP9-DL1) cell line OP9-DL1 cells were cultured in monolayer in OP9 medium, which is α-MEM supplemented with FCS (final concentration 20%), 2-mercaptoethanol (final concentration 0.1 mM), nonessential amino acids (final concentration 0.1 mM), sodium pyruvate (final concentration 1 mM), penicillin (final concentration 10 U / ml), L-glutamine (final concentration 1 mM), streptomycin (final concentration 100 μg / ml), and sodium bicarbonate (final concentration 2.2 g / liter).

[0365] Reverse lineage multipotent hematopoietic progenitor cells (also known as EB-derived progenitor cells as described above) were cultured at a cell density of 1–6 × 10 cells per well of a 6-well or 100 mm untreated dish. 5The iPSC-derived hematopoietic cells were plated onto an OP9-DL1 monolayer at 1000 μg / ml. The culture medium contained SCF (final concentration 30 ng / ml; R&D Systems), F1t3 ligand, and IL-7 (final concentrations of 5 ng / ml each; R&D Systems). On day 7 of culture, loosely adherent hematopoietic cells were harvested by gentle pipetting. Every 5 days thereafter, nonadherent iPSC-derived hematopoietic cells were harvested by vigorous pipetting, filtered through a 70 μm nylon mesh, and transferred to OP9 medium on the OP9-DL1 monolayer. All cytokines were added at all subsequent passages.

[0366] By day 14 of coculture with OP9-DL1 cells, the reverted lineage multipotent hematopoietic progenitor cells transformed into lymphoid cells. These cells expressed CD25 and / or CD44 by day 14 of coculture, suggesting that they differentiated into T lineages in the same manner as progenitor cells differentiate in the thymus. Expression of CD cell surface markers was verified using phycoerythrin-conjugated anti-CD8 (clone 53-6.7), anti-CD19 (clone 1D3), and anti-CD25 (clone 7D4) antibodies, as well as allophycocyanin-conjugated anti-CD4 (clone GK1.5), anti-CD11b (clone M1 / 70), and anti-CD44 (clone IM7) antibodies (all from Biolegend, Tokyo).

[0367] Rearrangements at the TCRβ locus (Tcrb) are a hallmark of T cell lineage commitment and are essential for progression of CD4 / CD8 double-negative thymocytes to the double-positive stage during normal αβ T cell development. To determine whether T cells arising from reverted lineage multipotent hematopoietic progenitors cultured on OP9-DL1 cells undergo normal rearrangements at the TCRβ locus, differentiated cells were stained at day 30 with various antibodies against the TCRβ chain. A fluorescein isothiocyanate-conjugated TCR panel (BD Biosciences) was used.

[0368] Diverse patterns of TcrVβ gene expression are expected to be detected in reverted lineage multipotent hematopoietic progenitor cells after differentiation. This diversity can be confirmed by genomic PCR. OP9-DL1 cells and adult mouse thymocytes can be used as positive controls for genomic PCR-based analysis. Previously reported PCR primers are used for the analysis of Tcr gene rearrangements (Ikawa T, Kawamoto H, Wright LY et al., "Long-term cultured E2A-deficient hematopoietic progenitor cells are pluripotent", Immunity, Vol. 20, pp. 349-360; Kawamoto H, Ohmura K, Fujimoto S et al., "Extensive proliferation of T cell lineage-restricted progenitors in the thymus: an essential process for clonal expression of diverse T cell receptor beta chains", Eur. J. Immunol., Vol. 33, pp. 606-615).

[0369] During normal thymocyte development, T cells bearing either TCRαβ or TCRγδ arise in the thymus. To determine whether both populations of T cells arise from revertant lineage multipotent hematopoietic progenitor cells cultured on OP9-DL1 cells, differentiated revertant lineage multipotent hematopoietic progenitor cells were analyzed for surface expression of TCRαβ or TCRγδ using an allophycocyanin-conjugated anti-TCRβ antibody (clone H57-597) and a phycoerythrin-conjugated anti-TCRγδ antibody (clone GL3). It is expected that both αβ and γδ T cells would be generated from differentiated cells in this coculture system.

[0370] Similarly, the differentiated cells after 20 days of co-culture are CD4 / CD8 double positive cells and CD8 single positive cells.

[0371] Confirmation of functional TCR expression on co-cultured differentiated cells. CD4+CD8+ differentiated cells in this co-culture system were sorted from the 21-day culture and 7.5 × 10 4 T cells were stimulated for 3 days with plate-bound anti-CD3 antibody (final concentration 10 μg / ml; clone 145-2C11) in the presence of IL-2 (final concentration 1 ng / ml) and anti-CD28 antibody (final concentration 1 μg / ml; clone 37.51) in differentiation medium. PMA / ionomycin was then added to the culture, and the cells were exposed to the added PMA / ionomycin for an additional 6 hours. Intracellular staining for IFN-γ was performed using Cytofix / Cytoperm® and GolgiStop® (BD Biosciences) according to the manufacturer's instructions. Phycoerythrin-conjugated anti-CD8 antibody (clone 53-6.7) and phycoerythrin-conjugated anti-IFN-γ antibody (clone XMG1.2) were used for IFN-γ production analysis. After staining, the cells were analyzed by flow cytometry. The CD4-CD8+ differentiated cells in this coculture system are expected to produce IFN-γ in response to TCR stimulation with anti-CD3 and anti-CD28 antibodies, indicating that functional TCRs are expressed in cells differentiated from reverted lineage multipotent hematopoietic progenitors.

[0372] Additionally, 7.5 x 10 CD4+CD8+ differentiated cells in this co-culture system 4The iPSC-derived T cells were cultured for 2 days with plate-bound anti-CD3 antibody (10 μg / ml; clone 145-2C11) in differentiation medium in the presence of IL-2 (2 ng / ml) and TGF-β1 (5 ng / ml). As observed in naive T cells derived from normal adult lymphoid tissues, the Foxp3-positive cell population, a hallmark of regulatory T cells, is expected to be enriched (Chen W, Jin W, Hardegen N et al., "Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3," J. Exp. Med., Vol. 198, pp. 1875-1886). These data indicate that the iPSC-derived T cells generated in this coculture can respond to stimulation via TCR or cytokine receptors.

[0373] T cell differentiation After 14 days of respecification, 1 × 10 5 5Fs were plated into OP9-DL1 stromal cocultures. Cells were cultured in α-MEM (Gibco), 1% penicillin / streptomycin, 20% FBS (Gemini), and 1 mM L-glutamine with 30 ng / mL SCF, 5 ng / mL FLT3, and 5 ng / mL ATP (all from R&D Systems) in the presence of 2 μg / mL Dox for 20 days, followed by Dox removal. Cells were collected by mechanical dissociation, filtered through a 40 μM cap, and passaged onto fresh stroma every 5–7 days. After 35 days, T cell development was assessed using CD45, CD7, CD3, CD4, and CD8.

[0374] Mouse YS or AGM cells were dissociated into single cells for 30 minutes in EB dissociation medium containing 250 mg of collagenase IV, 100 mg of hyaluronidase V, and 6.8 mg of DNase I in 50 mL of DMEM (10x) diluted to 1x with IMDM. Cells were pooled across multiple embryos and counted. 75,000 cells were seeded into one confluent well of OP9-DL1 in a 6-well plate. Cells were cultured for 12 days in aMEM, 20% serum, 1% Pen / Strep / L-glutamine, 5 ng / mL mIL-7 (R&D), and 5 ng / mL hFLT3 (R&D).

[0375] B cell differentiation After 14 days of respecification, 5 × 10 4 5F cells were plated in a single well of MS-5 stroma in a 6-well plate. Cells were cultured in Myelocult H5100 (Stem Cell Technologies) supplemented with 1% penicillin / streptomycin, 50 ng / mL SCF (R&D), 10 ng / mL FLT3 (R&D), 25 ng / mL IL-7 (R&D), and 25 ng / mL TPO (R&D) in the presence of 2 μg / mL Dox for 10 days, followed by Dox removal.

[0376] For mouse B cell differentiation, YS or EP pooled from multiple embryos of the same genotype were dissociated into single cells, and 75,000 cells were seeded onto confluent wells of OP9 stroma in 6-well plates. Cells were cultured in aMEM, 10% serum, 5 × 10 -5 M B-mercaptoethanol, 1% PSG, 50 U / mL mIL7 (R&D), and 10 ng / mL hFLT3 (R&D).

[0377] For class switching assays, B cell precursors were purified using B220 MACS or CD19 MACS microbead enrichment kits (Miltenyi Biotec) according to the manufacturer's recommendations. 5 × 10 cells were cultured in one well of a 6-well plate in complete RPMI medium supplemented with 0.5 μg / mL anti-CD40 (Ebioscience, Cat. #16-0402-86) and 25 ng / mL IL4 (Ebioscience, Cat. #14-8041). 5 B220 + cells or 5 x 10 5 CD19 + Cells were plated and analyzed for class switching by flow cytometry on day 4.

[0378] Colony assay After 14 days of respecification, 5 × 10 cells were cultured in 3 ml of complete methylcellulose H3434 (StemCell Technologies) supplemented with 10 ng / ml IL6 (Peprotech), 10 ng / ml FLT3 (R&D), and 50 ng / ml TPO (R&D). 4 The mixture was distributed into two 60 mm dishes and maintained in a humidified chamber for 14 days.

[0379] Mouse transplantation NOD / LtSz-scidIL2Rgnull (NSG) mice (Jackson Labs) were bred and maintained in the Boston Children's Hospital Animal Care and Use Facility. Animal experiments were performed in accordance with institutional guidelines approved by the Boston Children's Hospital Animal Care and Use Committee. Intravenous grafts were previously described. Briefly, 6- to 10-week-old mice were irradiated (275 rad) 24 hours before transplantation. To ensure consistency between experiments, only female mice were used. Cells were transplanted in a volume of 100 μL using a 28.5 g insulin needle. Sulfatrim was administered in drinking water to prevent infection after irradiation.

[0380] Flow cytometry The following antibodies were used for human cells: CD45 APC-Cy7 (557833, BD Biosciences), CD4 PE-Cy5 (IM2636U, Beckman Coulter Immunotech), CD8 BV421 (RPA-T8, BD Horizon), CD5 BV510 (UCHT2, BD Biosciences), TCRgd APC (555718, BD Biosciences), TCRab BV510 (T10B9.1A-31, BD Biosciences), CD3 PE-Cy7 (UCHT1, BD Pharmigen), CD7 PE (555361, BD Pharmigen), and CD1a APC (559775, BD Pharmigen) for T cell staining. For B cell staining, CD45 PE-Cy5 (IM2652U, Beckman Coulter Immunotech), CD19 PE (4G7, BD), CD56 V450 (B159, BD Biosciences), CD11b APC-Cy7 (557754, BD Biosciences), and for HSC / progenitor cell sorting, CD34 PE-Cy7 (8G12, BD), CD45 (557833, BD Biosciences), CD38 PE-Cy7 (IM2651U, BD), and DAPI. For myeloid and erythroid staining, CD11b APC-Cy7 (557754, BD Biosciences), GLYA PE-Cy7 (A71564, Beckman Coulter), CD71 PE (555537, BD Biosciences), and CD45 PE-Cy5 (IM2652U, Beckman Coulter Immunotech) were used. All staining was performed at RT in the dark for 30 minutes using <1 x 106 cells per 100 μl of staining buffer (PBS + 2% FBS) at a 1:100 dilution of each antibody. Compensation was performed using auto-compensation with anti-mouse Igk and negative beads (BD). All acquisitions were performed on a BD Fortessa or BD Aria cytometer.

[0381] The following antibodies were used for mouse cells: CD45.2 PE-Cy7 (104, eBiosci-ence), CD45.1 FITC (A20, eBioscience), B220 PB (RA3-6B2, BD Biosciences), Ter119 PE-Cy5 (Ter 119, eBioscience), GR1 (RB6-8C5, BD Bioscience), CD3 APC (145-2C11, eBioscience), CD19 APC-Cy7 (1D3, BD Bioscience), and MAC1 A700 (M1 / 70, BD Bioscience) for engraftment analysis. For HSC / progenitor cell staining, CD45.2 APC-Cy7 (104, BioLegend), Sca1 PE-Cy7 (D7, BD Bioscience), CD34 AF700 (RAM34, eBioscience), CD48 FITC (BioLegend), CD150 PE-Cy5 (TC15-12F12.2, BioLegend), cKit APC (2B8, eBioscience), Fcγ PE (93, eBioscience). For RNA-seq sorting, we used the following antibodies: CD16 / 32 (93, Bio-legend), Ter119 biotin (Ter119, eBioscience), GR1 biotin (RB6-8C, eBioscience), CD3 biotin (17A2, eBioscience), CD5 biotin (53-6.7, eBioscience), CD19 biotin (eBio1D3, eBioscience), streptavidin EF450 (eBioscience), CD45 PerCP-Cy5.5 (30-F11, eBioscience), CD144 EF660 (eBioBV13, eBioscience), CD117 APC-EF780 (2B8, eBioscience), and CD41 PE-Cy7 (eBioMWReg30, eBioscience).For B cell staining, CD45.2 APC-CY7 (104, BioLegend), CD23 PE-Cy7 (B3B4, eBioscience), Ter119 PE-Cy5 (Ter 119, eBioscience), IgM EF660 (eB121-15F9, eBioscience), MAC1 A700 (M1 / 70, BD Bioscience), CD5 BV510 (53-7.3 BD Biosciences), IgM (11 / 41, eBioscience), IgG1 FITC (A85-1, BD), B220 PE Cy5 (RA3-6B2, BD Biosciences). For T cell staining, CD45.2 APC-CY7 (104, BioLegend), TCRb PE-Cy5 (H57-597, BD Biosciences), CD8 APC-EF780 (53-6.7, eBioscience), CD4 APC (GK1.5, eBioscience), CD3 AF700 (17A2, BioLegend), and TCRgd FITC (GL3, BD Biosciences) were used. All staining was performed on ice in the dark for 30 minutes at a 1:100 dilution of each antibody, using <1 x 106 cells per 100 μl of staining buffer (PBS + 2% FBS). Compensation was performed using automated compensation with anti-mouse Igk and negative beads (BD). All acquisitions were performed on a BD Fortessa or BD Aria cytometer.

[0382] result We previously demonstrated that it is possible to respecify primitive progenitor cells with limited lymphoid differentiation potential. To expand the selection of candidate factors, we screened epigenetic modifiers to provide an additional layer of regulation for respecification. We employed a library of short hairpin RNAs (shRNAs) from the Broad RNAi Consortium to target 20 genes in DNA and histone methylation pathways previously used in laboratories to enhance reprogramming efficiency to pluripotency (Figure 7A) (Onder T. et al. 2012).

[0383] Using an established respecification platform, we differentiated two iPSC lines (MSC-IPS, CD45-IPS) into embryoid bodies (EBs) under hematopoietic-promoting conditions and obtained CD34 + CD45 + Myeloid progenitor cells were generated. Next, we transduced EB-derived progenitor cells with five transcription factors (5F cocktail: ERG, HOXA9, RORA, SOX4, and MYB), infected them with individual shRNAs targeting each epigenetic modifier, and screened them for T lymphocyte differentiation potential using the established OP9-DL1 coculture system. Results from three independent screens are summarized in Figures 1A-1F. After validation of the top hits, the lead candidate was EZH1.

[0384] EZH1 is a key repressor of definitive differentiation potential. To test the hypothesis that epigenetic factors act as a barrier to definitive differentiation potential, we employed a loss-of-function phenotype screen using an shRNA library targeting 20 DNA and histone methylation factors previously shown to affect somatic cell reprogramming (Figure 7A) (Onder et al. 2012). We cloned the library into primordial CD34 cells derived from embryoid body differentiation. + To facilitate phenotypic screening, we expanded these primitive progenitor cells using a defined set of five transcription factors (5F) (Doulatov et al., 2013). The expanded progenitor cells retained embryonic characteristics, including the lack of B- and T-cell differentiation potential and the expression of embryonic globins. We transduced 5F cells with individual hairpins (4 hairpins per gene) and screened them for the emergence of T-cell differentiation potential using the OP9-DL1 coculture system (Holmes and Zuniga-Pflucker, 2009) (Figures 1A and 1F). After 5 weeks of coculture, T-cell differentiation potential was analyzed by flow cytometry for CD7, CD3, CD4, and CD8. We found that knockdown of eight epigenetic regulators significantly reduced the CD4 expression from primitive 5F cells.+ CD8 + We found that these genes enhanced T cell differentiation potential (Figure 1B). Among the top hits were several members of methylated DNA-binding proteins, histone H3 lysine 9 (H3K9) methyltransferase, PRC2 components, and SMYD2, a SET domain-containing methyltransferase (Figure 1C). H3K9 and H3K27 methyltransferases have previously been associated with lineage commitment and self-renewal in fetal and adult HSCs (Ugarte et al. 2015, Chen et al. 2012, Xie et al. 2014, Hidalgo et al. 2012, Lee et al. 2015).

[0385] To prospectively validate these top candidates, we analyzed the T cell differentiation potential of 5F cells transduced with shRNAs targeting these eight factors (2x per gene). Independently, we tested their B lymphoid differentiation potential using coculture with MS-5, a mouse bone marrow-derived stroma that supports B cell differentiation (Nishihara et al. 1998, Ohkawara et al. 1998). 5F cells transduced with multiple shRNAs targeting a control luciferase gene showed no or negligible T and B cell differentiation potential (intervals, with standard deviations). Of the tested candidates, only knockdown of EZH1 induced robust T and B cell differentiation potential across independent hairpins and iPSC lines (Figures 2A, 2B, 2E). Flow cytometry (Figures 2C and 2D) and colony formation assays (Figure 2F) demonstrated that the myeloid-erythroid differentiation potential of progenitor cells transduced with shRNA for EZH1 (5F+shEZH1) was largely unchanged compared with that of shRNA targeting luciferase (5F+shLUC). These results demonstrate that EZH1 deletion confers multily lymphoid lineage differentiation potential to primitive hematopoietic progenitor cells.

[0386] Other PRC2 components do not phenocopy EZH1. EZH1 is a member of the Polycomb group of proteins. Polycomb repressive complex 2 (PRC2) mediates methylation of histone H3 lysine 27 (H3K27) and plays an important role in transcriptional regulation and stem cell development. EZH1 and EZH2 are closely related enzymatic subunits of PRC2. The well-characterized classical PRC2 is composed of EZH2, EED, and SUZ (Onder T. et al., 2012). EZH1 was identified as an EZH2 homolog and an interacting partner with EED (Jones CA et al. 1998 and Shen X. et al. 2008) and has been thought to play a redundant or complementary role to EZH2. However, recent studies have revealed a novel gene activation role for EZH1 in addition to its transcriptional repression function (Mousavi K. et al. 2012 and Xu J. et al. 2015).

[0387] EZH1 is a homolog of the Drosophila zeste E(z) enhancer (Abel et al. 1996), a catalytic component of PRC2 (Laible et al. 1997, Jones et al. 1998, Shen et al. 2008). PRC2 is composed of E(z), Eed, and Suz12, which mediate epigenetic silencing of developmentally regulated genes (Muller et al. 2002, Sparmann and van Lohuizen 2006, Simon and Kingston 2009). Although EZH2 is the primary catalytic component of PRC2, EZH1 can functionally substitute for Ezh2 (Shen et al. 2008), although Ezh1 has weaker methyltransferase activity (Magueron et al. 2008) and can promote RNA polymerase elongation (Mousavi et al. 2012). Growing evidence indicates that EZH1 and EZH2 have distinct molecular functions. Unlike Ezh2, Ezh1 is most frequently found by itself or in complex with the nucleosome-recognition protein Suz12. In contrast, Ezh2 is almost always complexed with Suz12 and the scaffolding protein Eed. Additionally, EZH2 is required for somatic reprogramming, and deletion of EZH1 enhances reprogramming (Onder et al. 2012, Cacchiarelli et al. 2015). These studies illustrate a complex mode of epigenetic regulation by PRC2 that depends on subunit interactions and holoenzyme composition.

[0388] To investigate the importance of PRC2 in definitive hematopoietic differentiation, we knocked down each component of PRC2 in 5F cells (two shRNAs per gene) and assessed T cell differentiation. We confirmed efficient knockdown by each shRNA (Figure 3A). Knockdown of EZH1 significantly reduced CD4 + CD8 +The percentage of T cells dramatically increased (5-fold compared to shLUC). Knockdown of SUZ12 also enhanced T cell differentiation, albeit to a lesser extent. However, knockdown of EED and EZH2 did not affect T cell differentiation (Figures 3A and 3C). Double knockdown of EZH1 and EZH2 phenocopied EZH2 depletion, indicating that EZH2 is superior to EZH1 (Figure 3B). Thus, EZH2 deletion does not phenocopy EZH1 in restoring definitive differentiation. To verify this result, we used the EZH2 / EZH1 dual inhibitor GSK126, which has 150-fold higher selectivity for EZH2 than EZH1 (McCabe et al. 2012) (Figures 3G, 3I, and 3J). At 3 μM GSK126, we observed a significant reduction in global H3K27me3 expression, with partial toxicity as assessed by colony formation assays (Figures 3I and 3J). To test the effect of EZH2 inhibition on T cell differentiation potential, we generated definitive hemogenic endothelium (HE) from hPSCs with T lymphocyte lineage potential via inhibition of Activin / Nodal signaling at the early stage of mesoderm differentiation (Kennedy et al. 2012). In DMSO-treated cells, we observed a robust CD4+CD8+ T cell population that was abolished by treatment with 3 μM GSK126, indicating that EZH2, unlike EZH1, is required for T cell differentiation (Figure 3H).

[0389] To determine whether the catalytic SET domain of EZH1 was required to restrict definitive hematopoietic differentiation, we performed rescue experiments using the full-length mouse Ezh1 open reading frame (ORF) (mEzh1) or Ezh1 lacking the catalytic SET domain (mEzh1ΔSET) to avoid targeting by shRNAs targeting human EZH1 (Figure 3D). We did not observe T cells in any of the 5F+shLUC conditions, and as before, we observed a robust population of CD4+CD8+ T cells in 5F+shEZH1 (Figures 3E and 3F). Coexpression of mEzh1 completely abolished the T cell differentiation potential of 5F+shEZH1 cells (Figures 3E and 3F), indicating that the mouse ORF is sufficient to functionally restrict definitive hematopoietic differentiation. In contrast, expression of mEzh1ΔSET did not suppress T cell differentiation (Figures 3E and 3F). Together, these data demonstrate that specific EZH1 inhibition, but not general PRC2 inhibition, releases definitive hematopoietic differentiation potential and that the catalytic SET domain is required to restrict this differentiation potential.

[0390] EZH1 directly regulates HSC and lymphoid genes. To understand the molecular basis for enhanced definitive differentiation potential, we investigated the role of CD34 + CD38 -We performed RNA sequencing analysis of 5F+shLUC and 5F+shEZH1 cells. Genes significantly upregulated after EZH1 knockdown (104 genes, >2-fold, t-test, p<0.1) were enriched for the gene ontology (GO) terms of defense response, immune response, and T cell costimulation (Figures 4A and 4B). To specifically analyze transcriptional changes associated with the human HSPC hierarchy, we performed GSEA using six signatures capturing the earliest patterns of lineage commitment: HSC, MLP (early lymphoid), HSC_MLP (stem and lymphoid), GMP (myeloid), CMP_MEP (erythroid), and progenitor (all progenitor cells) (Doulatov et al. 2010). Consistent with the acquisition of lymphoid lineage differentiation potential, the HSC_MLP and MLP signatures were highly enriched in 5F+shEZH1 (Figure 4C).

[0391] We next performed ATAC-sequencing to identify differential regions of chromatin accessibility (Buenrostro et al. 2013). Unbiased GREAT analysis (McLean et al. 2010) of 1,500 ATAC peaks significantly upregulated upon EZH1 knockdown revealed enrichment in pathways related to T cell development, lymphocyte activation, and immune responses (Figure 4E, Figure 9A). Conversely, downregulated peaks were enriched in pathways related to other cell developmental processes, such as reproductive processes, neural and lung development, and, importantly, embryonic hematopoiesis (Figure 4G, Figure 9B). Furthermore, HSC, HSC / MLP, B, and T cell signatures were all significantly enriched among the upregulated ATAC peaks (Figure 4F, Figure 9C), indicating that EZH1 knockdown induces epigenetic remodeling to deactivate accessibility to HSC- and lymphoid-related genes.

[0392] To determine whether changes in chromatin accessibility and gene expression were directly induced by EZH1, we overexpressed epitope-tagged EZH1 or EZH2 in 5F cells and subsequently defined its genome-wide occupancy by ChIP-sequencing. Comparison of EZH2- and EZH1-binding sites in promoter regions identified 1,069 unique EZH1-binding sites (Figure 5A), which were associated with repressive, bivalent, and activating marks (Figures 5B and 5C). To better annotate these EZH1-binding sites, we defined uniquely bound transcription factors (TFs; 152 of the 1,069 genes) and compared them to the TF signature of the early HSPC hierarchy (Laurenti et al. 2013). Strikingly, EZH1-bound TFs were highly enriched in HSCs, MLPs, and pro-B populations (Figures 5D and 5F), many of which were bivalently marked (Figure 5G). Among all EZH1-bound bivalent genes, a significant number were annotated as granulocyte / macrophage, NK cell, T cell, and B cell-specific genes (Novershtern et al. 2011) (Figure 5H). Although EZH1-bound TFs did not show significant changes in expression, the regulatory networks controlled by each EZH1-bound TF, as defined by CellNet, were significantly altered (Figure 5D). Specifically, EZH1-bound TFs and their networks were significantly enriched in the GRNs of HSPCs, B, and T cells (Figure 5E). A network of EZH1-bound TFs, including STAT5A, YAP1, NLRC5, ZNF697, and BACH2, were all significantly upregulated in GRNs of B and T cells (data not shown). Collectively, these data provide compelling evidence that EZH1 directly binds to HSC and MLP transcription factors and that inhibition of EZH1 abrogates definitive hematopoietic differentiation potential by derepressing stem cell and lymphoid gene regulatory networks.

[0393] Ezh1 deficiency enhances embryonic lymphopoiesis in vivo. To explore the role of Ezh1 in vivo, we first examined early lymphoid lineage development in mouse embryos. The extraembryonic yolk sac (YS), the earliest site of hematopoiesis, was thought to generate only primitive erythroid and myeloid-erythroid progenitor cells (Dzierzak and Speck 2008). However, recent studies have reported lymphoid differentiation potential in the yolk sac before the appearance of definitive HSCs (Boiers et al. 2013, Yoshimoto et al. 2012, Yoshimoto et al. 2011). To determine whether Ezh1 deficiency could enhance this early lymphoid differentiation potential, we performed lineage analysis of E9.5 yolk sacs (YS) from wild-type (WT) and Ezh1 knockout (Ezh1- / -) embryos. We found that CD19 expression in WT YS was significantly increased. + B220 + B cells (3.64%) and CD3 + CD5 + We detected a small population of T cells (0.45%) (Figures 11A-11C). These lymphoid populations tended to be more frequent in Ezh1- / - YS, but this increase was not significant (Figures 11B and 11C). Furthermore, we could not exclude the possibility that maternal circulation might confound our lineage analysis. Therefore, we compared the E9.5 WT and Ezh1- / - YS mice. - / - After dissecting the yolk sac from the embryo body and carrying out in vitro lymphoid differentiation, we detected a robust population of embryonic B cells (B-1 subtype; AA4.1 + CD19 + B220 lo-neg ), and adult-like B-2 cells (AA4.1 + CD19 + B220 + ) was hardly detected, which is consistent with previous results (Yoshimoto et al. 2012) (Figure 12A). - / - Although we did not find significant differences in B cell differentiation potential between WT and Ezh1 cells, we did notice an increase in class switching from germline IgM to IgG1 (Figure 12C). Similarly, we observed a significant difference in B cell differentiation potential between WT and Ezh1 cells in embryonic bodies (EPs). - / -No difference in T cell differentiation potential was observed between Ezh1 and Ezh1 (Figures 6A and 6B). - / - YS progenitors have 2-5 times more CD4 + CD8 + These early T cells expressed primarily fetal TCRγδ, although a small proportion expressed adult TCRβ (Figures 6B and 6D). These data demonstrate that Ezh1 deficiency enhances the lymphoid differentiation potential of YS progenitors. Together, these results suggest a role for EZH1 in suppressing lymphoid lineage fate as a surrogate measure of definitive differentiation potential.

[0394] Ezh1 haploinsufficiency promotes the generation of HSCs during ontogeny. The emergence of true HSCs, defined by their ability to repopulate adult recipients, marks the transition from embryonic to definitive hematopoiesis. We predicted that if EZH1 acts as a gatekeeper for definitive differentiation potential, HSCs might emerge earlier and exhibit high repopulation potential. Although HSCs appear in the AGM at approximately 10.5 dpc (Boisset et al. 2010), they are extremely rare (approximately one HSC per embryo) and do not strongly support engraftment of adult hosts (Bertrand et al. 2005, Muller et al. 1994, North et al. 2002). Therefore, focusing on this transitional point, we performed a multivariate analysis of E10.5 WT, Ezh1 + / - and Ezh1 - / - AGM and YS were isolated from embryos (Fig. 6E). Expression of Ezh1 and Suz12 decreased from YS to AGM, whereas Ezh2 and Eed were higher in AGM (Fig. 6F). We transfected whole AGM (3.5 embryo equivalents (ee)) or YS (5ee) with sublethally irradiated adult NOD / SCID-IL2Rγ cells. nullWe transplanted these into non-steroidal anti-inflammatory (NSG) mice and monitored hematopoietic reconstitution. After 4 weeks, engraftment from WT AGM was observed in 3 of 7 mice (11.9 ± 13.6%), but this decreased over time, with 2 of 7 mice engrafting after 16 weeks (12.2 ± 11.4%) (Figure 6G). This corresponds to one repopulating unit in approximately 10.4 embryonic equivalents (ee). Only 1 of 7 mice transplanted with WT AGM showed long-term multilineage chimerism, consistent with the very rare nature of HSCs in this study. In contrast, Ezh1 - / - Five of eight mice transplanted with AGM-derived cells engrafted after 4 weeks (36.7 ± 20.9%) and maintained stable chimerism over time (16 weeks; 34.3 ± 32.9%). Even more strikingly, mice transplanted with Ezh1 + / − cells had the highest initial chimerism (41.2 ± 29.2%; 4 of 5 mice engrafted), which increased over time (68.9 ± 35.6%) and was predominantly multilineage (3 of 5 mice) (Figures 6G and 6H). This corresponds to one repopulating unit in 3.6 Ezh1 − / − and 2.2 Ezh1 + / − embryo equivalents, a nearly fivefold increase in HSC frequency.

[0395] Like AGM, YS at E10.5 contain few, if any, HSCs. Consistent with this, we detected low levels of engraftment of WT YS cells in 5 of 7 recipients (3.4 ± 1.5%) after 4 weeks and in only 3 of 9 mice (4.3 ± 2.9%) after 16 weeks (Figure 6I). In contrast, most Ezh1 − / − (6.0 ± 4.9%, 5 of 7 engrafted) and all Ezh1 + / − YS-transplanted mice (8.8 ± 6.5%, 5 of 5 engrafted) showed stable, long-term engraftment (Figures 6I and 6M). The number of repopulating units was similar to that of AGM (approximately 1 in 8.9 WT; 1 in 4 Ezh1 − / −; 1 in <2 Ezh1 + / −). All engrafted mice were reconstituted for myeloid and lymphoid lineages (Figure 6J). We observed a significant increase in T cell engraftment in Ezh1- / - AGM graft recipients compared with WT AGM recipients (Figure 6J). Up to 80% of B cells in the peritoneal cavity of Ezh1+ / - AGM-engrafted mice were adult-like B-2 cells, as opposed to embryonic B-1 cells (Figure 13A). Furthermore, in Ezh1- / - and Ezh1+ / - AGM and YS-engrafted mice, >90% of donor-derived CD45.2+CD3+ T cells expressed adult TCRβ, as opposed to the embryonic TCRγδ configuration (Figure 13B). These data provide compelling evidence that Ezh1 deficiency, and particularly haploinsufficiency, stimulates the generation of definitive HSCs and adult-like lymphopoiesis.

[0396] To assess the extended self-renewal potential of Ezh1-deficient HSCs, we performed secondary transplants. Using 1% as the engraftment cutoff, we found no donor contribution in the peripheral blood after 4 weeks in mice transplanted with WT AGM (0 of 4 mice) or YS (0 of 7 mice). In contrast, 4 of 7 Ezh1- / - (4.4 ± 1.0%) and 9 of 9 Ezh1+ / - (57.8 ± 30.6%) AGM-transplanted secondary recipients engrafted (Figures 6K and 6L). - / - Although YS mice did not engraft (0 out of 10), the present inventors were able to confirm that Ezh1 + / -We observed chimerism from YS cells (5 of 7 mice engrafted, 1.5 ± 0.7%), which increased by 16 weeks post-transplant (6 of 7 mice engrafted, 5.3 ± 4.5%) (Figures 6M and 6N). Notably, all Ezh1-deficient AGM and YS secondary recipients exhibited multilineage engraftment involving B, T, and myeloid lineages (Figures 6L and 6N). Collectively, Ezh1 uniquely suppresses definitive differentiation potential during ontogeny, whereas Ezh1 deficiency promotes long-term multilineage differentiation and self-renewal of embryonic stem and progenitor cells.

[0397] References cited herein and throughout the specification are hereby incorporated by reference.

[0398] References TIFF0007635191000008.tif44164TIFF0007635191000009.tif232165TIFF0007635191000010.tif236165TIFF0007635191000011.tif227165

[0399] Table 1. Candidate target transcription factors for RNA interference and corresponding shRNAs TIFF0007635191000012.tif196139TIFF0007635191000013.tif201143TIFF0007635191000014.tif201119

[0400] Sequence information SEQUENCE LISTING <110> PRESIDENT AND FELLOWS OF HARVARD COLLEGE THE CHILDREN'S MEDICAL CENTER CORPORATION <120> IMMUNE CELLS DERIVED FROM INDUCED PLURIPOTENT STEM CELL <150> US 62 / 383,984 <151> 2016-09-06 <160> 111 <170> PatentIn version 3.5 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 1 ctatctggca gtgcgagaat g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 2 agacgtgcaa gcaggtcttt c 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 3 tggatgactt atgcgtgatt t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 4 caacagaact ttatggtaga a 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 ccgccgtggt ttgtattcat t 21 <210> 6 <211> 128 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 6 attacagcaa gatggaaata ccaaatcccc ctacctccaa atgtatcact tactggaaaa 60 gaaaagtgaa atctgaatac atgcgacttc gacaacttaa acggcttcag gcaaatatgg 120 gtgcaaag 128 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 7 tcgacaactt aaacggcttc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 8 tgcgacttcg acaacttaaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 9 cctccaaatg tatcacttac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 10 taaacggctt caggcaaata 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 11 aaacggcttc aggcaaatat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 12 catttggagg tagggggatt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 13 ccagtaagtg atacatttgg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 14 gtgatacatt tggaggtagg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 15 aagtgataca tttggaggta 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 16 agtgatacat ttggaggtag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 17 tttccagtaa gtgatacatt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 18 taagtgatac atttggaggt 20 <210> 19 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 19 gctttgtatg tggcaaattt tgcaaaggtt caagaaaaaa cccagatcct caatgaagaa 60 tggaagaagc ttcgtgtcca acctgttcag tcaatgaagc ctgtgagtgg acaccctttt 120 ctcaaaaag 1...

Claims

1. a) inhibiting Enhancer of Zeste Homolog 1 (EZH1) expression in multilineage hematopoietic progenitor cells (MHPCs), the MHPCs being differentiated from CD34+ hemogenic endothelial cells generated from induced pluripotent stem (iPS) cells; b) contacting said MHPCs with a notch ligand or stromal cells or both to promote differentiation into lymphoid lineages and production of T or B lymphocytes; c) isolating B220 or CD19 positive B lymphocytes, CD4 / CD8 double positive T cells, or CD8 single positive T cells; 2. An immune cell produced by a method comprising the steps of:

2. The immune cell according to claim 1 , wherein the immune cell is a CD4 / CD8 double positive or CD8 single positive T cell.

3. The immune cell according to claim 2 , wherein the immune cell is a CD8 single positive T cell.

4. The immune cell of claim 1 , wherein Enhancer of Zeste homolog 1 (EZH1) in the immune cell is inhibited.

5. The immune cell of claim 1 , wherein the immune cell comprises an exogenous copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC.

6. The immune cell of claim 1, wherein the immune cell has been further genetically modified to ablate the native T cell receptor (TCR) locus, to delete class I or class II major histocompatibility complexes or both, to express non-classical HLA-G or HLA-E or both, or to edit endogenous HLA therein.

7. A composition comprising the population of immune cells described in claim 1.

8. The composition of claim 7 further comprising a pharma- ceutically acceptable carrier.

9. a) transfecting in vitro myeloid progenitor cells that are CD34+ CD45+ with exogenous gene coding copies of each of the following transcription factors: ETS-related gene (ERG), homeobox A9 (HOXA9), and retinoic acid receptor (RAR)-related orphan receptor alpha (RORA), wherein the transcription factors are expressed in the transfected cells to produce a multilineage hematopoietic progenitor cell population with myeloid and erythroid differentiation potential and no or less than 5% lymphoid differentiation potential; b) inhibiting enhancer of Zeste homolog 1 (EZH1) expression in the resulting multilineage hematopoietic progenitor cell population to expand lymphoid differentiation potential; and c) differentiating the resulting population of multilineage hematopoietic progenitor cells in the presence of notch ligand or supportive stroma or both to promote lymphoid lineage differentiation and production of T or B lymphocytes; d) isolating B220 or CD19 positive B lymphocytes, CD4 / CD8 double positive T cells, or CD8 single positive T cells; 2. An immune cell produced by a method comprising the steps of:

10. The immune cell according to claim 9 , wherein the T lymphocyte is a CD4 / CD8 double positive or CD8 single positive T cell.

11. The immune cell of claim 9 , wherein EZH1 in the immune cell is inhibited.

12. The immune cell of claim 9 , wherein the immune cell comprises an exogenous copy of each of the following transcription factors: ERG, HOXA9, and RORA.

13. The immune cell of claim 12, wherein the immune cell further comprises an exogenous copy of each of the following reprogramming factors: SOX4 and MYB.

14. The immune cell of claim 12, wherein the immune cell further comprises an exogenous copy of each of the following reprogramming factors: NFIA and DACH1.

15. The immune cell of claim 12, wherein the immune cell further comprises an exogenous copy of each of the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC.

16. The immune cell of claim 9, wherein the immune cell has been further genetically modified to ablate the native T cell receptor (TCR) locus, to delete class I or class II major histocompatibility complexes or both, to express non-classical HLA-G or HLA-E or both, or to edit endogenous HLA therein.

17. A composition comprising the population of immune cells according to claim 10.

18. 20. The composition of claim 7, 8, or 17 for use in a method for treating cancer, an autoimmune disorder, a blood disorder, or other genetic diseases and disorders.

19. 20. The composition of claim 7, 8, or 17 for use in an ex vivo or in vitro method of improving in vivo engraftment of hematopoietic stem cells in a host.

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