Hematopoietic organoids

Abstract

The present disclosure provides cells and hematopoietic organoid compositions, and methods of making and using the same for bone marrow transplants, cell therapy and treatment of diseases or disorders associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency.

Description

Attorney Docket No. : AMTK.P0106WO PCTHEMATOPOIETIC ORGANOIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Application No. 63 / 690,473, entitled “METHODS OF GENERATING AN INJECTABLE A HEMATOPOIETIC ORGANOID,” filed September 4, 2025, the contents of which are expressly incorporated herein by reference in their entirety for all purposes.FIELD

[0002] Embodiments of the disclosure concern at least the fields of cell culture and development. In particular, it concerns the generation of three-dimensional cell organoids that can produce cells such as hematopoietic stem cells, progenitor cells, T cells, B cells, NK cells, macrophages, monocytes, neutrophils, platelets, red blood cells, myeloid derived suppressor cells, mast cells, eosinophils, basophils, dendritic cells, and / or the like.BACKGROUND

[0003] Hematopoiesis is the process that gives rise to blood cells, these include T cells, B cells, NK cells, macrophages, monocytes, neutrophils, platelets, red blood cells, myeloid derived suppressor cells, mast cells, eosinophils, basophils, dendritic cells begin[l]. In particular, the hematopoietic stem cells within the bone marrow are the key cell type that gives rise to these blood cellsfl ] . In some cases, such as congenital immunodeficiency, bone marrow failure, cancer treatment or autoimmune diseases, the ability of the hematopoietic stem cells to give rise to properly functioning immune cells may be impaired. One treatment approach is hematopoietic stem cell transplants in these patients[l-3]. This approach is limited due to difficulty of expanding large numbers of hematopoietic stem cells, aggressive leukoablative therapy required for engraftment, poor engraftment, graft rejection or graft vs host disease. In an allogeneic setting, deletion of HLA-I, HLA-II, Beta-2 microglobulin, T cell receptor, CD52, deoxycytidine kinase (dCK), CD7, 4-1BB Ligand, CIITA, CD155 or overexpression of tolerogenic HLA-E, HLA-G, Siglec-7, Siglec-9 may improve grafting[4]. Improved methods of engrafting stem cells and improved safety of stem cell engraftments are of great clinical interest.202693821.1 - 1 -SUMMARY

[0004] Some embodiments of the disclosure relate to a hematopoietic organoid comprising one or more fibroblasts; and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis, wherein the hematopoietic organoid produces a hematopoietic cell or a hematopoietic progenitor cell. Some embodiments relate to a hematopoietic organoid comprising one or more fibroblasts; and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis, optionally wherein the hematopoietic organoid produces a hematopoietic cell or a hematopoietic progenitor cell. Some embodiments of the disclosure relate to a population of cells comprising one or more fibroblasts; and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis, wherein the population of cells produce a hematopoietic cell or a hematopoietic progenitor cell. Some embodiments relate to a population of cells comprising one or more fibroblasts; and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis, optionally wherein the hematopoietic organoid produces a hematopoietic cell or a hematopoietic progenitor cell.

[0005] In some embodiments, the second cell type is selected from: (a) bone marrow cells or cells derived from bone marrow cells; (b) umbilical cord blood cells or cells derived from umbilical cord blood cells; (c) hematopoietic stem cells or cells derived from hematopoietic stem cells; (d) lineage committed lymphocyte progenitors or cells derived from lineage committed lymphocyte progenitors; (e) lineage committed myeloid progenitors or cells derived from lineage committed myeloid progenitors; (f) CAR-engineered cells or cells derived from CAR-engineered cells; or any combination of a), b), c), d), e), f).

[0006] In some embodiments, the organoid comprises at least 1% exogenous fibroblasts or between 1-99% exogenous fibroblasts.

[0007] In some embodiments, the ratio of fibroblasts to non-fibroblasts is at least 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 11, 1 : 12, 1 : 13, 1 :14, 1 : 15, 1 : 16, 1: 17, 1 : 18, 1 : 19, 1:20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, or 1 : 100.

[0008] In some embodiments, fibroblasts are incorporated throughout the organoid.

[0009] In some embodiments, the one or more fibroblasts are engineered fibroblasts.

[0010] In some embodiments, the one or more fibroblasts are genetically altered fibroblasts.202693821.1 - 2 -

[0011] In some embodiments, there are one or more fibroblasts, extracellular vesicles, exosomes, microvesicles, apoptotic bodies in the organoid.

[0012] In some embodiments, the engineered fibroblasts or the genetically altered fibroblasts encode and / or overexpress at least one gene product, optionally an exogenous gene product.

[0013] In some embodiments, the at least one gene product comprises one or more of Notch ligands, nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, antigens, antigen presenting molecules, co-stimulatory receptors, inhibitory receptors, any fragment or any derivative thereof, and any combination thereof.

[0014] In some embodiments, the at least one gene product comprises G-CSF, GM-CSF, survivin, CXCR4, SCF, VEGF, BMP4, bFGF, TPO, EPO, IL-3, IL-6, IL-15, SDF- 1, Hox genes, DLL4, DLL1, DLL3, X-delta 2, JAG1, JAG2, FOXN1, IL-7, FOXP3, FLT-3L, CCL25, CXCL12, CXCL19, Lymphotoxin-alpha / beta, insulin-growth factor-1 / 2, fibroblast growth factor-7 / 10, HoxAl, Eyal, Pax9, Sixl, Tbxl, Ripply3, E2F3, myc, miR-181a, Leptin receptor, ghrelin receptor, IL-22, fetal hemoglobin, hemoglobin, chimeric antigen receptor, or a fragment thereof, or a derivative thereof.

[0015] In some embodiments, the engineered fibroblast or genetically altered fibroblast has a disrupted expression of one or more endogenous genes.

[0016] In some embodiments, the one or more endogenous gene is of one or more of HLA-I, HLA-II, Beta-2 microglobulin, T cell receptor, CD52, dCK, CD7, 4-1BB Ligand, CIITA, CD 155 prostaglandin E2, CD26, androgen receptors (selected from Pgr, Nrli2, Ar, Esrl, Esr2, Gperl, Nr3cl, Nr3c2) or any combination thereof.

[0017] In some embodiments, the one or more of the second cell type is an engineered cell.

[0018] In some embodiments, the engineered cell expresses a gene product, optionally an exogenous gene product.

[0019] In some embodiments, the gene product is G-CSF, GM-CSF, survivin, CXCR4, SCF, VEGF, BMP4, bFGF, TPO, EPO, IL-3, IL-6, IL- 15, SDF-1, Hox genes, DLL4, DLL1, DLL3, X-delta 2, JAG1, JAG2, FOXN1, IL-7, FOXP3, FLT-3L, CCL25, CXCL12, CXCL19, Lymphotoxin-alpha / beta, insulin-growth factor-1 / 2, fibroblast growth factor-7 / 10, HoxAl, Eyal, Pax9, Sixl, Tbxl, Ripply3, E2F3, myc, miR-181a, Leptin receptor, ghrelin receptor, IL-22, fetal hemoglobin, hemoglobin, chimeric antigen receptor, HLA-E, HLA-G, Siglec-7, Siglec-9 or a fragment thereof, or a derivative thereof.202693821.1 - 3 -

[0020] In some embodiments, the engineered cell has a disrupted expression of one or more endogenous genes.

[0021] In some embodiments, the one or more endogenous gene is of one or more of HLA-I, HLA-II, Beta-2 microglobulin, T cell receptor, CD52, dCK, CD7, 4-1BB Ligand, CIITA, CD 155 prostaglandin E2, CD26, androgen receptors (selected from Pgr, Nrli2, Ar, Esrl, Esr2, Gperl, Nr3cl, Nr3c2) or any combination thereof.

[0022] In some embodiments, the one or more endogenous gene comprises an immune check point gene and the engineered cell is a lymphocyte precursor.

[0023] In some embodiments, the immune check point gene is selected from PD1, CTLA4, TIM3, TIGIT, CD96, BTLA, or LAG3.

[0024] In some embodiments, the ratio of the one or more fibroblasts to the one or more of the second cell type is in the range of about 0.0001 : 1 to 10000: 1, optionally about 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 11, 1 : 12, 1 : 13, 1 :14, 1 : 15, 1 : 16, 1: 17, 1 : 18, 1 : 19, 1:20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, or 1 : 100.

[0025] In some embodiments, the organoid has a size between 5um to 50,000um, optionally about 5 pm - 50 pm, about 50 pm - 150 pm, about 150pm - 250 pm, about 250 pm - 2,000 pm, about 2,000 pm - 10,000 pm, about 10,000 pm -50,000 pm in size.

[0026] In some embodiments, the one or more fibroblasts, the one or more of a second cell type, or any combination thereof, are autologous, allogeneic, xenogeneic, or syngenetic relative to a subject.

[0027] In some embodiments, the one or more fibroblasts, the one or more bone marrow cells, the one or more hematopoietic stem cells, the one or more umbilical cord blood cells, the one or more lymphoid progenitors or the one or more myeloid progenitors or the one or more umbilical cord blood cells or any combination thereof, are autologous, allogeneic, xenogeneic, or syngenetic relative to a subject.

[0028] In some embodiments, the one or more fibroblasts, the one or more of a second cell type, or any combination thereof, are activated with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof.

[0029] In some embodiments, the one or more fibroblasts, the one or more hematopoietic stem cells, the one or more umbilical cord blood cells ,the one or more lymphoid progenitors or the one or more myeloid progenitors, CAR-engineered cells, or any combination thereof are activated with one or more of nucleic acids, cytokines, chemokines,202693821.1 - 4 -transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof.

[0030] In some embodiments, the one or more fibroblasts are derived from one or more of bone marrow fibroblasts, skin fibroblasts, fibroblasts of other regions of the body, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, and / or progenitor cells.

[0031] In some embodiments, the hematopoietic cell or hematopoietic precursor cell is an immune cell, immune cell precursor or blood cell.

[0032] In some embodiments, the immune cell, immune cell precursor or blood cell is a T cell, B cell, NK cell, macrophage, monocyte, neutrophil, platelet, red blood cell, myeloid derived suppressor cell, mast cell, eosinophil, basophil, or dendritic cell.

[0033] Some embodiments of the disclosure relate to a pharmaceutical composition comprising any of the hematopoietic organoids or cell population disclosed herein.

[0034] Some embodiments of the disclosure relate to a method of generating a hematopoietic organoid in vitro, the method comprising culturing one or more fibroblasts; with one or more of a second cell type for a period of time in a culture medium, optionally on a low adhesion surface, wherein the second cell type is involved with hematopoiesis.

[0035] In some embodiments, the second cell type is: (a) bone marrow cells or cells derived from bone marrow cells; (b) umbilical cord blood cells or cells derived from umbilical cord blood cells; (c) hematopoietic stem cells or cells derived from hematopoietic stem cells; (d) lineage committed lymphocyte progenitors or cells derived from lineage committed lymphocyte progenitors; (e) lineage committed myeloid progenitors or cells derived from lineage committed myeloid progenitors; (f) CAR-engineered cells or cells derived from CAR-engineered cells; or any combination of a), b), c), d), e), f).

[0036] In some embodiments, the ratio of fibroblasts to second cell type is in the range of about 0.0001 : 1 to 10000: 1, optionally about 100: 1, 90: 1, 80: 1, 70:1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 11, 1 : 12, 1 : 13, 1 : 14, 1 :15, 1 : 16, 1 : 17, 1 : 18, 1 : 19, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, or 1 : 100.

[0037] In some embodiments, the culturing of the hematopoietic organoid does not require an exogenous extracellular matrix.

[0038] In some embodiments, the culturing of the hematopoietic organoid does not require exogenous growth factors.202693821.1 - 5 -

[0039] In some embodiments, the method does not include an exogenous growth factor.

[0040] In some embodiments, the method does not include addition of exogenous SCF, Rock-inhibitors, GSK-3 inhibitors, VEGF, BMP4, bfGF, IL-3, Flt-3L, TPO, MCSF, GMCSF, and / or IL-7.

[0041] In some embodiments, the culture medium is a serum-free medium.

[0042] In some embodiments, the culture medium comprises one or more nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or a combination thereof.

[0043] In some embodiments, the hematopoietic organoid can generate a hematopoietic cell or hematopoietic precursor cell.

[0044] In some embodiments, the hematopoietic cell or hematopoietic precursor cell is an immune cell, immune cell precursor, or blood cell.

[0045] In some embodiments, the immune cell, immune cell precursor, or blood cell is a T cell, B cell, NK cell, macrophage, monocyte, neutrophil, platelet, red blood cell, myeloid derived suppressor cell, mast cell, eosinophil, basophil, dendritic cell, TCR- engineered cell, or CAR-engineered cell.

[0046] In some embodiments, the period of time is a period of time sufficient for a hematopoietic organoid to self-assemble.

[0047] In some embodiments, the period of time is about 1, 2, 3, 4, 5, or 6 days.

[0048] In some embodiments, the organoid produced by the method comprises at least 1% exogenous fibroblasts; comprises fibroblasts incorporated throughout the organoid; and / or comprises a ratio of fibroblasts to non-fibroblasts is at least 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30:1, 20: 1, 10: 1, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 :11, 1 :12, 1 : 13, 1 : 14, 1 : 15, 1 : 16, 1 : 17, 1 : 18, 1 : 19, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, or 1 : 100.

[0049] Some embodiments of the disclosure relate to a hematopoietic organoid produced by any of the methods disclosed herein.

[0050] Some embodiments of the disclosure relate to a method of regenerating bone marrow tissue in a subject in need thereof, the method comprising administration to the subject any of the hematopoietic organoids, population of cells, and / or the pharmaceutical compositions disclosed herein in a pharmaceutically effective amount.

[0051] Some embodiments of the disclosure relate to a method of treating a disease or disorder in a subject in need thereof, the method comprising administration to the202693821.1 - 6 -subject any of the hematopoietic organoids, population of cells, and / or the pharmaceutical compositions disclosed herein in a pharmaceutically effective amount.

[0052] In some embodiments, the disease or disorder is associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency.

[0053] In some embodiments, the disease or disorder is selected from one or more of a malignancy (such as leukemia or lymphomas), bone marrow diseases (such as severe aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, megakaryocytes, congenital thrombocytopenia), immune deficiency (such as SCID, Wiskott-Aldrich syndrome), Hemoglobinopathies (such as beta thalassemia, sickle cell disease), Metabolic disorders (such as Krabbe disease, Hurler syndrome, adrenoleukodystrophy, metachromatic leukodystrophy), myelodysplastic syndrome, multiple myeloma, familial erythrophagocytic lymphohistiocytic disorders, age related immune deficiency, immune deficiency, bone marrow failure syndrome, Neuroblastoma, plasma cell disorders, POEMS syndrome, primary amyloidosis, autoimmune diseases.

[0054] In some embodiments, the hematopoietic organoid or population of cells is administered via an intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, intrathymic, perithymic, articular, renal subcapsular, hepatic, or injection into the bone marrow.

[0055] In some embodiments, administration of the hematopoietic organoid or population of cells (1) does not require a surgical procedure, and / or (II) does not require immune ablative therapy.

[0056] In some embodiments, the hematopoietic organoid or population of cells is administered via injection.

[0057] Some embodiments of the disclosure relate to a method of regenerating hematopoietic tissue in a subject in need thereof, the method comprising administering to the subject one or more fibroblasts and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis. Some embodiments of the disclosure relate to a method of treating or preventing a disease associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency in a subject in need thereof, the method comprising administering to the subject one or more fibroblasts and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis.

[0058] In some embodiments, the second cell type is selected from one of more of (a) bone marrow cells or cells derived from bone marrow cells; (b) umbilical cord blood cells or cells derived from umbilical cord blood cells; (c) hematopoietic stem cells or cells derived202693821.1 - 7 -from hematopoietic stem cells; (d) lineage committed lymphocyte progenitors or cells derived from lineage committed lymphocyte progenitors; (e) lineage committed myeloid progenitors or cells derived from lineage committed myeloid progenitors; (f) CAR- engineered cells or cells derived from CAR-engineered cells; or any combination of a), b), c), d), e), f).

[0059] In some embodiments, the one or more fibroblasts, the one or more or a second cell type, or any combination thereof are premixed and administered as a population of cells, or as a hematopoietic organoid, or co-administered as at least two populations of cells into a subject in need thereof.

[0060] In some embodiments, the at least two populations comprises a first cell population comprising the one or more fibroblasts and a second population comprising one or more of the second cell type.

[0061] In some embodiments, the first cell population is administered concurrently or sequentially with the second cell population.

[0062] In some embodiments, the subject is diagnosed with or is suspected of having a disease associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency.

[0063] In some embodiments, the disease is selected from a malignancy (such as leukemia or lymphomas), solid tumors, bone marrow diseases (such as severe aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, megakaryocytes, congenital thrombocytopenia), immune deficiency (such as SCID, Wiskott- Aldrich syndrome), Hemoglobinopathies (such as beta thalassemia, sickle cell disease), Metabolic disorders (such as Krabbe disease, Hurler syndrome, adrenoleukodystrophy, metachromatic leukodystrophy), myelodysplastic syndrome, multiple myeloma, familial erythrophagocytic lymphohistiocytic disorders, age related immune deficiency, immune deficiency, bone marrow failure syndrome, Neuroblastoma, plasma cell disorders, POEMS syndrome, primary amyloidosis, autoimmune diseases.

[0064] In some embodiments, the method further includes genetic editing of genes in hematopoietic stem cells, lymphoid progenitors, or myeloid progenitors.

[0065] In some embodiments, the method further includes administering to the subject at least one additional therapy.

[0066] In some embodiments, the at least one additional therapy is chemotherapy, radiation therapy, androgen deprivation therapy, antibody therapeutics, cell therapies,202693821.1 - 8 -immune activation therapy, checkpoint blockade therapy, immune suppression, leukobalative therapy, lymphodepletion therapy, immune tolerogenic therapy, or pain therapy.

[0067] Some embodiments of the disclosure relate to a kit comprising elements of any of the hematopoietic organoids, population of cells, and / or the pharmaceutical compositions disclosed herein in individual or bulk packaging.

[0068] Some embodiments of the disclosure relate to a kit comprising means for carrying out any of the methods disclosed herein.

[0069] Some embodiments of the disclosure relate to any of the hematopoietic organoids, population of cells, and / or the pharmaceutical compositions wherein the organoid, fibroblast or second cell type is engineered using or more of a biologic, chemical, mechanical or viral delivery method.BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Those with skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0071] FIG. 1 is a schematic showing a method of producing a three-dimensional hematopoietic organoid by mixing bone marrow cells with fibroblasts.

[0072] FIG. 2 provides microscopic images of images of a single hematopoietic organoid after 3 days of culture.

[0073] FIG. 3 provides FACS data to show viability of bone marrow cells and fibroblasts of a hematopoietic organoid. Cells were stained with 7AAD viability stain.

[0074] FIG. 4 provides data showing viability of fibroblasts and bone marrow cells after freezing the hematopoietic organoids. Cells were frozen in medium containing 10% DMSO, thawed after a few days and stained with 7AAD viability stain.

[0075] FIG. 5 provides representative images showing the expression of CD45.1 in donor organoid, wild-type mice, and wild-type mice that after transplantation with CD45.1 hematopoietic organoids.

[0076] FIG. 6 provides data showing CD45.1 cells in circulation in mice on days 7 and 14 following transplantation with CD45.1 hematopoietic organoids.

[0077] FIG. 7 provides data showing fibroblasts produce high levels of Stem Cell Factor (SCF).202693821.1 - 9 -DETAILED DESCRIPTION

[0078] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0079] The following description of various embodiments is exemplary and explanatory only and is not to be construed as limiting or restrictive in any way. Other embodiments, features, objects, and advantages of the present teachings will be apparent from the description and accompanying drawings, and from the claims.

[0080] The disclosure herein uses affirmative language to describe the numerous embodiments. The disclosure also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.

[0081] It should be understood that any use of subheadings herein are for organizational purposes and should not be read to limit the application of those subheaded features to the various embodiments herein. Each and every feature described herein is applicable and usable in all the various embodiments discussed herein and that all features described herein can be used in any contemplated combination, regardless of the specific example embodiments that are described herein. It should further be noted that exemplary description of specific features are used, largely for informational purposes, and not in any way to limit the design, subfeature, and functionality of the specifically described feature.I. Definitions of Terms

[0082] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. For purposes of the present disclosure, the following terms are explained below.

[0083] In keeping with long-standing patent law conventions, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some embodiments of the disclosure may consist of or202693821.1 - 10 -consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0084] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).

[0085] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicate the value plus or minus a range of 15%, 10%, 5%, or 1%. With respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise stated, the term 'about' means within an acceptable error range for the particular value.

[0086] As used herein, the term “activated cells” refers to cells treated with one or more stimuli capable of inducing one or more alterations in the cell: metabolic, immunological, epigenetic, growth factor secreting, surface marker expression, and production and excretion of microvesicles.

[0087] The term "administered" or "administering," as used herein, refers to any method of providing a composition to an individual such that the composition has its intended effect on the patient. For example, one method of administering is by an indirect mechanism using a medical device such as, but not limited to, a catheter, applicator gun, syringe, gel matrix, and a 3D matrix containing one or more cell types, cell-derived products, and or growth factors and or antibiotics, etc. A second exemplary method of administering is by a direct mechanism such as local tissue administration, oral ingestion, transdermal patch, topical, inhalation, suppository, etc.

[0088] The “term” therapeutically effective amount” refers to any amount of the organoid or cell population or composition that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can202693821.1 - 11 -be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0089] As used herein, “allogeneic” refers to tissues or cells from another body that, in a natural setting, are immunologically incompatible or capable of being immunologically incompatible, although from one or more individuals of the same species.

[0090] As used herein, “autologous” refers to tissues or cells that are derived or transferred from the same individual's body (z.e., autologous blood donation; an autologous bone marrow transplant).

[0091] As used herein, “agent” refers to nucleic acids, cytokines, chemokines, transcription factors, epigenetics factors, growth factors, hormones, or a combination thereof, including whole cell lysate.

[0092] As used herein, “xenogeneic” refers to tissues or cells from a species different from the patient.

[0093] Cell culture" is an artificial in vitro system containing viable cells, whether quiescent, senescent or (actively) dividing. In a cell culture, cells are grown and maintained at an appropriate temperature, typically a temperature of 37°C and under an atmosphere typically containing oxygen and CO2 at various concentrations. Culture conditions may vary widely for each cell type, though, and variation of conditions for a particular cell type can result in different phenotypes being expressed. The most commonly varied factor in culture systems is the growth medium and oxygen concentration during culturing. Growth media can vary in concentration of nutrients, growth factors, and the presence of other components. The growth factors used to supplement media are often derived from animal blood, such as calf serum.

[0094] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory but that no other202693821.1 - 12 -elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0095] The term "individual," as used herein, refers to a human or animal that may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may be receiving one or more medical compositions via the internet. An individual may comprise any age of a human or non-human animal and therefore includes both adults and juveniles (ie., children) and infants. It is not intended that the term "individual" connotes a need for medical treatment, therefore, an individual may voluntarily or involuntarily be part of experimentation, whether clinical or in support of basic science studies. The term “subject” or “individual” may be used interchangeably and refers to any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals.

[0096] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0097] The terms “reduce,” “inhibit," "diminish,” “suppress,” “decrease,” “prevent,” and grammatical equivalents (including “lower,” “smaller,” etc. when in reference to the expression of any symptom in an untreated subject relative to a treated subject, mean that the quantity and / or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one embodiment, the quantity and / or magnitude of the symptoms in the treated subject is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and / or at least 90% lower than the quantity and / or magnitude of the symptoms in the untreated subject.

[0098] As used herein, the term “transplantation” refers to the process of taking living tissue or cells and implanting it in another part of the body or into another body.202693821.1 - 13 -

[0099] Treatment,” “treat,” or “treating” means a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from pretreatment levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition. Therefore, in the disclosed methods, treatment” can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or the disease progression, including a reduction in the severity of at least one symptom of the disease. For example, a disclosed method for reducing the immunogenicity of cells is considered to be a treatment if there is a detectable reduction in the immunogenicity of cells when compared to pre-treatment levels in the same subject or control subjects. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. It is understood and herein contemplated that “treatment” does not necessarily refer to a cure of the disease or condition but an improvement in the outlook of a disease or condition. In specific embodiments, treatment refers to the lessening in severity or extent of at least one symptom and may alternatively or in addition refer to a delay in the onset of at least one symptom.

[0100] As used herein, the term “engineered” refers to refers to an aspect of having been manipulated and altered by the hand of man. In particular, the term “engineered cell” refers to a cell that has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, such as gene editing, base editing, and gene therapy. In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell.

[0101] As used herein, the terms “genetically altered” or “genetically modified” refer to a cell or organoid in which a genomic DNA sequence has been deliberately modified by recombinant technology.

[0102] As used herein the term “disrupted expression” encompasses both functional and structural disruption. Disrupted expression may refer to the permanent alteration or inactivation of a specific gene's function or expression, typically achieved through the introduction of insertions or deletions, or point mutations, at targeted genomic202693821.1 - 14 -locus / loci within the gene or outside. In an aspect, the disrupted expression may result in complete loss of expression of a gene (knockout). In an aspect, the disrupted expression may result in reduction in the level of the specific gene product. Thus, the gene may be expressed at less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of its endogenous expression level in the cell.

[0103] The term “organoid” as used herein refers to self-organized, three- dimensional tissue cultures that are derived from cells that have key functional, structural, and biological complexity of an organ, in addition to self-renewal and differentiation capacities. In specific cases, the term applies to organoids having specific shapes or substantially similar to specific shapes, such as spheroids. In specific cases, the cells that have self-renewal and differentiation capacities are fibroblasts.

[0104] The term “hematopoietic organoid” as used herein refers to an organoid that produces a hematopoietic cell or a hematopoietic progenitor cell.

[0105] The term “fibroblast-derived materials” as used herein refers to exosomes, cells separating from fibroblast organoids / spheroids, lysate, conditioned media, and / or apoptotic bodies, etc.

[0106] A cell may be "involved with" a cellular process if, for example, it is associated therewith, such as it being a causative factor or if it is affected by such a process. For example, a cell is "involved with hematopoiesis" if the cell is part of any of the processes during hematopoiesis or produced by a cell produced by hematopoiesis. A cell involved with hematopoiesis includes any cell or precursor cell that can give rise to a hematopoietic cell (blood cell) or an immune cell (including precursors). A cell involved with hematopoiesis also includes the hematopoietic cell (blood cell) or immune cell (including precursors of both). The term “involved with” can be used interchangeably with “associated with” or “contributes to.”

[0107] The term “blood cells” includes blood cell precursors. The terms “hematopoietic cell,” “hemocyte,” “hemacyte, and “blood cell” can be used interchangeably. The term “immune cells” includes immune cell precursors.

[0108] The terms “cell population” and “population of cells” can be used interchangeably.202693821.1 - 15 -

[0109] The term “CAR-engineered” refers to a cell engineered to express a chimeric antigen receptor. The cell can be any immune cell, including but not limited to T- cells, NK cells, macrophages, and the like.II. Overview

[0110] The current disclosure is based in part on the unexpected discovery that fibroblasts mixed with a second cell involved with hematopoiesis are capable of generating functional hematopoietic organoids. The second cell type can include, but not be limited to, bone marrow cells; umbilical cord blood, hematopoietic stem cells; lineage committed lymphocyte progenitors; lineage committed myeloid progenitors; CAR-engineered cells; and or the like; and / or any combination thereof. Embodiments of the disclosure related to a hematopoietic organoid or cell population comprising fibroblasts. As used herein, a hematopoietic organoid refers to a three-dimensional cell organoid that can contribute to hematopoiesis or processes involving hematopoiesis. For example, the organoid described herein can produce blood cells, and / or immune cells, and or precursor cells thereof. For example, the organoid described herein can produce hematopoietic stem cells, progenitor cells, T cells, B cells, NK cells, macrophages, monocytes, neutrophils, platelets, red blood cells, myeloid derived suppressor cells, mast cells, eosinophils, basophils, CAR-engineered cells, and / or dendritic cells and / or any combination. The production of such cells can occur and / or continue after the organoid is transplanted into a living organism. Functionality of the organoid may include the production of such cells or growth factors, cytokines, or chemokines associated with those cells.

[0111] The organoids may be generated without the need of added extracellular matrix, for example Matrigel™ or a Matrigel-like material. Similarly, the organoids may be generated without exogenous growth factors commonly used in the art. These organoids may be injected into a subject in need thereof, and do not necessarily require surgical transplantation. In some aspects, injection / transplantation does not require immune ablation therapy, This is in contrast with, for example, current methods of patients receiving bone marrow transplant that must undergo aggressive chemo / radiation to allow for the graft to be accepted. The organoids or cell populations can also be easily stored and have high post storage viability. The organoids may comprise or be developed from genetically or chemically modified cells. The ease of developing and using these hematopoietic organoids make them a compelling and much needed clinical product. Additionally, the disclosed202693821.1 - 16 -methods may be expanded to development of other organoids and tissue structures for clinical and non-clinical use.

[0112] Disclosed herein are compositions including cells and hematopoietic organoids and method of making and using the same for clinical and non-clinical purposes.III. Compositions., cells, and organoids

[0113] In an aspect, the current disclosure encompasses hematopoietic organoid or population of cells comprising one or more fibroblasts, or one or more of a second cell type involved with hematopoiesis (including cells produced by a cell produced by hematopoiesis) or both. Aspects of the disclosure relate to the formation of the three dimensional (3D) hematopoietic organoid using fibroblasts alone or in combination with dissociated single cell involved with hematopoiesis. In some aspects, fibroblasts in combination with bone marrow cells, umbilical cord blood, hematopoietic stem cells, lineage committed lymphocyte progenitors, lineage committed myeloid progenitors, and / or CAR- engineered cells are used.

[0114] The second cell type can include bone marrow cells, umbilical cord blood cells, hematopoietic stem cells; lineage committed lymphocyte progenitors; lineage committed myeloid progenitors; and / or the like; and / or any combination thereof.

[0115] The disclosed organoid can include fibroblasts incorporated throughout the organoid. The organoid can include at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 40% or more fibroblasts. The ratio of fibroblasts to other cell types can be about 0.0001:1, 0.001:1, 0.01:1, 0.1:1, 1:1, 10:1, 100:1, 1000:1, 10000:1, 0.0002:1, 0.002:1, 0.02:1, 0.2:1, 2:1, 20:1, 200:1, 2000:1, 20000:1, 0.0005:1, 0.005:1, or any value within the range. For example, the ratio can be between 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1,1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100 or any range therebetween.

[0116] In some aspects, the disclosure encompasses a population of cells comprising one or more fibroblasts, or one or more cells involved with hematopoiesis, or both. In some aspects, the disclosure encompasses at least two populations of cells, a first population with fibroblasts and the second population with the second cell type involved with hematopoiesis.

[0117] In some aspects, the disclosure encompasses a composition including the any of the organoids or population of cells described herein.202693821.1 - 17 -

[0118] As used herein, the term fibroblast is applied broadly to include fibroblast cells and fibroblast-like cells. In general fibroblasts are connective tissue cells that synthesize extracellular matrix and collagen, thereby providing a structural framework. Therefore, the fibroblasts for use in the current disclosure encompass any cell, or derivatives thereof, capable of providing an extracellular structural framework. The fibroblast may be isolated from a subject or may be a cultured fibroblast. Fibroblasts may be isolated from an autologous, allogeneic, xenogeneic, syngenetic, or a combination thereof, with respect to the subject.

[0119] In an aspect, the fibroblast may be differentiated in vitro from an autologous, allogeneic, xenogeneic, syngenetic cell, for example a stem cell. In an aspect, the stem cell may be a pluripotent stem cell or a progenitor stem cell. In an aspect, the stem cells may be embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, bone marrow- derived mesenchymal stromal cells, tissue plastic-adherent placental stem cells (PDACC), umbilical cord stem cells, amniotic fluid stem cells, amnion derived adherent cells (AMDACs), osteogenic placental adherent cells (OPACs), limbal stem cells, dental pulp stem cells, endothelial progenitor cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells, amnion derived adherent cells, or side population stem cells. In an aspect, the stem cells may be engineered to encode or overexpress at least one gene product (optionally, an exogenous gene product) or have disrupted expression of an endogenous gene or both. The gene product may be notch ligands, nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, and any combination thereof. Non-limiting examples of one or more encoded gene products include DLL4, DLL1, DLL3, X-delta 2, JAG1, JAG2, FOXN1, IL-7, FLT-3L, SCF, CCL25, CXCL12, CXCL19, Lymphotoxin-alpha / beta, insulingrowth factor-1 / 2, fibroblast growth factor-7 / 10, HoxAl, Eyal, Pax9, Sixl, Tbxl, Ripply3, E2F3, myc, miR-181a, Leptin receptor, ghrelin receptor, IL-22, or a fragment thereof, or a derivative thereof. The stem cell may have disrupted expression of one or more endogenous genes, non-limiting examples of which include Pgr, Nrli2, Ar, Esrl, Esr2, Gperl, Nr3cl, Nr3c2 androgen receptor.

[0120] As used throughout the disclosure, the term disrupted expression encompasses both functional and structural disruption. Disrupted expression may refer to the permanent alteration or inactivation of a specific gene's function or expression, typically achieved through the introduction of insertions or deletions, or point mutations, at targeted202693821.1 - 18 -genomic locus within the gene or outside. In an aspect, the disrupted expression may result in complete loss of expression of a gene (knockout). In an aspect, the disrupted expression may result in reduction in the level of the specific gene product. Thus, the gene may be expressed at less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of its endogenous expression level in the cell. In an aspect, the disrupted expression may be a functional disruption, in that the gene product does not perform one or more of its endogenous functions or exhibits reduced functionality. For example, for an enzyme, this could manifest as an alteration in enzyme kinetics as measured by methods known in the art.

[0121] In an aspect, the fibroblasts may be isolated from an autologous, allogeneic, xenogeneic, syngenetic, or a combination thereof, with respect to the subject. The fibroblast may be isolated from any organ of the body. Fibroblasts may be derived from various tissues or organs, including, but not limited to skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, foreskin, which can be obtained by biopsy (where appropriate) or upon autopsy. In some aspects, the cells comprise fibroblasts, which can be from a fetal, neonatal, adult origin, or a combination thereof. In an aspect, the one or more fibroblast is isolated from thymic tissue. In an aspect the thymic fibroblasts may include fibroblasts type 1 (Fbl) or fibroblasts type 2 (Fb2). The one or more fibroblasts may be a modified fibroblast, or an unmodified fibroblast. In an aspect, the one or more fibroblasts may be an engineered fibroblast. In an aspect, the fibroblast may be engineered to encode and / or overexpress at least one gene product (optionally an exogenous gene product) or have disrupted expression of an endogenous gene or both. The gene product that is encoded or overexpressed may be notch ligands, nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, and any combination thereof. Nonlimiting examples of one or more encoded gene products include G-CSF, GM-CSF, survivin, CXCR4, SCF, VEGF, BMP4, bFGF, TPO, EPO, IL-3, IL-6, IL- 15, SDF-1, Hox genes, DLL4, DLL1, DLL3, X-delta 2, JAG1, JAG2, FOXN1, IL-7, FOXP3, FLT-3L, CCL25, CXCL12, CXCL19, Lymphotoxin-alpha / beta, insulin-growth factor-1 / 2, fibroblast growth factor-7 / 10, HoxAl, Eyal, Pax9, Sixl, Tbxl, Ripply3, E2F3, myc, miR-181a, Leptin receptor, ghrelin receptor, IL-22, fetal hemoglobin, hemoglobin, chimeric antigen receptor, HLA-E, HLA-G, Siglec-7, Siglec-9 or the like, or a fragment thereof, or a derivative thereof. In an aspect, the one or more fibroblasts may comprise an activated fibroblast. The fibroblast cells may be activated, such as having activated or engineered surface markers, nucleic acid202693821.1 - 19 -modification, and / or expression or excretion of one or more chemokines, cytokines, exosomes, and / or growth factors. Methods of activating fibroblasts are well known in the art and may encompass contacting the one or more fibroblasts with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof.

[0122] In an aspect, the disclosed organoid or population of cells comprising a fibroblast may further comprise one or more second cell types, for example cells involved in hematopoiesis. Non-limiting examples of cells involved in hematopoiesis include, but are not limited to, bone marrow cells; umbilical cord blood, hematopoietic stem cells; lineage committed lymphocyte progenitors; lineage committed myeloid progenitors; CAR- engineered cells; and or the like; and / or any combination thereof. In an aspect, the second cell type is engineered cell. In an aspect, the engineered cell is engineered to express or overexpress a gene product. Examples of such gene products, include, but are not limited to, G-CSF, GM-CSF, survivin, CXCR4, SCF, VEGF, BMP4, bFGF, TPO, EPO, IL-3, IL-6, IL- 15, SDF-1, Hox genes, DLL4, DLL1, DLL3, X-delta 2, JAG1, JAG2, FOXN1, IL-7, FOXP3, FLT-3L, CCL25, CXCL12, CXCL19, Lymphotoxin-alpha / beta, insulin-growth factor-1 / 2, fibroblast growth factor-7 / 10, HoxAl, Eyal, Pax9, Sixl, Tbxl, Ripply3, E2F3, myc, miR- 181a, Leptin receptor, ghrelin receptor, IL-22, fetal hemoglobin, hemoglobin, chimeric antigen receptor, HLA-E, HLA-G, Siglec-7, Siglec-9, or the like, or a fragment thereof, or a derivative thereof. In an aspect, the engineered cells are engineered have disrupted expression of one or more endogenous gene products. Examples of endogenous gene products, include, but are not limited to, HLA-I, HLA-II, Beta-2 microglobulin, T cell receptor, CD52, dCK, CD7, 4-1BB Ligand, CIITA, CD155 prostaglandin E2, CD26, androgen receptors (selected from, for example, Pgr, Nrli2, Ar, Esrl, Esr2, Gperl, Nr3cl, Nr3c2k, or the like), or the like or any combination thereof.

[0123] In some aspects, the endogenous gene comprises an immune check point gene and the engineered cell is a lymphocyte precursor. Non-limiting examples of immune check point genes, include, but are not limited to PD1, CTLA4, TIM3, TIGIT, CD96, BTLA, LAG3 and the like. Other immune check point genes are known in the art.

[0124] In an aspect, the one of more second cell type may be activated, for example, by adding activating antigens, mitogens, having activated or engineered surface markers, nucleic acid modification, and / or expression or excretion of one or more chemokines, cytokines, exosomes, and / or growth factors. Methods of activating cells are known in the art and may encompass contacting the one or more cells with one or more of202693821.1 - 20 -nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or the like any combination thereof.

[0125] In an aspect, the ratio of the fibroblast to the one or more second cell type may range from about 0.0001:1 to 10000:1 in the organoid or population of cells. Thus, in an aspect, the fibroblast to second cell type ratio may vary from about 0.0001:1, 0.001:1, 0.01:1, 0.1:1, 1:1, 10:1, 100:1, 1000:1, 10000:1, 0.0002:1, 0.002:1, 0.02:1, 0.2:1, 2:1, 20:1, 200:1, 2000:1, 20000:1, 0.0005:1, 0.005:1, or any value within the range. For example, the ratio can be between 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1,1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100 or any range therebetween.

[0126] The disclosed organoid may be about 5 pm to 50,000 pm in size. In an aspect, the disclosed organoid may range in size from about 5 pm - 50 pm, about 50 pm - 60 pm, about 60 pm - 70 pm, about 70 pm - 80 pm, about 80 pm - 90 pm, about 90 pm - 100 pm, about 100 pm - 1,500 pm, about 1,500 pm - 2,000 pm, about 2,000 pm - 3,000 pm, about 3,000 pm - 4,000 pm, about 4,000 pm - 5,000 pm, about 5,000 pm - 6,000 pm, about 6,000 pm - 7,000 pm, about 7,000 pm - 8,000 pm, about 8,000 pm - 9,000 pm, about 9,000 pm - 10,000 pm, about 10,000 pm - 20, 000 pm, about 20,000 pm - 30,000 pm, about 30,000 pm - 40,000 pm, about 40,000 pm - 50,000 pm in size.

[0127] In as aspect, the organoid can give rise to a functional hematopoietic cell, for example, an immune cell, immune cell precursor, blood cell precursor, blood cell, or the like. For example, the organoid can produce and continue to produce an immune cell, immune cell precursor, blood cell precursor, blood cell, and / or the like in culture or in vivo (e.g., after transplantation). Non-limiting examples of immune cell, or immune cell precursor, blood cell precursor, or blood cell include, but are not limited to, a T cell, B cell, NK cell, macrophage, monocyte, neutrophil, platelet, red blood cell, myeloid derived suppressor cell, mast cell, eosinophil, basophil, dendritic cell, or the like. As used herein, the term “functional” refers to the cells ability to respond to activation or suppression cues, cytokine production, and / or potential to modulate disease outcomes.

[0128] In an aspect, the disclosed organoid or population of cells may be for clinical or experimental use in a subject. In an aspect, the disclosed organoid or population of cells may be developed for clinical use in a subject in need thereof. In an aspect, the one or more cells used to develop the organoid, for example, the one or more fibroblasts, the one or more second cell type associated with hematopoiesis, or any combination thereof may be autogenic, allogeneic, xenogeneic, or syngenetic to the subject.202693821.1 -21 -

[0129] In specific aspects, the organoids or population of cells of the disclosure are specifically formulated for administration into a subject in need thereof. They may or may not be formulated as a cell suspension. The cells / organoids may be formulated specifically for intravenous administration, comprising one or more pharmaceutically acceptable carriers, excipients, or vehicles suitable for delivering organoids to a subject. Such formulations may further comprise additives such as stabilizers, preservatives, or penetration enhancers to enhance the stability, bioavailability, or targeted delivery of the organoids / cells. In an aspect, the disclosed compositions may be formulated to maintain cellular osmolarity, pH, and metabolic activity during storage. Salts, sugars like glucose, and buffering agents such as HEPES ensure the physiological integrity of organoids / cells, minimizing ischemic damage and preserving their viability until transplantation or administration.

[0130] For injections of the organoids or population of cells, excipients are tailored to ensure optimal delivery and performance. Physiological saline solutions are frequently utilized as a vehicle for injections, maintaining tissue hydration and osmolarity while minimizing cellular stress. Buffered solutions, such as phosphate-buffered saline (PBS), Plasma-lyte A, Ringers solution or HEPES-buffered saline, serve to stabilize pH levels and osmotic balance, crucial for maintaining tissue integrity during the injection process.

[0131] In some aspects, the organoid and / or second cell type may be engineered using or more of a biologic, chemical, mechanical, or viral delivery method.

[0132] To enhance stability and efficacy, stabilizers may be incorporated into injection formulations. Compounds like albumin or gelatin aid in preventing tissue aggregation or degradation, safeguarding the structural and functional integrity of injected tissues. The cell / organoid formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin. Furthermore, penetration enhancers, such as surfactants or liposomes, may be included to facilitate tissue penetration and uptake of therapeutic agents, ensuring targeted delivery and efficacy.

[0133] To further prevent reduce potential innate or adaptive immune response, and prevent coagulation, the organoid or cell population maybe either pre-treated with heparin or administered along with heparin in order to block tissue factor receptor on cell surfaces of the organoid.

[0134] Overall, the selection and formulation of excipients for organoid and tissue storage and injection are guided by principles of biocompatibility, tissue compatibility, and regulatory requirements. Careful consideration of these factors, alongside thorough validation202693821.1 - 22 -and testing, is essential to developing safe and effective formulations for preserving and delivering organs and tissues in clinical and research settings. The cells / organoids may be formulated specifically for intravenous administration; for example, they may be formulated for intravenous administration over less than one hour. The disclosed formulations find application in various therapeutic, diagnostic, or research contexts, including but not limited to tissue repair, organ regeneration, disease treatment, and imaging.

[0135] Additionally, methods of preparing, administering, and using said formulations, as well as kits comprising the same, are encompassed within the scope of the present disclosure. In specific cases they are formulated in a single dose form or multiple dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may comprise one or more cryopreservation agents, such as DMSO (for example, in 5% DMSO), methylcellulose, ethylene glycol, or any combination thereof. In particular aspects the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from time of thawing.

[0136] In an aspect, the organoid can be supplied with instrumentation required for any of the methods disclosed herein in a kit form.III. Methods of cell and organoid culture

[0137] In an aspect, the current disclosure also encompasses a method of generating the disclosed hematopoietic organoid. The method may comprise co-culturing the one or more fibroblasts, and one or more of a second cell type involved in hematopoiesis, optionally, on a low adhesion surface. The second cell type can be bone marrow cells; umbilical cord blood, hematopoietic stem cells; lineage committed lymphocyte progenitors; lineage committed myeloid progenitors; CAR-engineered cells; and or the like; and / or any combination thereof. As disclosed herein, any of the one or more fibroblasts, or second cell type, may be obtained from an endogenous source or cultured, and / or differentiated in vitro prior to mixing the cells with the disclosed fibroblasts. In an aspect, the method may comprise culturing the one or more fibroblasts as provided herein, with dissociated hematopoietic tissue / source from an autogenic, allogeneic, xenogeneic, or syngenetic source. Cells obtained from the dissociated hematopoietic tissue may be selectively used in the culture or may be used as a hematopoietic tissue extract. Where the cells are selectively used, one or more isolated cells may be further proliferated, differentiated, or both prior to mixing the fibroblasts. In an aspect, the one or more additional cells may not be of hematopoietic202693821.1 - 23 -tissue origin. As such the cells may be isolated or derived from cells isolated from various tissues or organs, including, but not limited to skin, heart, skeletal muscle, liver, pancreas, brain, foreskin, and / or the like which can be obtained by biopsy (where appropriate) or upon autopsy. Derivative cells may also originate from cell-lines.Cell culture methods

[0138] As provided above, certain aspects of the method concern culturing the disclosed cells for incorporation into compositions and / or use in methods described herein. In some aspects, organoids or cells are grown and maintained at an appropriate temperature, typically a temperature of 37°C and under an atmosphere typically containing oxygen and CO2. Culture conditions may vary widely for each cell type though, and variation of conditions for a particular cell type can result in different phenotypes being expressed. The most commonly varied factor in culture systems is the growth medium. Growth media can vary in concentration of nutrients, growth factors, and the presence of other components. The growth factors used to supplement media are often derived from animal blood, such as calf serum.

[0139] In some aspects, organoids or cells may be cultured for at least between about 10 days and about 40 days, for at least between about 15 days and about 35 days, for at least between about 15 days and 21 days, such as for at least about 15, 16, 17, 18, 19 or 21 days. In some aspects, the organoids or cells of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The organoids or cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days. The organoids or cells may be cultured in the presence of a liquid culture medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture cells herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured. In some aspects, a culture medium formulation may be explants medium (CEM) which is composed of IMDM202693821.1 - 24 -supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 pg / ml streptomycin and 2 mmol / L L-glutamine. Other aspects may employ further basal media formulations, such as chosen from the ones above.

[0140] Any medium capable of supporting organoids or cells in vitro may be used to culture the cells. Media formulations that can support the growth of organoids or cells include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimal Essential Medium (aMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, up to 20% fetal bovine serum (FBS) or 1- 20% horse serum is added to the above medium in order to support the growth of cells. However, use of serum-free media, or media comprising cell extracts with no added serum is also envisaged. A defined medium can also be used if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells. The cells may be grown at temperatures between 27° C to 40° C, such as 31° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1% and 22%. The disclosure, however, should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure. For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian202693821.1 - 25 -plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated.

[0141] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. In particular aspects, cells are cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, in particular a cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and / or inducing in an unspecific or specific reprogramming.Organoid generation

[0142] In an aspect, the current disclosure encompasses a method of generating a hematopoietic organoid in vitro, the method may include culturing one or more fibroblasts, and one or more of a second cell type, for example bone marrow cells, umbilical cord blood, hematopoietic stem cells; lineage committed lymphocyte progenitors, lineage committed myeloid progenitors, CAR-engineered cells; any cell derived therefrom bone marrow cells, umbilical cord blood, hematopoietic stem cells; lineage committed lymphocyte progenitors, lineage committed myeloid progenitors, or CAR-engineered cells; and or the like, and / or any combination thereof, in a culture medium. In some aspects, the culturing can be a low adhesion surface. In an aspect, the fibroblasts may be combined with enzymatically or non- enzymatically, or both, dissociated hematopoietic tissue / source and can be cultured together in vitro to form a hematopoietic organoid. One of more cell types from the dissociated hematopoietic tissue / source may be depleted, and other cell types may be added.

[0143] The one or more fibroblasts and the one or more of a second cell type can be co-cultured for a period of time sufficient to induce formation of a hematopoietic organoid. For example, the period of time can be less than about 12, 24, 36, 48 hours or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 days. In some aspects, the organoid self-assembles. In some aspects, the period of time is sufficient to form a functional organoid (e.g., one that produces blood cells and / or immune cells).202693821.1 - 26 -

[0144] The disclosed methods can be used with current protocols in organoid formation. For example, a current protocol can be used with the modification of adding fibroblasts and / or the subtraction of the need to use growth factors or inhibitors. For example, in some embodiments, the claimed method does not include the addition of SCF, Rock-inhibitors, GSK-3 inhibitors, VEGF, BMP4, bfGF, IL-3, Flt-3L, TPO, MCSF, GMCSF, IL-7, and / or the like.

[0145] In an aspect, the organoids may be developed in a coated dish, wherein the coating makes the dish low adhesion. Non-limiting examples of low adhesion cell culture dish coatings include polyethylene glycol (PEG), polyvinyl alcohol (PVA), Pluronic F-127, and hydrogels like agarose or alginate. Any suitable apparatus / dish may be used for developing the disclosed organoids. Non-limiting examples include petri dishes, multi-well plates, cell culture flasks, roller bottles, and bioreactors. Non-limiting examples of low adhesion dish include ELPLASIA® plates, ELPLASIA® flasks, Coming® Ultra-Low Attachment Plates, Greiner CELLSTAR® Low Binding Plates, Thermo Scientific™ Nunc™ Edge 2.0 Plates, Sarstedt Low Binding Cell Culture Plates, and Fisher brand™ Low Binding Plates.

[0146] Methods of generating organoids are known in the art. In an aspect, the methods may use an extracellular matrix to develop the organoids. Non-limiting examples of extracellular matrices for use in the disclosed method include Matrigel, collagen I, laminin, fibronectin, and hyaluronic acid-based matrices. In an aspect, the disclosed method does not require an exogenous extracellular matrix. In an aspect, the disclosed fibroblasts and / or the second cell type associated with hematopoiesis may serve as a source of extracellular matrix, thereby foregoing the need for additional anchor.

[0147] In an aspect, the disclosed organoid may be cultured in any suitable media. The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal, and in an aspect, the same subject. The serum- free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors). The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid- rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors,202693821.1 - 27 -trace elements, 2-mercaptoethanol, 3'-thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98 / 30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically- defined Lipid concentrated (Gibco), and Glutamax (Gibco). In certain aspects, the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components such as corticosterone; D-galactose; ethanolamine hcl; glutathione (reduced); 1- carnitine hcl; linoleic acid; linolenic acid; progesterone; putrescine 2hcl; sodium selenite; and / or t3 (Triiodo-L- thyronine). In specific aspects, one or more of these may be explicitly excluded.

[0148] In some aspects, the medium further comprises one or more vitamins. In some aspects, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some aspects, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some aspects, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some aspects, the medium further comprises proteins. In some aspects, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some aspects, the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or Triiodo-L-thyronine, or combinations thereof. In some aspects, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, or combinations thereof. In some aspects, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some aspects, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine,202693821.1 - 28 -glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some aspects, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some aspects, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain aspects, the medium comprises or consists essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or Triiodo-L-thyronine„ a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific aspects, one or more of these may be explicitly excluded.

[0149] The medium can also contain one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), antioxidant substances, 2- mercaptoethanol, pyruvic acid, buffering agents, and / or inorganic salts. In specific aspects, one or more of these may be explicitly excluded. In an aspect, the medium may also contain growth one or more nucleic acids, cytokines, chemokines, growth factors, transcription factors, epigenetic factors, hormones, or any combination thereof. In an aspect, the medium may further contain agents to activate immune cells, for example thymocytes.

[0150] One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, pg / ml, mg / ml, or any range derivable therein.

[0151] The culturing conditions can vary depending on the specific method and components of the culture. In some aspect, the organoid may be cultures at a fixed or variable temperature ranging from about 20° to about 40 °C, or about 25 °C to about 38 °C, or about 37 °C. In an aspect, the organoid may be cultured at a fixed, or variable carbon dioxide concentration ranging from 1-10%, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% carbon dioxide concentration. In an aspect, the oxygen tension can range from 1-20%, for example about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about202693821.1 - 29 -10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% and any combination of ranges derivable therein.

[0152] In specific aspects, the cells or organoids of the disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In specific cases they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may comprise one or more cryopreservation agents, such as DMSO (for example, in 5% DMSO), methylcellulose, ethylene glycol, or any combination thereof. In some aspects, the organoid may be cryopreserved for at least a day, at least a week, at least 2 weeks, at least 1 months, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years or more before use.

[0153] In particular embodiments, for formation of the organoid, the fibroblasts and / or second cell type may be cultured for 12-72, 24-48, 24-44, 24-40, 24-36, 24-30, 24-28, 28-48, 28-44, 28-40, 28-36, 28-30, 30-48, 30-44, 30-40, 30-36, 36-48, 36-44, 36-40, 40-48, 40-44, 44-48, 24-72, 28-72, 30-72, 36-72, 40-72, or 44-72 hours, including any value or range derivable therein, in an incubator of suitable conditions. In particular embodiments, for formation of the organoid, the fibroblasts and / or second cell type may be cultured for about 1-13, 1-10, 2-10, 2-8, 2-6 days including any value or range derivable therein, in an incubator of suitable conditions. In some aspects, organoids may be cultured for at least between about 10 days and about 40 days, for at least between about 15 days and about 35 days, for at least between about 15 days and 21 days, such as for at least about 15, 16, 17, 18, 19 or 21 days. In some aspects, the organoid of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The organoid may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days in an incubator of suitable conditions. In specific embodiments, the incubator may be set at a particular temperature, such as at about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37°C, including any value or range derivable therein. A range of temperatures includes about 20-37, 20-35, 20-30, 20-27, 20-25, 20-22, 22-37, 22-35, 22-33, 22-30, 22-27, 22-25, 25-37, 25-35, 25-33, 25-30, 25-27, 27-37, 27-35, 27-33, 27-30, 30-37, 30-35, 30-33, 33-37, 33-35, or 35-37°C, including any value or range derivable therein.202693821.1 - 30 -

[0154] In specific embodiments, the incubator may utilize a certain percentage of a gas, such as carbon dioxide, oxygen, and / or nitrogen. The carbon dioxide may be at 3, 4, 5, 6, 7, or 8%. Ranges of carbon dioxide include about 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8%, including any value or range derivable therein. In specific embodiments, the incubator may be set at a particular percentage of humidity, such as about65, 70, 75, 80, 85, or 90% humidity, including any value or range derivable therein. The oxygen in the incubator may be 0.5-25% oxygen, including 0.5-25, 0.5-20, 0.5-15, 0.5- 10, 0.5-5, 0.5-1, 1-25, 1-20, 1-15, 1-10, 1-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, 10-15, 15- 25, 15-20, or 20-25%, including any value or range derivable therein. The oxygen in the incubator may be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, including any value or range derivable therein. The nitrogen in the incubator may be 1-75% nitrogen, including 1-75, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 5-75, 5- 70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-75, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20- 75, 20-70, 20-60, 20-50, 20-40, 20-30, 30-75, 30-70, 30-60, 30-50, 30-40, 40-75, 40-70, 40- 60, 40-50, 50-75, 50-70, 50-60, 60-75, 60-70, or 70-75% nitrogen, including any value or range derivable therein. The nitrogen in the incubator may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75% nitrogen, including any value or range derivable therein.

[0155] A range of humidities may be utilized, such as about 65-90, 65-85, 65-80, 65-75, 65-70, 70-90, 70-85, 70-80, 70-75, 75-90, 75-85, 75-80, 80-90, 80-85, or 85-90%, including any value or range derivable therein. Other incubator conditions and culture durations may be present or required.

[0156] In various embodiments, the size of the produced organoid is able to be controlled at least in part. The desired size may be dictated for one or more purposes, such as a desired size for production purposes, a desired size for transportation purposes, and / or a desired size for a research and / or therapeutic application. In some embodiments, a plurality of organoids to be utilized comprise substantially the same size. In some embodiments, a first plurality of organoids may be combined with a second plurality of organoids of a different size before transportation and / or research and / or therapeutic application; in such cases, the first and second pluralities may have been produced under different conditions, such as different culture parameters, including different durations of culture time. The produced size of the organoids may be dictated by adjusting the concentration of fibroblast202693821.1 - 31 -cells used to initiate organoid formation. Such adjusting of the concentration may or may not occur by dilution; by the selection of the base media; by the selection of one or more additives to the media (such as sugar, serum, non-essential amino acids, L-glutamine, a combination thereof, etc.); by selection of the amount of carbon dioxide in the incubator; by the temperature in the incubator; by the duration of time of production of the organoid; using an outside source of a gas or gas mixtures to feed the incubator; or a combination thereof. The concentration may be increased for certain purposes and decreased for certain purposes. The adjustment of the concentration may increase the size of the organoid by an approximate certain fold level, such as about 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, etc., including any value or range derivable therein. The adjustment of the concentration may decrease the size of the organoid by an approximate certain fold level, such as about 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, etc., including any value or range derivable therein.

[0157] In some embodiments, the size of the organoids can be modified to accommodate varying clinical needs (also see below), such as adjusting the concentration of the cells and / or adjusting the duration of time of growth of the organoid. For example, in embodiments wherein a tissue is in need of repair, regeneration and / or reversal of involution, the size of the organoid may be particularly produced, depending on the tissue site in need of the organoids. In some embodiments, the organoid is utilized as a therapeutic delivery agent wherein the therapeutic(s) delivered by the organoid is the fibroblasts themselves and / or one or more agents derived from the fibroblasts. In such cases, the organoid may act as an extended time release mechanism that allows for the release of the therapeutic(s) over a desired period of time. The desired period of time may be when there is a detectable improvement at the site (either by visual or assaying means). The desired period of time may be when there is complete healing at the site (either by visual or assaying means). In some cases, the desired period of time may be the time to be able to initiate or maintain immune modulation as a result of the extended time release. In some cases, the desired period of time may be the time to be able to initiate or maintain activated or inactivated fibroblasts and / or fibroblast-derived material upon delivery and as a result of the extended time release. In some cases, the desired period of time may be the time to initiate or maintain migration and / or proliferation to a targeted tissue or organ for any purpose (including for any purpose, such as repair, regeneration, and / or reversal of involution) and as a result of the extended time release. In some cases, the desired period of time may be the time to repair, regenerate and / or reverse involution.202693821.1 - 32 -

[0158] In some embodiments, the organoids following production are stored under suitable conditions. In at least specific cases, the temperature of the storage environment is about 2°C to about 37°C. In specific embodiments, the storage environment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37°C, including any value or range derivable therein. The storage temperature may be in range of suitable temperatures, including at least about 2-37, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-37, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-37, 10-35, 10-30, 10-25, 10-20, 20-37, 20-35, 20-30, 20-25, 25-37, 25-35, 25-30, 30-37, 30-35, or 35- 37°C, including any value or range derivable therein. In some aspects, the organoids following production are cryopreserved as described above.Generation of engineered cells

[0159] The one or more cells, including fibroblasts, and second cell type involved in hematogenesis (e.g., bone marrow cells; umbilical cord blood, hematopoietic stem cells; lineage committed lymphocyte progenitors; lineage committed myeloid progenitors; CAR- engineered cells; or the like) may be engineered as disclosed herein above. Genetic modification methods or compositions may be used to introduce nucleic acids into cells, edit genomic DNA or transcripts using gene editing, homologous and non-homologous recombination, TALENS, CRISPR, zinc finger nucleases or any combination thereof. These genetic modifications may introduce exogenous genes, delete, or mutate endogenous genes, introduce selection or identification markers or any combination thereof. These methods are known in the art and may be used in isolation, or a combination of these methods may be used to obtain cells with desired properties.IV. Methods of Using

[0160] The organoids or cell populations of the disclosure can be used in treatment, prevention and / or studying or modeling any disease or disorder associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency, for which the organoids ability to produce and maintain hematopoietic cells and / or immune cells is particularly advantageous and renders them applicable to a wide range of conditions. The organoids or cell populations of the disclosure can be used in a method to regenerate bone marrow tissue. In some embodiments, the methods include administering any of the organoids or cell compositions disclosed herein. Also disclosed herein are the organoids or202693821.1 - 33 -cell compositions disclosed herein for use in the manufacture of a medicament for the treatment of an immune disease or symptom.

[0161] In an aspect, the organoids, cell populations or compositions disclosed herein can be used in methods to treat or prevent any disease or disorder associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency, and / or any disease or disorder where the regeneration of bone marrow tissue or immune cells would be beneficial. In an aspect, the organoids or cell populations disclosed herein are used in a method to regenerate bone marrow tissue. The organoids or cell populations are transplanted or administered in a therapeutically effective amount.

[0162] As used herein, the terms “associated with hematopoiesis or immunity” or “associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency” refers to any disease or symptom where hematopoietic cells or immune cells play a role in the development of the disease or is impacted by the disease. Non- limiting examples of such diseases include, but are not limited to, malignancy (such as leukemia or lymphomas or the like), bone marrow diseases (such as severe aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, megakaryocytes, congenital thrombocytopenia, or the like), immune deficiency (such as SCID, Wiskott- Aldrich syndrome, or the like), hemoglobinopathies (such as beta thalassemia, sickle cell disease, or the like), metabolic disorders (such as Krabbe disease, Hurler syndrome, adrenoleukodystrophy, metachromatic leukodystrophy, or the like), myelodysplastic syndrome, multiple myeloma, familial erythrophagocytic lymphohistiocytic disorders, age related immune deficiency, immune deficiency, bone marrow failure syndrome, Neuroblastoma, plasma cell disorders, POEMS syndrome, primary amyloidosis, autoimmune diseases, or the like.

[0163] For example, the organoid can be transplanted or administered into a subject having any or suspected to have any of the disorders described above. The organoid can be transplanted in a subject. Following transplantation, the subject can have increased survival rate and or alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of a disorder or disease, stabilization of state of disease, prevention of development of a disorder or disease, prevention of spread of a disorder or disease, delay or slowing of disorder or disease progression, delay or slowing of onset of a disorder or disease, amelioration or delay of a disorder or disease state, and remission (whether partial or total).

[0164] In an aspect, the organoid may be formed using any of the methods disclosed herein and then immediately injected into a subject. For example, the organoid may202693821.1 - 34 -be formed within 1, 2, 3, 4, 5, 6, or more days of co-culture and then injected into the subject, for example, the three-dimensional organoid may be immediately injected into a subject within 3 days of setting up the culture. In another aspect, the organoid can be cryopreserved for later use. The organoid can be cryopreserved for days, weeks, months, or years. In an aspect, after the organoid is thawed after cryopreservation and still capable of producing blood cells and / or immune cells. In an aspect, the organoid continues to produce blood cells and / or immune cells after transplantation or administration, for example the organoid may continue to produce blood cells and / or immune cells after transplantation for up to 10, 20, 30, days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or 1, 1.5, 2, or more years.. In some aspects, the methods are dynamic and the organoid may recruit additional hematopoietic stem cells from the subject to maintain the generation of more immune cells.

[0165] For example, the cell population can be administered into a subject having any or suspected to have any of the disorders described above. Following administration, the subject can have increased survival rate and or alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of a disorder or disease, stabilization of state of disease, prevention of development of a disorder or disease, prevention of spread of a disorder or disease, delay or slowing of disorder or disease progression, delay or slowing of onset of a disorder or disease, amelioration or delay of a disorder or disease state, and remission (whether partial or total). A single cell population can be used, or at least two cell populations can be used. For example, a first cell population may include any of the fibroblasts disclosed herein and a second cell population may include any of the second cell types disclosed herein. The first cell population can be administered concurrently with the second population or sequentially in any order. In some aspects, the first cell population is premixed with the second cell population prior to administration. Administration can be intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, intrathymic, perithymic, articular, renal subcapsular, hepatic, injection into the bone marrow, or the like.

[0166] In an aspect, a therapeutically effective amount of the organoid or population of cells is administered in a single dose or various dose.

[0167] In an aspect, the method for treating or preventing the disease or disorder associated with hematopoietic cell and / or immune cell disfunction or deficiency or the method for regenerating hematopoietic tissue can include one or more additional therapies. Non-limiting examples of additional therapies can include chemotherapy, radiation therapy, androgen deprivation therapy, antibody therapeutics, cell therapies, immune activation therapy, checkpoint blockade therapy, immune suppression, leukobalative therapy,202693821.1 - 35 -lymphodepletion therapy, immune tolerogenic therapy, pain therapy, or the like, or any combination thereof. The administration of the organoid or cell population can occur concurrently with the additional therapy or sequentially.

[0168] In another aspect, the hematopoietic organoid or population of cells can be used an in vitro human model system for studying hematopoiesis or immune cell function, studying diseases associated with hematopoietic cell or immune cell disfunction or deficiency, identifying and / or screening for therapeutic targets, and / or identifying therapeutic compounds and / or compositions effective in treating diseases associated with hematopoietic cell or immune cell disfunction or deficiency. Accordingly, the hematopoietic organoids population of cells of the disclosure can allow for new developments in the treatment and study of diseases associated with hematopoietic cell or immune cell disfunction or deficiency.

[0169] In an aspect, the hematopoietic organoid can be supplied with instrumentation required to carry out any of the methods disclosed herein in a kit form.

[0170] Having described the disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLES

[0171] The following non-limiting examples are provided to further illustrate embodiments of the disclosure disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.EXAMPLE 1Generation of a hematopoietic organoid

[0172] A hematopoietic organoid was formed by combining mouse fibroblasts with mouse bone marrow cells at a ratio of 2: 1 in a low-binding Elplasia plate. After 3 days in culture, the plate was imaged (FIG. 2). The size of the diameter was measured to be ~170uM.202693821.1 - 36 -EXAMPLE 2Viability of cells in the hematopoietic organoid

[0173] A hematopoietic organoid was formed by combining mouse fibroblasts with mouse bone marrow cells at a ratio of 2: 1 in a low-binding Elplasia plate. Fibroblasts and bone marrow cells were co-cultured in the Elplasia low binding plates on day 0 and organoids are formed by day 3. On day 3, for FACS analysis, cells were disassociated into single cells using enzymatic digestion with Accumax for 45 minutes. The cells were then washed and stained for viability using 7AAD, then the cells were analyzed on a flow cytometer for viability (FIG. 3). Results show viability of both bone marrow cells and fibroblasts.EXAMPLE 3Viability of hematopoietic organoids after a freeze-thaw cycle

[0174] A hematopoietic organoid was formed by combining mouse fibroblasts with mouse bone marrow cells at a ratio of 2: 1 in a low-binding Elplasia plate. The organoids are harvested after day 3, washed in Media (DMEM / F12 with 10% FBS), then resuspended in freezing medium (DMEM / F12 + 10% FBS + 10% DMSO) and stored in -80 degrees freezer. The cells are then thawed, dissociated into single cells, and stained with 7AAD and compared with equivalent cells that were not frozen (FIG. 4).EXAMPLE 4Functional transplanted organoids

[0175] Organoids were developed according to the disclosed methods. The organoids were transplanted into wild-type mice preconditioned with low dose cyclophosphamide (150mg / kg). Organoids were generated by combining bone marrow cells from CD45.1 congenic mice with fibroblasts. The organoids were transplanted into mice and CD45.1 hematopoietic cells from the organoids were monitored in peripheral blood of the recipient CD45.2 mice. The organoid was shown to produce CD45.1 immune cells after transplantation. Representative images showing the expression of CD45.1 in donor organoid, wild-type mice (express CD45.2), and wild-type mice that were transplanted with CD45.1 organoids (FIG. 5). In Figure 5, The “donor organoid” represents the drug product. It represents what comes out of the culture and has not yet been injected into a mouse. Therefore, the majority of the cells are CD45.1 positive. In Figure 5, the wild-type + donor202693821.1 - 37 -refers to a representative mouse that has been transplanted with the donor organoid (drug product). Hence the majority of the cells (in the third plot) are from the mouse (CD45.2) and CD45.1 cells are produced by the transplanted donor organoid. The frequency of CD45.1 cells in circulation was monitored on day 7 and 14 following transplantation (FIG. 6).EXAMPLE 5Fibroblasts produce growth factors

[0176] Experiments were done to test fibroblast production of growth factors. One million Fibroblasts were cultured for 3 days. Supernatants were harvested and the levels of Stem Cell Factor (SCF) were quantified using an ELISA assay. It was found that the fibroblasts produce high levels of (SCF). SCF is an essential hematopoietic cytokine that plays an important role in maintaining hematopoietic stem and progenitor cells.

[0177] The various methods and techniques described above provide a number of ways to carry out the disclosure. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[0178] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0179] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the disclosure extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications and equivalents thereof.202693821.1 - 38 -

[0180] In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the application are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0181] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0182] Preferred embodiments of this application are described herein. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.202693821.1 - 39 -

[0183] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and / or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and / or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and / or the use of the term in the present document shall prevail.

[0184] In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the disclosure. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.References1. Pinho, S. and P. S. Frenette, Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol, 2019. 20(5): p. 303-320.2. Shoemaker, D., et al., HEMATOPOIETIC STEM AND PROGENITOR CELL THERAPY. 2020: US.3. Pelus Louis, M., J. Hoggatt, and P. Singh, Materials and methods to enhance hematopoietic stem cells engraftment procedures. 2020: AU.4. Sun, W ., et al., Universal chimeric antigen receptor T cell therapy - The future of cell therapy: A review providing clinical evidence. Cancer Treat Res Commun, 2022. 33: p. 100638.202693821.1 - 40 -

Claims

CLAIMSWhat is claimed is:

1. A hematopoietic organoid comprising one or more fibroblasts; and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis, wherein the hematopoietic organoid produces a hematopoietic cell or a hematopoietic progenitor cell.

2. The organoid of claim 1, wherein the second cell type is selected from: a) bone marrow cells or cells derived from bone marrow cells; b) umbilical cord blood cells or cells derived from umbilical cord blood cells; c) hematopoietic stem cells or cells derived from hematopoietic stem cells; d) lineage committed lymphocyte progenitors or cells derived from lineage committed lymphocyte progenitors; e) lineage committed myeloid progenitors or cells derived from lineage committed myeloid progenitors; f) CAR-engineered cells or cells derived from CAR-engineered cells; or g) any combination of a), b), c), d), e), f)3. The hematopoietic organoid of claim 1 or 2, wherein the organoid comprises at least 1% exogenous fibroblasts or between 1-99% exogenous fibroblasts.

4. The hematopoietic organoid of any one of claims 1-3, wherein the ratio of fibroblasts to non-fibroblasts is at least 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100.

5. The hematopoietic organoid of any one of claims 1-4, wherein fibroblasts are incorporated throughout the organoid.

6. The hematopoietic organoid of any one of claims 1-5, wherein the one or more fibroblasts are engineered fibroblasts.

7. The hematopoietic organoid of any one of claims 1-6, wherein the one or more fibroblasts are genetically altered fibroblasts.

8. The hematopoietic organoid of any one of claims 1-7, wherein there are one or more fibroblasts, extracellular vesicles, exosomes, microvesicles, apoptotic bodies in the organoid.202693821.1 -41 -9. The hematopoietic organoid of any one of claim 6-8, wherein the engineered fibroblasts or the genetically altered fibroblasts encode and / or overexpress at least one gene product, optionally an exogenous gene product.

10. The hematopoietic organoid of claim 9, wherein the at least one gene product comprises one or more of Notch ligands, nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, antigens, antigen presenting molecules, co-stimulatory receptors, inhibitory receptors, any fragment or any derivative thereof, and any combination thereof.

11. The hematopoietic organoid of any one of claims 9-8, wherein the at least one gene product comprises G-CSF, GM-CSF, survivin, CXCR4, SCF, VEGF, BMP4, bFGF, TPO, EPO, IL-3, IL-6, IL-15, SDF-1, Hox genes, DLL4, DLL1, DLL3, X-delta 2, JAG1, JAG2, FOXN1, IL-7, FOXP3, FLT-3L, CCL25, CXCL12, CXCL19, Lymphotoxin- alpha / beta, insulin-growth factor-1 / 2, fibroblast growth factor-7 / 10, HoxAl, Eyal, Pax9, Sixl, Tbxl, Ripply3, E2F3, myc, miR-181a, Leptin receptor, ghrelin receptor, IL-22, fetal hemoglobin, hemoglobin, chimeric antigen receptor, or a fragment thereof, or a derivative thereof.

12. The hematopoietic organoid of any one of claims 9-11, wherein the engineered fibroblast or genetically altered fibroblast has a disrupted expression of one or more endogenous genes.

13. The hematopoietic organoid of claim 12, wherein the one or more endogenous gene is of one or more of HLA-I, HLA-II, Beta-2 microglobulin, T cell receptor, CD52, dCK, CD7, 4-1BB Ligand, CIITA, CD155 prostaglandin E2, CD26, androgen receptors (selected from Pgr, Nrli2, Ar, Esrl, Esr2, Gperl, Nr3cl, Nr3c2) or any combination thereof.

14. The hematopoietic organoid of any one of claims 1-13, wherein the one or more of the second cell type is an engineered cell.

15. The hematopoietic organoid of claim 14, wherein the engineered cell expresses a gene product, optionally an exogenous gene product.

16. The hematopoietic organoid of claim 15, wherein the gene product is G-CSF, GM-CSF, survivin, CXCR4, SCF, VEGF, BMP4, bFGF, TPO, EPO, IL-3, IL-6, IL-15, SDF- 1, Hox genes, DLL4, DLL1, DLL3, X-delta 2, JAG1, JAG2, FOXN1, IL-7, FOXP3, FLT-3L, CCL25, CXCL12, CXCL19, Lymphotoxin-alpha / beta, insulin-growth factor-1 / 2, fibroblast growth factor-7 / 10, HoxAl, Eyal, Pax9, Sixl, Tbxl, Ripply3, E2F3, myc, miR-181a, Leptin receptor, ghrelin receptor, IL-22, fetal hemoglobin, hemoglobin, chimeric antigen receptor, HLA-E, HLA-G, Siglec-7, Siglec-9 or a fragment thereof, or a derivative thereof.202693821.1 - 42 -17. The hematopoietic organoid of any one of claim 14-16, wherein the engineered cell has a disrupted expression of one or more endogenous genes.

18. The hematopoietic organoid of claim 17, wherein the one or more endogenous gene is of one or more of HLA-I, HLA-II, Beta-2 microglobulin, T cell receptor, CD52, dCK, CD7, 4-1BB Ligand, CIITA, CD155 prostaglandin E2, CD26, androgen receptors (selected from Pgr, Nrli2, Ar, Esrl, Esr2, Gperl, Nr3cl, Nr3c2) or any combination thereof.

19. The hematopoietic organoid of any one of claims 17-18, wherein the one or more endogenous gene comprises an immune check point gene and the engineered cell is a lymphocyte precursor.

20. The hematopoietic organoid of claim 19, wherein the immune check point gene is selected from PD1, CTLA4, TIM3, TIGIT, CD96, BTLA, or LAG3.

21. The hematopoietic organoid of any one of claims 1-20, wherein the ratio of the one or more fibroblasts to the one or more of the second cell type is in the range of about 0.0001 :1 to 10000: 1, optionally about 100: 1, 90: 1, 80:1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 11, 1 : 12, 1 : 13, 1 : 14, 1 : 15, 1 : 16, 1 : 17, 1 : 18, 1 : 19, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, or 1 : 100.

22. The hematopoietic organoid of any one of claims 1-21, wherein the organoid has a size between 5um to 50,000um, optionally about 5 pm - 50 pm, about 50 pm - 150 pm, about 150pm - 250 pm, about 250 pm - 2,000 pm, about 2,000 pm - 10,000 pm, about 10,000 pm -50,000 pm in size.

23. The hematopoietic organoid of any one of claims 1-22, wherein the one or more fibroblasts, the one or more of a second cell type, or any combination thereof, are autologous, allogeneic, xenogeneic, or syngenetic relative to a subject.

24. The hematopoietic organoid of any one of claims 2-23, wherein the one or more fibroblasts, the one or more bone marrow cells, the one or more hematopoietic stem cells, the one or more umbilical cord blood cells, the one or more lymphoid progenitors or the one or more myeloid progenitors or the one or more umbilical cord blood cells or any combination thereof, are autologous, allogeneic, xenogeneic, or syngenetic relative to a subject.

25. The hematopoietic organoid of any one of claims 1-24, wherein the one or more fibroblasts, the one or more of a second cell type, or any combination thereof, are activated with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof.202693821.1 - 43 -26. The hematopoietic organoid of any one of claims 2-25, wherein the one or more fibroblasts, the one or more hematopoietic stem cells, the one or more umbilical cord blood cells ,the one or more lymphoid progenitors or the one or more myeloid progenitors, CAR-engineered cells, or any combination thereof are activated with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof.

27. The hematopoietic organoid any one of claims 1-26, wherein the one or more fibroblasts are derived from one or more of bone marrow fibroblasts, skin fibroblasts, fibroblasts of other regions of the body, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, and / or progenitor cells.

28. The hematopoietic organoid of any one of claims 1-27, wherein the hematopoietic cell or hematopoietic precursor cell is an immune cell, immune cell precursor or blood cell.

29. The hematopoietic organoid of claim 28, wherein the immune cell, immune cell precursor or blood cell is a T cell, B cell, NK cell, macrophage, monocyte, neutrophil, platelet, red blood cell, myeloid derived suppressor cell, mast cell, eosinophil, basophil, or dendritic cell.

30. A pharmaceutical composition comprising the hematopoietic organoid of any one of claims 1-29.

31. A population of cells comprising one or more fibroblasts; and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis, wherein the population of cells produce a hematopoietic cell or a hematopoietic progenitor cell.

32. The population of cells of claim 31, wherein the second cell type is selected from: a) bone marrow cells or cells derived from bone marrow cells; b) umbilical cord blood cells or cells derived from umbilical cord blood cells; c) hematopoietic stem cells or cells derived from hematopoietic stem cells; d) lineage committed lymphocyte progenitors or cells derived from lineage committed lymphocyte progenitors; e) lineage committed myeloid progenitors or cells derived from lineage committed myeloid progenitors; f) CAR-engineered cells or cells derived from CAR-engineered cells; or g) any combination of a), b), c), d), e), f)202693821.1 - 44 -33. The population of cells of any one of claim 31 or 32, wherein the ratio of the one or more fibroblasts to the one or more of the second cell type is in the range of about 0.0001:1 to 10000:1, optionally about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100.

34. The population of cells of any one of claims 31-33, wherein the one or more fibroblasts are engineered fibroblasts.

35. The population of cells of any one of claims 31-34, wherein the one or more fibroblasts are genetically altered fibroblasts.

36. The population of cells of any one of claims 31-35, wherein there are one or more fibroblasts, extracellular vesicles, exosomes, microvesicles, apoptotic bodies in the organoid or population of cells.

37. The population of cells of any one of claims 31-36, wherein the engineered fibroblasts or the genetically altered fibroblasts encode and / or overexpress at least one gene product, optionally an exogenous gene product.

38. The population of cells of claim 37, wherein the at least one gene product comprises one or more of Notch ligands, nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, antigens, antigen presenting molecules, co-stimulatory receptors, inhibitory receptors, any fragment or any derivative thereof, and any combination thereof.

39. The population of cells of any one of claims 31-38, wherein the at least one gene product comprises G-CSF, GM-CSF, survivin, CXCR4, SCF, VEGF, BMP4, bFGF, TPO, EPO, IL-3, IL-6, IL-15, SDF-1, Hox genes, DLL4, DLL1, DLL3, X-delta 2, JAG1, JAG2, FOXN1, IL-7, FOXP3, FLT-3L, CCL25, CXCL12, CXCL19, Lymphotoxin- alpha / beta, insulin-growth factor-1 / 2, fibroblast growth factor-7 / 10, HoxAl, Eyal, Pax9, Sixl, Tbxl, Ripply3, E2F3, myc, miR-181a, Leptin receptor, ghrelin receptor, IL-22, fetal hemoglobin, hemoglobin, chimeric antigen receptor, or a fragment thereof, or a derivative thereof.

40. The population of cells of any one of claims 31-39, wherein the engineered fibroblast or genetically altered fibroblast has a disrupted expression of one or more endogenous genes.

41. The population of cells of claim 40, wherein the one or more endogenous gene is of one or more of HLA-I, HLA-II, Beta-2 microglobulin, T cell receptor, CD52, dCK,202693821.1 -45 -CD7, 4-1BB Ligand, CIITA, CD155 prostaglandin E2, CD26, androgen receptors (selected from Pgr, Nrli2, Ar, Esrl, Esr2, Gperl, Nr3cl, Nr3c2) or any combination thereof.

42. The population of cells of any one of claims 31-41, wherein the one or more of a second cell type is an engineered cell.

43. The population of cells of claim 42, wherein the engineered cell expresses a gene product, optionally an exogenous gene product.

44. The population of cells of claim 43, wherein the gene product is G-CSF, GM- CSF, survivin, CXCR4, SCF, VEGF, BMP4, bFGF, TPO, EPO, IL-3, IL-6, IL-15, SDF-1, Hox genes, DLL4, DLL1, DLL3, X-delta 2, JAG1, JAG2, FOXN1, IL-7, FOXP3, FLT-3L, CCL25, CXCL12, CXCL19, Lymphotoxin-alpha / beta, insulin-growth factor-1 / 2, fibroblast growth factor-7 / 10, HoxAl, Eyal, Pax9, Sixl, Tbxl, Ripply3, E2F3, myc, miR-181a, Leptin receptor, ghrelin receptor, IL-22, fetal hemoglobin, hemoglobin, chimeric antigen receptor, HLA-E, HLA-G, Siglec-7, Siglec-9or a fragment thereof, or a derivative thereof.

45. The population of cells of any one of claim 31-44, wherein the engineered cell has a disrupted expression of one or more endogenous genes.

46. The population of cells of claim any one of claims 45, wherein the one or more endogenous gene is of one or more of HLA-I, HLA-II, Beta-2 microglobulin, T cell receptor, CD52, dCK, CD7, 4-1BB Ligand, CIITA, CD155 prostaglandin E2, CD26, androgen receptors (selected from Pgr, Nrli2, Ar, Esrl, Esr2, Gperl, Nr3cl, Nr3c2) or any combination thereof.

47. The population of cells of any one of claims 45-46, wherein the one or more endogenous gene comprises an immune check point gene and the engineered cell is a lymphocyte precursor.

48. The population of cells of claim 47, wherein the immune check point gene is selected from PD1, CTLA4, TIM3, TIGIT, CD96, BTLA, or LAG3.

49. The population of cells of any one of claims 31-48, wherein the ratio of the one or more fibroblasts to the one or more of the second cell type is in the range of about 0.0001 :1 to 10000: 1.

50. The population of cells of any one of claims 31-49, wherein the one or more fibroblasts, the one or more of the second cell type or any combination thereof, are autologous, allogeneic, xenogeneic, or syngenetic relative to a subject.

51. The population of cells of any one of claims 32-50, wherein the one or more fibroblasts, the one or more bone marrow cells, the one or more hematopoietic stem cells, the one or more umbilical cord blood cells, the one or more lymphoid progenitors or the one or202693821.1 - 46 -more myeloid progenitors or the one or more umbilical cord blood cells or any combination thereof, are autologous, allogeneic, xenogeneic, or syngenetic relative to a subject.

52. The population of cells of any one of claims 31-51, wherein the one or more fibroblasts, the one or more of the second cell type, or any combination thereof are activated with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof.

53. The population of cells of any one of claims 32-52, wherein the one or more fibroblasts, the one or more hematopoietic stem cells, the one or more umbilical cord blood cells ,the one or more lymphoid progenitors or the one or more myeloid progenitors, CAR- engineered cells, or any combination thereof are activated with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof.

54. The population of cells any one of claims 31-53, wherein the one or more fibroblasts are derived from one or more of bone marrow fibroblasts, skin fibroblasts, fibroblasts of other regions of the body, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, and / or progenitor cells.

55. The population of cells of any one of claims 31-54, wherein the population of cells can give rise to a hematopoietic organoid.

56. The population of cells of claim 55, wherein the organoid can give rise to an immune cell or immune cell precursor or blood cell.

57. The population of cells of claim 56, wherein the immune cell or immune cell precursor or blood cell is a T cell, B cell, NK cell, macrophage, monocyte, neutrophil, platelet, red blood cell, myeloid derived suppressor cell, mast cell, eosinophil, basophil, or dendritic cell.

58. A pharmaceutical composition comprising the organoid or any one of claims 1-29 or the population of cells of any one of claims 31-57.

59. A method of generating a hematopoietic organoid in vitro, the method comprising culturing one or more fibroblasts; with one or more of a second cell type for a period of time in a culture medium, optionally on a low adhesion surface, wherein the second cell type is involved with hematopoiesis.

60. The method of claim 55, wherein the second cell type is a) bone marrow cells or cells derived from bone marrow cells; b) umbilical cord blood cells or cells derived from umbilical cord blood cells; c) hematopoietic stem cells or cells derived from hematopoietic stem cells;202693821.1 - 47 -d) lineage committed lymphocyte progenitors or cells derived from lineage committed lymphocyte progenitors; e) lineage committed myeloid progenitors or cells derived from lineage committed myeloid progenitors; f) CAR-engineered cells or cells derived from CAR-engineered cells; or g) any combination of a), b), c), d), e), f).

61. The method of any one of claims 59-60, wherein the ratio of fibroblasts to second cell type is in the range of about 0.0001:1 to 10000:1, optionally about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100.

62. The method of any one of claims 59-61, wherein the culturing of the hematopoietic organoid does not require an exogenous extracellular matrix.

63. The method of any one of claims 59-62 wherein the culturing of the hematopoietic organoid does not require exogenous growth factors.

64. The method of any one of claims 59-63 wherein the method does not include an exogenous growth factor.

65. The method of any one of claims 59-64 wherein the method does not include addition of exogenous SCF, Rock-inhibitors, GSK-3 inhibitors, VEGF, BMP4, bfGF, IL-3, Flt-3L, TPO, MCSF, GMCSF, and / or IL-7.

66. The method of any one of claims 59-65, wherein the culture medium is a serum-free medium.

67. The method of any one of claims 59-66, wherein the culture medium comprises one or more nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or a combination thereof.

68. The method of any one of claims 59-67, wherein the hematopoietic organoid can generate a hematopoietic cell or hematopoietic precursor cell.

69. The method of claim 68, wherein the hematopoietic cell or hematopoietic precursor cell is an immune cell, immune cell precursor, or blood cell.

70. The method of claim 69, wherein the immune cell, immune cell precursor, or blood cell is a T cell, B cell, NK cell, macrophage, monocyte, neutrophil, platelet, red blood cell, myeloid derived suppressor cell, mast cell, eosinophil, basophil, dendritic cell, or CAR- engineered cell.202693821.1 -48 -71. The method of any one of claims 72, where in the period of time a period of time sufficient for a hematopoietic organoid to self-assemble.

72. The method of any one of claims 71, where in the period of time is about 1, 2, 3, 4, 5, or 6 days.

73. The method of any one of claims 59-72, wherein the organoid:- comprises at least 1% exogenous fibroblasts;- comprises fibroblasts incorporated throughout the organoid; and / or- comprises a ratio of fibroblasts to non-fibroblasts is at least 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10:1, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 : 11, 1 : 12, 1 : 13, 1 : 14, 1 : 15, 1 : 16, 1 : 17, 1 : 18, 1 : 19, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, or 1 : 100.

74. A hematopoietic organoid produced by the method of any one of claims 59- 73.

75. A method of regenerating bone marrow tissue in a subject in need thereof, the method comprising administration to the subject the hematopoietic organoid of any one of claims 1-29 or the population of cells of any one of claims 31-57 or the pharmaceutical composition of claim 30 or 58 in therapeutically effective amounts.

76. A method of treating a disease or disorder in a subject in need thereof, the method comprising administration to the subject the hematopoietic organoid of any one of claims 1-29 or the population of cells of any one of claims 31-57 or the pharmaceutical composition of any one of claims 30 or 58.

77. The method of claim 76, wherein the disease or disorder is associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency.

78. The method of claim 76 or 77, wherein the disease or disorder is selected from one or more of a malignancy (such as leukemia or lymphomas), bone marrow diseases (such as severe aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, megakaryocytes, congenital thrombocytopenia), immune deficiency (such as SCID, Wiskott-Aldrich syndrome), Hemoglobinopathies (such as beta thalassemia, sickle cell disease), Metabolic disorders (such as Krabbe disease, Hurler syndrome, adrenoleukodystrophy, metachromatic leukodystrophy), myelodysplastic syndrome, multiple myeloma, familial erythrophagocytic lymphohistiocytic disorders, age related immune deficiency, immune deficiency, bone marrow failure syndrome, Neuroblastoma, plasma cell disorders, POEMS syndrome, primary amyloidosis, autoimmune diseases. The method of any one of claims 67-68, wherein the hematopoietic organoid or population of cells is202693821.1 - 49 -administered via an intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, intrathymic, perithymic, articular, renal subcapsular, hepatic, or injection into the bone marrow.

79. The method of any one of claims 75-78, wherein administration of the hematopoietic organoid or population of cells (1) does not require a surgical procedure, and / or (II) does not require immune ablative therapy.

80. The method of any one of claims 75-79, wherein the hematopoietic organoid or population of cells is administered via injection.

81. A method of regenerating hematopoietic tissue in a subject in need thereof, the method comprising administering to the subject one or more fibroblasts and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis.

82. The method of claim 81, wherein the second cell type is selected from one of more of a) bone marrow cells or cells derived from bone marrow cells; b) umbilical cord blood cells or cells derived from umbilical cord blood cells; c) hematopoietic stem cells or cells derived from hematopoietic stem cells; d) lineage committed lymphocyte progenitors or cells derived from lineage committed lymphocyte progenitors; e) lineage committed myeloid progenitors or cells derived from lineage committed myeloid progenitors; f) CAR-engineered cells or cells derived from CAR-engineered cells; or g) any combination of a), b), c), d), e), f).

83. The method of any one of claims 81-82, wherein the one or more fibroblasts, the one or more or a second cell type, or any combination thereof are premixed and administered as a population of cells, or as a hematopoietic organoid, or co-administered as at least two populations of cells into a subject in need thereof.

84. The method of claim 83, wherein the at least two populations comprises a first cell population comprising the one or more fibroblasts and a second population comprising one or more of the second cell type.

85. The method of claim 84, wherein the first cell population is administered concurrently or sequentially with the second cell population.

86. The method of any one of claims 81-85, wherein the subject is diagnosed with or is suspected of having a disease associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency.202693821.1 - 50 -87. The method of claim 86, wherein the disease is selected from a malignancy (such as leukemia or lymphomas), bone marrow diseases (such as severe aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, megakaryocytes, congenital thrombocytopenia), immune deficiency (such as SCID, Wiskott- Aldrich syndrome), Hemoglobinopathies (such as beta thalassemia, sickle cell disease), Metabolic disorders (such as Krabbe disease, Hurler syndrome, adrenoleukodystrophy, metachromatic leukodystrophy), myelodysplastic syndrome, multiple myeloma, familial erythrophagocytic lymphohistiocytic disorders, age related immune deficiency, immune deficiency, bone marrow failure syndrome, Neuroblastoma, plasma cell disorders, POEMS syndrome, primary amyloidosis, autoimmune diseases.

88. The method of any one of claims 81-87 further comprising genetic editing of genes in hematopoietic stem cells, lymphoid progenitors, or myeloid progenitors.

89. The method of any one of claims 81-88 further comprising administering to the subject at least one additional therapy.

90. The method of claim 89, wherein the at least one additional therapy is chemotherapy, radiation therapy, androgen deprivation therapy, antibody therapeutics, cell therapies, immune activation therapy, checkpoint blockade therapy, immune suppression, leukobalative therapy, lymphodepletion therapy, immune tolerogenic therapy, or pain therapy.

91. A method of treating or preventing a disease associated with hematopoietic cell disfunction or deficiency and / or immune cell disfunction or deficiency in a subject in need thereof, the method comprising administering to the subject one or more fibroblasts and one or more of a second cell type, wherein the second cell type is involved in hematopoiesis.

92. The method of claim 82, wherein the second cell type is selected from one of more of a) bone marrow cells or cells derived from bone marrow cells; b) umbilical cord blood cells or cells derived from umbilical cord blood cells; c) hematopoietic stem cells or cells derived from hematopoietic stem cells; d) lineage committed lymphocyte progenitors or cells derived from lineage committed lymphocyte progenitors; e) lineage committed myeloid progenitors or cells derived from lineage committed myeloid progenitors; f) CAR-engineered cells or cells derived from CAR-engineered cells; or g) any combination of a), b), c), d), e), f)202693821.1 - 51 -93. The method of any one of claims 81-92, wherein the one or more fibroblasts, the one or more or a second cell type, or any combination thereof are premixed and administered as a population of cells, or as a hematopoietic organoid, or co-administered as at least two populations of cells into a subject in need thereof.

94. The method of claim 93, wherein the at least two populations comprises a first cell population comprising the one or more fibroblasts and a second population comprising one or more of the second cell type.

95. The method of claim 94, wherein the first cell population is administered concurrently or sequentially with the second cell population.

96. The method of any one of claims 91-95, wherein the disease is selected from a malignancy (such as leukemia or lymphomas), bone marrow diseases (such as severe aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, megakaryocytes, congenital thrombocytopenia), immune deficiency (such as SCID, Wiskott- Aldrich syndrome), Hemoglobinopathies (such as beta thalassemia, sickle cell disease), Metabolic disorders (such as Krabbe disease, Hurler syndrome, adrenoleukodystrophy, metachromatic leukodystrophy), myelodysplastic syndrome, multiple myeloma, familial erythrophagocytic lymphohistiocytic disorders, age related immune deficiency, immune deficiency, bone marrow failure syndrome, Neuroblastoma, plasma cell disorders, POEMS syndrome, primary amyloidosis, autoimmune diseases.

97. The method of any one of claims 91-96 further comprising genetic editing of genes in hematopoietic stem cells, lymphoid progenitors, or myeloid progenitors.

98. The method of any one of claims 91-97 further comprising administering to the subject at least one additional therapy.

99. The method of claim 98, wherein the at least one additional therapy is chemotherapy, radiation therapy, androgen deprivation therapy, antibody therapeutics, cell therapies, immune activation therapy, checkpoint blockade therapy, immune suppression, leukobalative therapy, lymphodepletion therapy, immune tolerogenic therapy, or pain therapy.

100. A kit comprising elements of the hematopoietic organoid of any one of claims 1-29 or the population of cells of any one of claims 31-57 or the pharmaceutical composition of any one of claims 30 or 58 in individual or bulk packaging.

101. A kit comprising means for carrying out the method of any one of claims 59-99.202693821.1 - 52 -102. The organoid, population of cells or methods of any one of the preceding claims wherein the organoid, fibroblast or second cell type is engineered using or more of a biologic, chemical, mechanical or viral delivery method.202693821.1 - 53 -

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