Organoid compositions for production of hematopoietic stem cells and derivatives thereof

Abstract

To provide methods for the manufacture of hematopoietic stem cells (HSCs) or derivative immune cells.SOLUTION: The present invention provides a method comprising: a. contacting definitive endoderm derived from a precursor cell with a wnt signaling pathway activator and an FGF signaling pathway activator until foregut cells are formed; and b. culturing the foregut cells in the absence of retinoic acid to form a liver organoid producing hematopoietic cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 730,061, filed September 12, 2018, which is incorporated by reference herein in its entirety for all purposes. [Background technology]

[0002] Currently, bone marrow transplantation is the primary means of treatment for individuals who require reconstitution of the hematopoietic system. Stem and progenitor cells in donated bone marrow can proliferate and replace blood cells involved in protective immunity, tissue repair, clotting, and other functions of blood. After a successful bone marrow transplant, blood, bone marrow, spleen, thymus, and other organs of immunity can be repopulated with cells derived from the donor. Bone marrow has been used with high success rates for a variety of diseases, including aplastic anemia, Fanconi anemia, immunodeficiencies, cancers such as lymphoma or leukemia, carcinomas, various solid tumors, and inherited disorders of hematopoiesis. Bone marrow transplantation has also been applied to treat hereditary storage diseases, thalassemia major, sickle cell disease, and osteoporosis.

[0003] Hematopoietic stem cells (HSCs) have the potential to differentiate into all types of blood cells and can be transplanted to treat blood disorders, but obtaining large quantities of HSCs is difficult due to a shortage of donors. Furthermore, perfectly matched (genetically identical) donors are rare, severely limiting the use of bone marrow transplants to obtain immune cells for individuals in need.

[0004] Therefore, there is still a need in the art for HSC composition suitable for transplantation and the method that can provide HSC.Furthermore, the development of such composition would be useful for research purposes where modern HSC is not available in sufficient quantity.The present disclosure aims to address one or more of the above-mentioned needs in the art. Summary of the Invention

[0005] The present disclosure relates to compositions derived from progenitor cells for the manufacture of hematopoietic stem cells (HSCs) or derived immune cells, and methods of using such compositions.More specifically, methods are disclosed for obtaining hematopoietic stem cells from organoid tissue or cultures comprising organoids, the organoid tissue or cultures comprising liver or colon tissues derived from progenitor cells (such as embryonic stem cells or induced pluripotent stem cells) via differentiation induction. [Brief description of the drawings]

[0006] Those skilled in the art will understand that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way.

[0007] [Figure 1] Characterization of human liver organoid (HLO) gene expression at day 21 of culture. A. Albumin expression is moderately decreased and alpha-fetoprotein (AFP) expression is increased compared to previous methods of differentiating mature liver organoids (Ouchi et al., 2019). B. Endothelial markers CD34 and KDR (VEGFR2) are increased. C. Both erythropoietin (EPO) and hemoglobin gamma (HBG) are increased. [Diagram 2] Myeloid lineage differentiation from HLO cultures. A. HLO cultures were dissociated into single cell suspensions on days 8-20 and added to methylcellulose supplemented with cytokines to promote myeloid differentiation, and colonies were analyzed after 7-14 days. B. Representative Giemsa staining showing the multiple cell types generated. C. Quantification of CFC colonies comparing cells from HLO cultures to umbilical cord blood (UCB) CD34+ cells and undifferentiated iPSCs (ND = not detectable). [Diagram 3] Differentiation of B cells from HLO cultures. A. HLO cultures were dissociated into single cell suspensions on days 8–20 and co-cultured with confluent MS-5 cells. B. Flow cytometry of UCB CD34+ cells and HLO after co-culture with MS-5. Cells were first gated on CD45, then CD19 and CD11b to separate B cells and myeloid cells, respectively. [Figure 4] Hemogenic endothelial cells co-arise in human colonic organoid cultures. (A) RUNX1 (red), ENDOMUCIN (green), and CDX2 (white) staining of whole-tissue preparations of e10.5 mouse embryo nuclear RUNX1 staining in the dorsal aorta (n=3). (B,C) Optical slices from (A). (D) Whole-tissue preparation RUNX1 (red), CD34 (green), and CDH1 (white) staining of day 22 HCO showing nuclear RUNX1 staining within CD34+ endothelial tubes (n=3). (E,F) Optical slices from (D). DA=dorsal aorta. (G) TPM (transcripts per million) graphs of RNAseq data from day 21 HIO and HCO (n=3 for each group). (H) Flow cytometry plots of gated CD34+ cells stained with CD45 and CD73. CD34+ / CD45- / CD73- cells are boxed in black. CD34+ / CD45+ / CD73- cells are boxed in green. [Diagram 5] Erythroid, myeloid and lymphoid progenitors are generated in HCO cultures. (A) Micrograph of cytospun cells from HCO cultures. (B) Example of colonies formed in Methocult™ medium. (C) Quantification of colony formation in Methocult™ of cells from HCO cells derived from H1 human embryonic stem cells and (D) IPSC 263-10. (E) Flow cytometry plot of CD45 gated cells stained for CD3 and CD4, treated or untreated by cytokine induced T cell differentiation. [Figure 6]HCOs contain co-occurring macrophages. (A) Immunofluorescent staining of human colon biopsies for CD163 (red) and CDH1 (green) counterstained with DAPI. (A') Inset of boxed region in (A). (B) HIO and (C) HCO whole tissue preparations stained for CD163 (red) and CDH1 (green) counterstained with DAPI. Graphs of TPM (transcripts per million) for (D) SPI1 (PU.1) and (E) CD163 from RNAseq data obtained from HIOs and HCOs at day 35 (n=3 for each group). Immunofluorescent staining of (F) mouse colon, (G) human colon biopsy and explanted HCO counterstained with DAPI and stained for hCD163 (red) and F4 / 80 (green). [Figure 7] HCOs harbor inflammatory macrophages that secrete pro-inflammatory cytokines. (A) Heatmap of inflammation-related genes generated from RNAseq data of 35dHIOs and HCOs. (B) Immunofluorescence staining of day 35 HCOs for CD163 (green), iNOS (red), and CDH1 (white) counterstained with DAPI. (B') Inset of boxed region in B, excluding DAPI. (C-D) Luminex array data for IL-6, IL-8, MIP1A (CCL3), and MIP1B (CCL4). Each point represents Luminex values ​​from an individual differentiation. Paired HIO and HCO samples (from the same differentiation) are indicated by lines. [Figure 8] HCO macrophages are functional. (A) Time lapse micrographs of live imaging of HCOs treated with LPS. (B-E) Luminex array data for IL-6, IL-8, MIP1A (CCL3), MIP1B (CCL4) of HCOs or HCOs treated with LPS. (F) Immunofluorescence staining of HCOs at day 35 for - / + pHRODO E.coli particles (green) and CD14 (red). (G) Quantification of phagocytosed particles (green) (n=3 organoid wells per group). [Figure 9]HCOmacs migrate into the organoid lumen in response to bacteria. Immunofluorescence staining of HCOs at day 35 for CDH1 (green) and MUC2 (red) 24 h after injection of (A) PBS, (B) commensal E. coli, and (C) EHEC, counterstained with DAPI. Immunofluorescence staining of HCOs at day 35 for CDH1 (green), HAM56 (red), and E. coli (white) 24 h after injection of (D) PBS, (E) commensal E. coli, and (F) EHEC, counterstained with DAPI. (G) Quantification of MUC2 fluorescence intensity (n=3 per group). (H) Quantification of E. coli fluorescence intensity (n=3 per group). (I) Quantification of HAM56 macrophage distribution (n=3 organoids per group). [Figure 10] Experimental workflow. [Figure 11] BMP signaling specifies hemogenic endothelial cells. [Figure 12] Endothelial and hematopoietic cells co-develop within HCO cultures. [Figure 13] Hemoglobin expression in red blood cells derived from HCO cultures. [Figure 14] Cytof analysis of immune cells present in HCO. [Figure 15] WELLS Fig. S6. Macrophages persist within HCO after transplantation into the mouse kidney capsule. [Figure 16] WELLS Fig. S7. Gene ontology analysis reveals parallel cell differentiation, macrophage maturation, and inflammation in HCO. [Figure 17] Macrophages within HCOs can extend filopodia in response to E. coli particles. [Figure 18] Images of the placenta, liver, lungs, and colon. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] definition Unless otherwise specified, terms should be understood according to conventional usage by those skilled in the relevant art. In case of conflict, the present document including definitions shall prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used to practice or test the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0009] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, and reference to "a dosage" includes reference to one or more dosages and equivalents thereof known to those of skill in the art, and so forth.

[0010] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly 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. When a particular value is described in the present application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0011] As used herein, the term "totipotent stem cells" (also known as omnipotent stem cells) are stem cells that can differentiate into embryonic and extraembryonic cell types. Such cells are capable of constructing a complete, viable organism. These cells are produced from the fusion of an egg and a sperm cell. The cells produced by the first few divisions of a fertilized egg are also totipotent.

[0012] As used herein, the term "pluripotent stem cells (PSCs)", also commonly known as PS cells, encompasses any cell that can differentiate into nearly any cell, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (stomach lining, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), and ectoderm (epidermal tissue and nervous system). PSCs can be derived from embryonic stem cells (including embryonic germ cells) or can be the progeny of totipotent cells obtained through the induction of non-pluripotent cells, such as adult somatic cells, by forcing the expression of certain genes.

[0013] As used herein, the term "induced pluripotent stem cells (iPSCs)," commonly abbreviated as iPS cells, refers to a type of pluripotent stem cell that is artificially derived from a normally non-pluripotent cell, such as an adult somatic cell, by inducing "forced" expression of certain genes.

[0014] As used herein, the term "embryonic stem cells (ESCs)", commonly abbreviated as ES cells, refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For purposes of the present invention, the term "ESCs" is sometimes used broadly to encompass embryonic germ cells as well.

[0015] As used herein, the term "progenitor cell" encompasses any cell that can be used in the methods described herein, through which one or more progenitor cells acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, progenitor cells are subject to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, progenitor cells can be totipotent (or omnipotent) stem cells, pluripotent (induced or non-induced) stem cells, multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells can be formed from embryos, infants, children, or adults. In some embodiments, progenitor cells can be somatic cells that are subject to treatment such as through genetic manipulation or protein / peptide treatment to confer pluripotency.

[0016] As used herein, the term "cellular constituent" refers to individual genes, proteins, mRNA expression genes, and / or other variable cellular constituents, or protein activities, such as the degree of protein modification (e.g., phosphorylation), that are commonly measured in biological experiments (e.g., by microarray or immunohistochemistry) by those skilled in the art. Important discoveries related to the complex network of biochemical processes that underlie biological systems, common human diseases, and gene discovery and structure determination can now be attributed to the application of cellular constituent data as part of the research process. The cellular constituent data can be used to identify biomarkers, distinguish disease subtypes, and identify mechanisms of toxicity.

[0017] FGF signaling pathway activator: Fibroblast growth factor (FGF) is a family of growth factors involved in angiogenesis, wound healing, and embryonic development. In some embodiments, any of the FGFs can be used in combination with proteins from the Wnt signaling pathway, as will be understood by those skilled in the art. Exemplary FGF signaling pathway activators can include small molecule or protein FGF signaling pathway activators, FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, and combinations thereof. siRNA and / or shRNA that target cellular components related to FGF signaling pathway can be used to activate these pathways. Those skilled in the art will easily understand the appropriate amount and duration.

[0018] WNT signaling pathway activators: Modulators / activators of Wnt signaling pathway can include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. In some embodiments, regulation of pathway can be through the use of small molecule or protein modulators that activate the aforementioned pathways, or proteins that activate the aforementioned pathways. For example, small molecule modulators of the Wnt pathway include, but are not limited to, lithium chloride, 2-amino-4,6-disubstituted pyrimidine (hetero)arylpyrimidine, IQ1, QS11, NSC668036, DCAβ-catenin, 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl)pyrimidine. In some embodiments, exogenous molecules include WAY-316606, SB-216763, or BIO (6-bromoindirubin-3'-oxime). In some embodiments, siRNA and / or shRNA targeting cellular components related to the Wnt and / or FGF signaling pathways can be used to activate these pathways. It will be understood by those skilled in the art that target cellular components include, but are not limited to, SFRP proteins GSK3, Dkk1, and FrzB. Additional modulators include molecules or proteins that inhibit GSK3, which activates the Wnt signaling pathway. Exemplary GSK3 inhibitors can include Chiron / CHIR99021, for example, inhibit GSK3β.Those skilled in the art will recognize suitable GSK3 inhibitors for carrying out the disclosed method.WNT signaling pathway activator can be administered in an amount sufficient to carry out the disclosed method.Those skilled in the art will easily understand the appropriate amount and duration.

[0019] BMP Activators: Exemplary BMP signaling pathway activators may be selected from BMP2, BMP4, BMP7, BMP9, small molecules that activate the BMP pathway, proteins that activate the BMP pathway, including noggin, dorsomorphin, LDN189, DMH-1, ventromorphin, and combinations thereof.

[0020] Organoid technology is a developing field. Briefly, organoids are "organ-like tissues", or three-dimensional tissues with similar structural organization to the corresponding native organ. Organoids can be derived from progenitor cells, such as embryonic stem cells or induced pluripotent stem cells. Organoids are usually cultured in vitro using a time sequence of growth factor manipulations that mimic embryonic development for the desired organ tissue - a process commonly referred to as differentiation induction of progenitor cells. In general, organoids can often contain differentiated cell types that are functional, such as gastric parietal cells that can secrete acid. Thus, the organoids currently reported in the literature are not the same in scope as those of naturally derived organ tissues. For example, organoids may lack the vascular system or one or more other features of the native organ that they may be intended to mimic. To date, organoids have not been recognized to have a developed hematopoietic system or to generate significant amounts of immune cells. The present disclosure attempts to address one or more such needs in the art.

[0021] As described herein, methods and systems have been established using sequential growth factor manipulation to mimic the embryonic development of tissues in culture, with modifications that allow for the development of immune system cells, variations of which are described herein.

[0022] Hematopoietic Stem and Progenitor Cells Blood cell formation originates from a single type of cell, the hematopoietic stem cell, and through proliferation and differentiation, develops into the entire hematopoietic system. Hematopoietic stem cells are thought to be capable of self-renewal, expanding their own population of stem cells, and they are multipotent (capable of differentiating into any cell in the hematopoietic system). From this rare population of cells, the entire mature hematopoietic system is formed, including lymphocytes (B and T cells of the immune system) and myeloid cells (erythrocytes, megakaryocytes, granulocytes, and macrophages). The lymphoid lineage, including B and T cells, produces antibodies, regulates the cellular immune system, detects foreign substances in the blood, detects cells that are foreign to the host, etc. The myeloid lineage, including monocytes, granulocytes, megakaryocytes, and other cells, monitors for the presence of foreign substances, provides defense against neoplastic cells, removes foreign substances, produces platelets, etc. The erythroid lineage produces red blood cells that function as oxygen carriers.

[0023] As mentioned above, the current treatment for replacing HSC includes bone marrow transplantation.Patient "matching" can be difficult, so there is a need in the art for compositions and methods that address this need.To this end, disclosed herein is a hematopoietic cell generating organoid composition and a method for producing hematopoietic cell generating organoid.

[0024] The disclosed compositions and methods can be used to generate hematopoietic cells, and can be used for the treatment of any disease state where administration of hematopoietic cells is advantageous.Therefore, the method can further comprise isolating or collecting hematopoietic cells from the disclosed organoid composition.The disease state that can be treated using hematopoietic cells derived from the disclosed organoid includes, for example, beta-thalassemia, sickle cell anemia, adenosine deaminase deficiency, recombinase deficiency, recombinase regulatory gene deficiency through the introduction of wild-type genes into stem cells, for example, using CRISPR technology.The organoid and / or hematopoietic cells disclosed herein can be used in certain embodiments to reconstitute irradiated hosts or hosts undergoing chemotherapy.

[0025] composition Also disclosed herein is a hematopoietic stem cell composition that is substantially free of differentiated or specific hematopoietic cells, for example, highly enriched hematopoietic stem cell composition. Substantially free can mean that less than 10%, or less than 5%, or less than 1% of cells are present in the population. The hematopoietic cells derived from the organoid composition can be a substantially homogenous viable mammalian or human hematopoietic cell composition, and can be generated for various purposes, for example, bone marrow transplantation, where the cells can be largely free of neoplastic cells or other cells that are pathogenic, for example, HIV-infected cells, transplantation where avoidance of graft-versus-host disease is desired. Substantially homogenous can mean that the majority of the cells in the composition are of the same cell type, for example, at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or in certain circumstances more than 95% of the desired cells, and the cell type can be hematopoietic stem cells. In certain embodiments, hematopoietic cells can be modified by appropriate recombination, either homologous or non-homologous, to correct genetic defects or provide genetic capabilities that stem cells naturally lack, either for the individual or for stem cells in general. Such genetically modified cells (i.e., using CRISPR methods well known in the art) can be further administered to individuals in need thereof.

[0026] HCO culture to generate immune cells In the first aspect, the method of producing hematopoietic stem cell (HSC) or its derived cell is disclosed.The method can include: contacting the definitive endoderm derived from progenitor cell with wnt signaling pathway activator and FGF signaling pathway activator until foregut cell is formed; and culturing foregut cell in the absence of retinoic acid to form the liver organoid that produces hematopoietic cell.

[0027] In one aspect, the progenitor cells of any preceding paragraph may be selected from one or both of embryonic stem cells and induced pluripotent stem cells (iPSCs).

[0028] In one aspect, the wnt signaling pathway activator of any preceding paragraph is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, small molecule activators of the wnt signaling pathway (e.g., lithium chloride, 2-amino-4,6-disubstituted pyrimidine (hetero)arylpyrimidines, IQ1 , QS11, NSC668036, DCA β-catenin, 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl)pyrimidine), WAY-316606, SB-216763, or BIO (6-bromoindirubin-3'-oxime)), siRNA and / or shRNA activators of the Wnt signaling pathway, GSK3 inhibitors (e.g., Chiron / CHIR9902), and combinations thereof.

[0029] In one aspect, the FGF signaling pathway activator of any preceding paragraph may be selected from small molecule or protein FGF signaling pathway activators, FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, siRNA and / or shRNA activators of the FGF signaling pathway, and combinations thereof.

[0030] In one embodiment, any of the preceding paragraphs may further comprise forming spheroids from foregut cells before forming liver organoids.In other embodiments, foregut cells may generate spheroids before generating liver organoids, and the method may further comprise fragmenting the spheroids to generate a plurality of cells derived from the spheroids.Fragmentation may be achieved through one or both of chemical and / or mechanical disruption.For example, in one embodiment, fragmentation may comprise treatment with enzymes such as enzymes having one or both of proteolytic enzyme activity and collagenolytic enzyme activity, for example, one or more enzymes selected from Accutase, trypsin, collagenase, hyaluronidase, DNase, papain, Trypzean (Sigma), or combinations thereof.

[0031] In one embodiment, any of the preceding paragraphs may further comprise culturing the foregut in the presence of a cytokine.The cytokine may be any cytokine accepted in the art, such as a cytokine selected from transferrin, stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and combinations thereof.In certain embodiments, the foregut may be dissociated into single cells before culturing. Culturing with cytokines may be carried out for a period of time, for example, about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 9 weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or more than 12 weeks.

[0032] In one embodiment, the method of any preceding paragraph includes contacting liver organoids, e.g., human liver organoids, with one or both of thrombopoietin (TPO) and stem cell factor (SCF), wherein the contacting with one or both of thrombopoietin (TPO) and stem cell factor (SCF) is for a period of time, e.g., about 1 day, about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or The method is carried out for a period selected from about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 9 weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or more than 12 weeks.

[0033] In one embodiment, the liver organoid of any preceding paragraph can be a human liver organoid derived from human progenitor cells, such as organoids that are understood to be in fetal state, for example, organoids that contain fetal liver tissue.For example, in one embodiment, liver organoids can produce reduced albumin compared to human liver organoids that are treated with retinoic acid.In one embodiment, liver organoids produce alpha-fetoprotein (AFP).In one embodiment, liver organoids increase endothelial markers CD34 and KDR compared to liver organoids that are treated with retinoic acid.In one embodiment, liver organoids increase erythropoietin (EPO) and hemoglobin gamma (HBG) compared to liver organoids that are treated with retinoic acid.

[0034] In one aspect, the method can include the steps of any preceding paragraph and further suspending the foregut cells in a basement membrane matrix, e.g., Matrigel™. The foregut cells can be further cultured with a stromal cell line, e.g., a stromal cell line derived from bone marrow.

[0035] In the method of claim 1, the derivative cells are selected from myeloid cells (such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes up to platelets), lymphoid cells (such as T cells, B cells, and natural killer cells), and combinations thereof.

[0036] HCO and HCO culture to generate immune cells In one embodiment, human colon organoid (HCO) comprising hemogenic endothelium and the method of making it are disclosed.In one embodiment, the hemogenic endothelium of HCO described herein produces immune cells, such as one or more of erythroid-myeloid progenitor cells, lymphoid progenitor cells, and macrophages.In one embodiment, the hemogenic endothelium of disclosed HCO produces macrophages that secrete proinflammatory cytokines.Disclosed HCO can further comprise hematopoietic progenitor cells, said progenitor cells being CD34+, said CD34 progenitor cells being in organoid mesenchyme, said hematopoietic progenitor cells being capable of producing T cells.In another embodiment, disclosed HCO can comprise endothelial duct, said endothelial duct (ET) being positive for CD34+, said ET comprising RUNX1+ cells.

[0037] Colon organoid can be derived from progenitor cell such as human progenitor cell and can be used to obtain immune cells.In this aspect, a method is disclosed, comprising: culturing colon organoid to form organoid culture, and collecting one or more immune cells from the colon organoid culture.

[0038] In this embodiment, colon organoids can be cultured for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 9 days several weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or for more than 12 weeks, or until the colon organoids comprise one or more of hemogenic endothelium and endothelial ducts that generate hematopoietic progenitor / stem cells.

[0039] In one embodiment, the method can comprise separating mesenchyme from the colon organoid culture and culturing the mesenchyme.In one embodiment, the mesenchyme culturing step can be carried out for a period of about 4 days to 3 months, or about 5 days to 2 months, or about 6 days to about 1 month, or about 7 days to about 21 days.In a further embodiment, the mesenchyme culture can be suspension culture.

[0040] In one embodiment, the colon organoid can comprise mesenchyme, and the culturing step can be carried out for a certain period of time, for example, about 4 days to 3 months, or about 5 days to 2 months, or about 6 days to about 1 month, or about 7 days to about 14 days. The culturing step can be carried out as suspension culture for a period of about 1 week to 4 weeks, or about 1 week, to allow the expansion of mesenchyme.

[0041] In one embodiment, the immune cells of the disclosed method can be selected from erythrocytes, myeloid, and mixed myeloid colonies. In other embodiments, the immune cells can be one or more of macrophages, neutrophils, eosinophils, basophils, erythrocytes, leukocytes, and monocytes.

[0042] In one aspect, colon organoids can be derived from definitive endoderm derived from progenitor cells as described herein.In one aspect, progenitor cells are embryonic stem cells or induced pluripotent stem cells.

[0043] In one embodiment, the method can further comprise culturing the organoid culture with T cell-inducing growth factor.In a further embodiment, the method can comprise disrupting the culture to disperse colonic organoid into single colonic organoid and disrupting the mesenchyme of the culture.This step can be by culturing the resulting disrupted organoid and mesenchyme in basement membrane matrix (e.g., Matrigel) for a period of about 1 week to about 4 weeks, or about 2 weeks to about 3 weeks.

[0044] In further embodiments, colon organoids, particularly human colon organoids, can be used to model pathologies.For example, methods are disclosed for modeling pathologies selected from necrotizing enterocolitis, very early onset IBD30, infection from bacterial pathogens (such as Clostridium difficile), infection from viral pathogens (such as HIV, which easily infects fetal intestinal macrophages).In this embodiment, the method can include initiating pathologies in colon organoids, such as human colon organoids, produced according to the methods disclosed herein.

[0045] In one aspect, a method of making a HCO or HIO capable of generating hematopoietic stem cells (HSCs) is disclosed, the method comprising contacting definitive endoderm derived from progenitor cells with one or more factors for a period of time sufficient to generate midgut / hindgut spheroids, optionally embedding the midgut / hindgut spheroids in a basement membrane matrix, and contacting the DE with a combination of factors including FGF, CHIR, Noggin, and a SMAD inhibitor in an amount and for a period of time sufficient to generate anterior foregut spheroids, wherein the midgut / hindgut spheroids or anterior foregut spheroids generate HSCs.

[0046] In yet another aspect, a method for treating an individual who needs immune cells is disclosed. The method comprises collecting hematopoietic stem cells (HSCs) or derived cells from HCO or HLO according to any of the above paragraphs, and administering the HSCs or derived cells to an individual who needs them, and the administration comprises transplanting the HSCs into the bone marrow of the individual. In one aspect, the treatment can be for anemia (including aplastic anemia, Fanconi anemia), immune deficiency, cancer (lymphoma, leukemia, carcinoma, solid tumor, etc.), genetic disorders of hematopoiesis, inherited storage diseases, thalassemia major, sickle cell disease, osteoporosis, or a combination thereof.

[0047] Progenitor cells In some embodiments, stem cells that are pluripotent or can be induced to become pluripotent can be used. In some embodiments, pluripotent stem cells are derived from embryonic stem cells, which are in turn derived from the totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of early embryos, blastocysts. Methods for deriving embryonic stem cells from blastocysts are well known in the art. For example, three cell lines (H1, H13, and H14) had normal XY karyotypes, and two cell lines (H7 and H9) had normal XX karyotypes. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described in this application, but it will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells. Additional stem cells that can be used in embodiments of the present invention include, but are not limited to, those described in databases provided by the National Stem Cell Bank (NSCB), the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF), the WISC cell Bank at the Wi Cell Research Institute, the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC), Novocell, Inc. (San Diego, Calif.), Cellartis AB (Gothenburg, Sweden), ES Cell International Pte Ltd (Singapore), the Technion at the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania.Exemplary embryonic stem cells that can be used in embodiments according to the present invention include, but are not limited to, SA01 (SA001), SA02 (SA002), ES01 (HES-1), ES02 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6), BG01 (BGN-01), BG02 (BGN-02), BG03 (BGN-03), TE03 (I3), TE04 (I4), TE06 (I6), UC01 (HSF1), UC06 (HSF6), WA01 (H1), WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14). In some embodiments, the stem cells are further modified to incorporate additional properties. Exemplary modified cell lines include, but are not limited to, H1 OCT4-EGFP, H9 Cre-LoxP, H9 hNanog-pGZ, H9 hOct4-pGZ, H9 inGFPhES, and H9 Syn-GFP.For details on embryonic stem cells, see, for example, Thomson et al., 1998, "Embryonic Stem Cell Lines Derived from Human Blastocysts," Science 282(5391):1145-1147, Andrews et al., 2005, "Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin," Biochem Soc Trans 33:1526-1530, Martin 1980,"Teratocarcinomas and mammalian embryogenesis,".Science 209(4458):768-776, Evans and Kaufman,1981,"Establishment in culture of pluripotent cells from mouse embryos,"Nature 292(5819):154-156, Klimanskaya et al.,2005,"Human embryonic stem cells derived without feeder Alternatively, pluripotent stem cells can be derived from embryonic germ cells (EGCs), which are the cells that give rise to the gametes of sexually reproducing organisms. EGCs are derived from primordial germ cells found in the gonadal ridges of the late embryo and have many of the characteristics of embryonic stem cells. Primordial germ cells in the embryo develop into stem cells that generate reproductive gametes (sperm or eggs) in the adult. In mice and humans, embryonic germ cells can be grown in tissue culture under appropriate conditions. Both EGCs and ESCs are pluripotent. For purposes of the present invention, the term "ESCs" is sometimes used to encompass EGCs.

[0048] Induced pluripotent stem cells (iPSC) In some embodiments, iPSCs are derived by transfection of specific stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection may be accomplished via a viral vector, such as a retrovirus. Transfected genes include the master transcriptional regulators Oct-3 / 4 (Pouf51) and Sox2, although other genes have been suggested to increase the efficiency of induction. After 3-4 weeks, a small number of transfected cells begin to resemble morphologically and biochemically pluripotent stem cells, and are typically isolated by morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include, but are not limited to, first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells. In some embodiments, non-viral based techniques may be used to generate iPSCs. In some embodiments, adenoviruses can be used to transport the required four genes into the DNA of mouse skin and liver cells, resulting in cells identical to embryonic stem cells. Adenovirus does not combine its own genes with the target host, thus eliminating the risk of creating tumors. In some embodiments, reprogramming can be achieved through plasmids without using any viral transfection system, albeit with very low efficiency. In other embodiments, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viral or genetic modification. In some embodiments, generation of mouse iPSCs is possible using similar methodology. Repeated treatment of cells with specific proteins delivered to cells via polyarginine anchors was sufficient to induce pluripotency. In some embodiments, expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.For details of embryonic stem cells, see, for example, Kaji et al., 2009, "Virus free induction of pluripotency and subsequent excision of reprogramming factors," Nature 458:771-775; Woltjen et al., 2009, "piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells," Nature 458:766-770; Okita et al., 2008, "Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors," Science 322(5903):949-953; Stadtfeld et al., 2008, "Induced Pluripotent Stem Cells Generated without Viral Integration," Science 322(5903):945-949; and Zhou et al., 2009, "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins," Cell Stem Cell 4(5):381-384, each of which is incorporated herein in its entirety. In some embodiments, exemplary iPS cell lines include, but are not limited to, iPS-DF19-9, iPS-DF19-9, iPS-DF4-3, iPS-DF6-9, iPS(Foreskin), iPS(IMR90), and iPS(IMR90).

[0049] Definitive endoderm. The spheroids, organoids, and / or tissues described herein may be derived from a simple sheet of cells called definitive endoderm (DE). Methods for deriving definitive endoderm from progenitor cells are well known in the art, as taught by D'Armour et al. 2005 and Spence et al. Any method for generating definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) is applicable to the methods described herein. In some embodiments, the pluripotent cells are derived from morulae. In some embodiments, the pluripotent stem cells are stem cells. The stem cells used in these methods may include, but are not limited to, embryonic stem cells. Embryonic stem cells can be derived from the inner cell mass of an embryo or the gonadal ridge of an embryo. Embryonic stem cells or germ cells can be derived from various animal species, including, but not limited to, various mammalian species, including humans. In some embodiments, human embryonic stem cells are used to produce definitive endoderm. In some embodiments, human embryonic germ cells are used to produce definitive endoderm. In some embodiments, iPSC cells are used to produce definitive endoderm. In some embodiments, one or more growth factors are used in the differentiation process from pluripotent stem cells to DE cells. The one or more growth factors used in the differentiation process can include growth factors from the TGF-beta superfamily. In such embodiments, the one or more growth factors can include the Nodal / Activin and / or BMP subgroups of the TGF-beta superfamily of growth factors. In some embodiments, the one or more growth factors are selected from the group consisting of Nodal, Activin A, Activin B, BMP4, Wnt3a, or any combination of these growth factors. In some embodiments, the embryonic stem cells or germ cells and iPSCs are treated with one or more growth factors for 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 84 hours or more, 96 hours or more, 120 hours or more, 150 hours or more, 180 hours or more, or 240 hours or more.In some embodiments, the embryonic stem cells or germ cells and iPSCs are treated with one or more growth factors at a concentration of 10ng / ml or more, 20ng / ml or more, 50ng / ml or more, 75ng / ml or more, 100ng / ml or more, 120ng / ml or more, 150ng / ml or more, 200ng / ml or more, 500ng / ml or more, 1,000ng / ml or more, 1,200ng / ml or more, 1,500ng / ml or more, 2,000ng / ml or more, 5,000ng / ml or more, 7,000ng / ml or more, 10,000ng / ml or more, or 15,000ng / ml or more. In some embodiments, the concentration of the growth factor is maintained at a constant level throughout the treatment. In other embodiments, the concentration of the growth factor is changed during the treatment process. In some embodiments, the growth factor is suspended in a medium containing fetal bovine serine (FBS) with various HyClone concentrations. Those skilled in the art will understand that the regimen described herein is applicable to any known growth factor, alone or in combination. When using two or more growth factors, the concentration of each growth factor can be varied independently. In some embodiments, a population of cells that is enriched for definitive endoderm cells is used. In some embodiments, the definitive endoderm cells are isolated or substantially purified. In some embodiments, the isolated or substantially purified definitive endoderm cells express SOX17, FOXA2, and / or CXRC4 markers to a greater extent than OCT4, AFP, TM, SPARC, and / or SOX7 markers. Methods for enriching a cell population with definitive endoderm are also contemplated. In some embodiments, definitive endoderm cells can be isolated or substantially purified from a mixed cell population by contacting the cells with a reagent that binds to a molecule present on the surface of the definitive endoderm cells but not on the surface of other cells, and then separating the cells that are bound to the reagent. In certain embodiments, the cellular component present on the surface of the definitive endoderm cells is CXCR4.Additional methods for obtaining or generating DE cells that can be used in the present invention include, but are not limited to, those described in U.S. Pat. No. 7,510,876 to D'Amour et al., U.S. Pat. No. 7,326,572 to Fisk et al., Kubol et al., 2004, "Development of definitive endoderm from embryonic stem cells in culture," Development 131:1651-1662, D'Amour et al., 2005, "Efficient differentiation of human embryonic stem cells to definitive endoderm," Nature Biotechnology 23:1534-1541, and Ang et al., 1993, "The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3 / forkhead proteins," Development 119:1301-1315, each of which is incorporated herein by reference in its entirety. In some embodiments, soluble FGF and Wnt ligands are used to mimic early hindgut specification in culture, and through differentiation induction, DE generated from iPSCs or ESCs is converted into hindgut epithelium that efficiently gives rise to all major intestinal cell types. In humans, differentiation induction of DE is achieved by selectively activating certain signaling pathways that are important for intestinal development. It will be understood by those skilled in the art that altering the expression of any Wnt signaling protein in combination with any FGF ligand can cause differentiation induction as described herein.In some embodiments, the DE culture is treated with one or more modulators of a signaling pathway described herein for 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 84 hours or more, 96 hours or more, 120 hours or more, 150 hours or more, 180 hours or more, 200 hours or more, 240 hours or more, 270 hours or more, 300 hours or more, 350 hours or more, 400 hours or more, 500 hours or more, 600 hours or more, 700 hours or more, 800 hours or more, 900 hours or more, 1,000 hours or more, 1,200 hours or more, or 1,500 hours or more.

[0050] In some embodiments, the DE culture may be treated with one or more modulators of a signaling pathway described herein at a concentration of 10 ng / ml or more, 20 ng / ml or more, 50 ng / ml or more, 75 ng / ml or more, 100 ng / ml or more, 120 ng / ml or more, 150 ng / ml or more, 200 ng / ml or more, 500 ng / ml or more, 1,000 ng / ml or more, 1,200 ng / ml or more, 1,500 ng / ml or more, 2,000 ng / ml or more, 5,000 ng / ml or more, 7,000 ng / ml or more, 10,000 ng / ml or more, or 15,000 ng / ml or more. In some embodiments, the concentration of the signaling molecule is kept constant throughout the treatment. In other embodiments, the concentration of the modulator of the signaling pathway is changed during the treatment process. In some embodiments, the signaling molecule according to the present invention is suspended in a medium comprising DMEM and fetal bovine serine (FBS). FBS can be at a concentration of 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 50% or more. One of skill in the art will appreciate that the regimens described herein are applicable to any known modulators of the signaling pathways described herein, alone or in combination, including but not limited to molecules in the Wnt and FGF signaling pathways.

[0051] In embodiments in which more than one signaling molecule is used to treat a DE culture, the signaling molecules can be added simultaneously or separately. When more than one molecule is used, the concentration of each can be varied independently. EXAMPLES

[0052] The following non-limiting examples are provided to further illustrate the embodiments of the present invention disclosed herein. Those skilled in the art should understand that the techniques disclosed in the following examples represent methods that are recognized to function well in the practice of the present invention, and therefore can be considered to constitute examples of the manner of its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed, and still obtain the same or similar results, without departing from the spirit and scope of the present invention.

[0053] Establishment of functional immune cells in colonic organoid cultures derived from human pluripotent stem cells Adult hematopoietic progenitors arise from hemogenic endothelium that develops in close proximity to the embryonic colon. Here, applicants engineer colonic organoid cultures derived from human pluripotent stem cells that co-generate hemogenic endothelium and hematopoietic progenitors capable of forming myeloid and lymphoid derivatives. BMP signaling can be used to generate hindgut mesenchyme that is capable of forming hemogenic endothelium and gives rise to RUNX1-expressing hematopoietic progenitors. Up to 3 weeks of culture, a diverse array of myeloid cell types and lymphoid cells are present. Macrophages can be maintained within the developing mesenchyme of HCOs after extended in vitro culture. Following transplantation of HCOs and 3 months of growth in vivo, human macrophages derived from PSCs established a close association with the colonic epithelium and were not replaced by host-derived macrophages. HCO-associated macrophages were functional, responded to both LPS and pathogenic bacteria by producing proinflammatory cytokines, underwent transepithelial migration, and phagocytosed bacteria, all characteristics of tissue-resident macrophages. As in the embryo, Applicant's engineered human hindgut / colon organoid cultures contain macrophages that support the formation of hemogenic endothelium that gives rise to myeloid and lymphoid lineages, and establish long-term residence in the developing human colon.

[0054] There is a diverse array of immune cells present in the adult intestinal tract. These include both myeloid and lymphoid cell types, which cooperate with the epithelium and ENS to maintain barrier function, communicate with the microbiome, and distinguish between helpful and harmful antigens. Most diseases of the intestine, particularly inflammatory bowel disease (IBD), involve the immune system. Dogma holds that all immune cells in the intestine are derived from hematopoietic stem cells (HSCs) that are derived from the bone marrow. However, increasing evidence from animal studies supports the conclusion that some organs contain populations of tissue-resident macrophages that co-arise during embryonic development. 1-3 Recently, it has been shown that the colon contains a stable, self-maintaining population of macrophages that are derived from both embryonic progenitors and adult HSCs. 4 .

[0055] Hematopoietic cells arise from three sites: primitive hematopoietic cells arise during gastrulation, migrate to the yolk sac, and are short-lived 5 Adult hematopoietic progenitor cells (HPCs) are derived from the hemogenic endothelium either in the yolk sac or in the aorta-gonad-mesonephros (AGM) region of the embryo adjacent to the developing colon. One distinguishing feature of primitive hematopoietic cells is that they have limited differentiation potential and lack lymphoid lineage potential. Intraembryonic HPCs derived from the AGM region express markers such as Runx1 and Tek and emerge from the endothelium of the aorta and surrounding blood vessels adjacent to the hindgut. 6 The development of this posterior region of the embryo requires BMP signaling 7-10 In addition, BMP signaling regulates the expression of GATA2, a transcription factor required for hemogenic endothelial formation. 11 .

[0056] Engineering human colon organoids (HCOs) by inducing differentiation of human pluripotent stem cells has already been achieved by the applicant. 10 Such HCOs contain both colonic epithelium and surrounding mesenchymal derived cells including fibroblasts, myofibroblasts, and smooth muscle cells. Bioinformatics analysis of the transcriptional changes that occurred during the stages of HCO differentiation revealed a surprising enrichment in genes associated with hematopoietic development. Disclosed herein is a method of processing the cultures to identify the range of cell types present within the HCO, as shown in FIG. 10.

[0057] Hematopoietic progenitors arise from the caudal mesoderm. To determine whether the caudal mesoderm is specified, applicants tested the expression of anteriorly and posteriorly expressed HOX genes. Consistent with previous results, posterior HOX genes were significantly upregulated by BMP treatment (Figure 11, panel D). Furthermore, anterior HOX factors, including HOXA3, which inhibits EHT12, were downregulated in HCOs compared to HIOs. When formation of the hindgut and colon fates is induced by transient activation of BMP signaling (BMP2 treatment occurs on days 7-10 of the protocol), expression of hematopoietic vascular markers, including GATA2, KDR / FLK1, and pan-endothelial markers CD34 and VEGFR1, is observed, similar to that observed during developing mouse embryonic hindgut (Figure 11).

[0058] The presence of immune cells in HCO cultures raises the question of whether these cells are primitive or derived from hemogenic endothelium. No expression of GYPA (CD235), a marker of primitive hematopoietic progenitor cells (Figure ​(Figure11,F), was observed. 13 , suggesting that the source of hematopoietic cells was hemogenic endothelium. RUNX1-expressing hematopoietic progenitor cells have been shown to emerge from the endothelium of the AGM region in e10.5 mouse embryos (Figure 4(A-C)). HCO cultures similarly contained CD34+ endothelial tubes with associated clusters of RUNX1+ cells (Figure 4(D-G)). Hemogenic endothelium can be further distinguished from non-hemogenic endothelium by the lack of CD73 expression. 14 Analysis of day 21 HCO cultures by flow cytometry revealed CD34 + / CD73 - revealed the presence of endothelial cells, suggesting the presence of hemogenic endothelium (Figure 4(H)). By 21 days of culture, the transcriptional profile of HCOs revealed pathway relationships related to immune cells and their functions compared to HIOs. These included neutrophil degranulation, the innate immune system, platelet activation, and transendothelial migration of leukocytes. Analysis of HCO cultures at day 21 by immunofluorescence staining (IF) revealed the expression of PU.1, an endothelial protein embedded within the mesenchyme of colonic organoids but not in intestinal organoids. + Cells and CD34+ The presence of endothelial tubes was confirmed (Figure 12). Brightfield live imaging of HCO cultures at day 22 revealed refractory cells migrating within endothelial tubes, appearing and visible as both mesenchymal and floating in the medium. Taken together, these data suggest that HCO cultures contain hemogenic endothelium capable of generating hematopoietic cells.

[0059] To determine the cell types in the HCO cultures, the medium was sampled and cytospins and Giemsa staining were performed to identify cells resembling macrophages, neutrophils, eosinophils, and basophils (Figure 5, A). No exogenous factors or mouse bone marrow stromal cells were added to the cultures, suggesting that the mesenchymal cell types in the HCO were able to support the differentiation of myeloid cell types. To determine whether there were erythromyeloid progenitors generated by the HCO, a Methocult™ assay was performed. HCO contained progenitors capable of generating erythromyeloid, myeloid, and mixed myeloid colonies, whereas HIO cultures did not (Figure 5, B). HCO from human embryonic and induced pluripotent stem cell lines were capable of generating erythromyeloid derivatives, indicating that the method is robust across PSC lines. Erythrocytes generated from HCO expressed fetal (HBG1 and 2) and fetal / adult (HBA1, HBA2) hemoglobins but no appreciable levels of embryonic hemoglobin (HBE1, HBZ), suggesting that HCO cultures contain adult erythroid myeloid progenitors.

[0060] [Table 1]

[0061] One of the hallmarks of definitive hematopoietic cells in the embryo is their ability to give rise to lymphoid cell types such as T cells. 13、15Because hematopoietic progenitors with lymphoid potential appear late in embryonic development, applicants hypothesized that these progenitors would emerge following long-term culture of HCO mesenchyme. Applicants therefore developed a culture method that allows for the long-term maintenance of intact hemogenic endothelial tubes (Figure 10, C). HCO cultures were grown for an additional week to expand the mesenchyme, which was scraped off the plate and grown in suspension culture for up to an additional 3 weeks. IL7 and FLT3 were added to test for lymphoid potential T cell-inducing growth factors. In the absence of T cell induction, HCO cultures contained 0.2% CD3+ / CD4+ cells. Addition of T cell-inducing growth factors increased the number of T cells by 4-fold (Figure 5, E). The presence of T cells with potential further supports the conclusion that the hematopoietic cells formed from HCO cultures are of the adult type.

[0062] Although definitive hematopoiesis migrates during development to other organs, including the fetal liver and then bone marrow, tissue-resident macrophages can colonize organs early in development and persist until emergence. In some organs, such as the lung and liver, embryonic macrophages persist throughout life. 5 In other organs, HSCs in the postnatal bone marrow give rise to macrophages that replace embryonic macrophages. In the colon, some data suggest that embryonic macrophages are replaced by macrophages derived from HSCs. 16、17 However, recent lineage tracing of embryonic macrophages suggests that they persist postnatally along with macrophages derived from HSCs. Colon organoids were passaged at day 21 by trituration, which results in the disruption of mesenchyme and dispersion of individual HCOs. HCOs were then replated in Matrigel and cultured for an additional 14 days. Transcriptional profiles were examined with gene ontology analysis and observed enrichment for myeloid cell types, such as leukocytes, neutrophils, as well as GO terms associated with defense and inflammatory responses. [Table 2]

[0063] Immunostaining of HCOs on day 35 revealed macrophages expressing the markers CD68 and HAM56 (data not shown), as well as the tissue-resident macrophage marker CD163 (Figure 12) 18、19 CD163 is expressed on several tissue-resident macrophage populations, including alveolar macrophages, Kupffer cells in the liver, and Hofbauer cells in the placenta. 20-22 The presence of inflammatory macrophages was investigated by co-staining for CD163 and iNOS, a known marker of inflammatory macrophages (Figure 7). Interestingly, the majority of CD163+ macrophages were also positive for iNOS, suggesting that these cells are inflammatory. Taken together, these data suggest that HCOs contain co-occurring macrophages that co-express tissue-resident and inflammatory macrophage markers.

[0064] Multiple subtypes of macrophages exist in the human intestine. To determine whether macrophages in HCOs are heterogeneous, we investigated the expression of cell surface markers using CYTOF. CYTOF analysis revealed the presence of at least four distinct monocyte populations, including a CD11bhi population, a CD14- / CD16+ population, a CD14+ / CD16+ population, and a CD14+ / CD16- population. These data suggest that HCO cultures can generate a diverse set of monocytes / macrophages similar to the native human intestine.

[0065] It has been hypothesized that tissue-resident macrophages in the colon are continually replenished by bone marrow-derived monocytes (BMDMs). 16、17 However, recent studies have challenged that paradigm, suggesting that some subtypes of macrophages are continuously replenished by BMDMs, whereas others have an embryonic source that is long-lived and self-maintaining. 4、23、24To determine whether HCO macrophages ("HCOMac") could be maintained long term, the presence of human CD163+ macrophages was examined after transplantation of HCO into mouse kidney capsule (FIG. 8). Applicant hypothesized that the short-lived macrophages would be replaced by host-derived murine macrophages expressing the mouse-specific marker F / 480. Only a few hCD163+ cells were detected in control but not NOG HIO transplants, and F / 480+ macrophages infiltrated all mesenchymal layers up to the top of the villi (FIG. 5). In contrast, hCD163 macrophages were readily detectable in HCO even 12 weeks after transplantation. These macrophages were primarily located in the lamina propria, which lacked infiltration of F / 480+ macrophages. In the muscularis layer, hCD163+ cells were interspersed with F / 480+, suggesting that host macrophages have colonized these tissue layers. Examination of blood and bone marrow from HCO-implanted mice revealed a lack of human-derived cells, suggesting that hCD163 macrophages were self-maintained within HCO and were not replenished by human cells that had colonized the bone marrow (Figure 14). These data suggest that HCO generate self-maintaining macrophages independently of BMDM.

[0066] Examination of RNAseq data from 35-day HCOs revealed inflammatory signs compared to HIOs. To confirm that HCOs indeed exhibited functional inflammation, secretion of pro-inflammatory cytokines into the medium of HCOs was examined using Luminex multiplex ELISA (Figure 13). IL1B, IL6, and IL8 have all been reported to be expressed by epithelial cells in vitro, suggesting that epithelial cells may contribute to the inflammatory signs exhibited in HCOs. To confirm that macrophages were inflammatory, secretion of macrophage inflammatory protein 1A (MIP1A) and 1B (MIP1B) was examined. HCOs secreted significantly higher levels of MIP1A and MIP1B, suggesting that macrophages within HCOs exhibit a basal level of inflammation.

[0067] Adult colonic macrophages are normally resistant to stimulation by lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria. 25 In contrast, fetal macrophages respond to LPS stimulation, suggesting that tolerance is achieved postnatally. 26 To determine whether macrophages within HCOs were sensitive to LPS stimulation, HCOs were treated with LPS and cell motility and secretion of inflammatory cytokines were examined. Live imaging revealed that macrophages increased their motility in response to LPS and were able to undergo chemotaxis to foci within the organoids (Figure 8(A)). Examination of cytokine secretion revealed significant increases in IL6, IL8, MIP1A, MIP1B, and TNFA, suggesting that cytokine production may be a driver of macrophage motility (Figure 8(B-E)).

[0068] Direct stimulation of HCOs with LPS suggested that macrophages within organoids are able to respond to cytokines and bacterial factors. Macrophages play a direct role in innate immunity by phagocytosing bacteria. To determine whether HCO macrophages ("HCOmacs") can phagocytose bacteria, HCOs were treated with E. coli particles labeled with a pH-sensitive fluorophore. Live imaging revealed that HCOmacs continuously extend filopodia and survey the microenvironment. HCOmacs phagocytose bacterial particles within acidic phagolysosomes, as evidenced by an increase in pH-sensitive fluorescence (Figure 8(F-G)). In addition, microinjection of live commensal E. coli and EHEC induced macrophage migration into the lumen of HCOs (Figure 9(A-F)), similar to what has been observed in Salmonella-infected mice. 27 Moreover, introduction of bacteria into the lumen of HCOs resulted in a decrease in MUC2 staining (Figure 9(G)), likely due to the degradation of mucus by the bacteria. Taken together, our data suggest that HCO Macrophages are functional resident-like macrophages capable of responding to bacterial particles and live bacteria.

[0069] Development of the mammalian hindgut and aorta-gonad-mesonephros regions occurs in close proximity to one another. BMP signaling has been shown to activate both posterior HOX patterns of hindgut endoderm and mesenchyme, as well as expression of the hemogenic endothelial transcription factor GATA2. Using the methods described above to generate HCOs, BMP signaling also specifies hemogenic endothelium with definitive hematopoietic potential. This is consistent with normal human development, where definitive hematopoietic progenitors are formed from the ventral posterior mesoderm. Thus, it appears that the HCO culture described herein mimics most of the posterior embryo more closely than originally thought.

[0070] Hematopoietic progenitors from HCO cultures have erythroid-myeloid and lymphoid lineage potential even in the absence of hematopoietic growth factors and mouse-derived bone marrow stromal cells (OP9-DLL4 cells). This suggests that the mesoderm that co-occurs in HCO cultures compensates for the lack of these signals and cell types. Interestingly, hematopoietic growth factors are expressed in HIO cultures, suggesting that the development of cells expressing these factors is not dependent on BMP signaling. These cell types may be an alternative to using OP9-DLL4 cells, which are murine and therefore likely confer immunogenic properties to human hematopoietic progenitors. 28、29 Moreover, recent studies have shown that a subset of macrophages is self-maintained in the intestine, so these cell types may also be present in normal intestinal tissue. 4 .

[0071] The presence of co-occurring macrophages within HCOs brings a new tool to examine the interactions between innate immune cells and the colonic epithelium. Furthermore, HCOs can be used to determine the niche factors that allow the maintenance of tissue-resident macrophages. HCOs will allow for the modeling of inflammatory diseases such as necrotizing enterocolitis, very early onset IBD, bacterial pathogens such as Clostridium difficile, and viral pathogens such as HIV, which readily infect fetal intestinal macrophages. 31Furthermore, the incorporation of other immune cell types can be used to study other innate immune mechanisms, such as neutrophil-induced inflammatory hypoxia. 32 .

[0072] method DE induction. Human ES and iPS cells were plated as single cells at 150,000 cells per well in mTesR1 medium supplemented with ROCK inhibitor Y27632 (10 μM, Stemgent) in Matrigel (BD Biosciences)-coated 24-well plates. Starting the next day, cells were treated with Activin A (100 ng / ml, Cell Guidance Systems) for 3 days in RPMI1640 (Invitrogen) containing increasing concentrations of non-purified fetal bovine serum (dFBS, Invitrogen) at 0%, 0.2%, and 2.0%. Endoderm patterning and gut morphogenesis. After DE induction, cells were treated with growth factors / antagonists for 3 days in RPMI1640 supplemented with 2.0% dFBS. To generate posterior foregut spheroids, DEs were treated with FGF4 (500 ng / ml, R&D Systems) and CHIR99021 (3 μM, Stemgent) for 4 days. Three-dimensional growth. Mid-hindgut spheroids were embedded in Matrigel (BD Biosciences) as previously described. 10、12 , and subsequently expanded in Advanced DMEM / F12 (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen), L-glutamine, 10 μM HEPES, penicillin / streptomycin, and EGF (100 ng / ml, R&D Systems). For proximal intestinal specification, Noggin (100 ng / ml, R&D Systems) was added for the first 3 days of three-dimensional growth. For colonic specification, BMP2 (100 ng / ml, R&D Systems) was added for the first 3 days of three-dimensional growth.

[0073] A method for generating human intestinal organoids (HIOs) with increased immune cell production Human embryonic stem cells and induced pluripotent stem cells are maintained on Matrigel (BD Biosciences) in feeder-free mTesR1 medium. Differentiation to definitive endoderm was performed as previously reported (D'Amour KA, et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005;23:1534-1541). Briefly, a 3-day Activin A (R&D Systems) differentiation protocol was used. Cells are treated with Activin A (100 ng / mL) for 3 consecutive days in RPMI 1640 medium (Invitrogen) supplemented with increasing concentrations of 0%, 0.2%, and 2% HyClone depurated fetal bovine serum (dFBS) (Thermo Scientific). For hindgut differentiation, DE cells are incubated in 2% dFBS-DMEM / F12 supplemented with 500ng / ml FGF4 and 500ng / ml Wnt3a (R&D Systems) for up to 4 days. Three-dimensional floating spheroids form within 2-4 days of treatment with growth factors and are then transferred to three-dimensional cultures that have previously been shown to promote intestinal growth and differentiation (Gracz AD, Ramalingam S, Magness ST. Sox9-Expression Marks a Subset of CD24-Expressing Small Intestine Epithelial Stem Cells that Form Organoids in vitro. Am J Physiol Gastrointest Liver Physiol. 2010;298:G590-600, 16. Sato T,et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459:262-265).Briefly, spheroids were embedded in Matrigel (BD Bioscience) containing 500ng / mL R-Spondin1 (R&D Systems), 100ng / mL Noggin (R&D Systems), and 50ng / mL EGF (R&D Systems). After Matrigel solidified, they were covered with growth factor-containing medium (Advanced DMEM / F12 (Invitrogen) supplemented with L-glutamine, 10μM Hepes, N2 supplement (R&D Systems), B27 supplement (Invitrogen), and Pen / Strep) and replaced every 4 days.

[0074] A method for generating human colon organoids (HCO) with increased immune cell production Human embryonic stem cells and induced pluripotent stem cells are grown under feeder-free conditions in 6-well Nunclon surface plates (Nunc) coated with Matrigel (BD Biosciences) and maintained in mTESR1 medium (Stem Cell Technologies). For definitive endoderm (DE) induction, human ES or iPS cells are passaged with Accutase (Invitrogen) and added to Matrigel-coated Nunclon surface 24-well plates at a density of 100,000 cells per well. For Accutase split cells, 10 μM Y27632 compound (Sigma) was added to the medium on the first day. After the first day, the medium was replaced with mTESR1 and the cells were allowed to grow for another 24 hours. The cells are then treated with 100 ng / mL activin A for 3 days as previously reported (Spence et al., 2011). After DE induction, DE were treated with hindgut induction medium (RPMI1640, 2mM L-glutamine, 2% heat-inactivated FBS, penicillin-streptomycin) for 4 days with 500ng / mL FGF4 (R&D) and 3μM Chiron 99021 (Tocris, WNT pathway activator, inhibits GSK3) to induce the formation of mid-hindgut spheroids. Midgut / hindgut spheroids were collected from 24-well plates and added to Matrigel (BD) as previously described, followed by growth in Advanced DMEM / F12 (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen), L-glutamine, 10μM HEPES, penicillin / streptomycin, and EGF (100ng / ml, R&D Systems). To generate human colon organoids (HCO), spheroids were covered with 100ng / mL EGF plus 100ng / mL BMP (BMP2 or 4, R&D, or other BMP pathway activators may also be used) for at least 3 days. Media was changed after 3 days, maintaining only EGF in the media for all patterning conditions. Media was then changed twice weekly.

[0075] Isolation of human hematopoietic cells from HCO cultures At least 3 days after activation of the BMP pathway (e.g., day 9), HCOs begin to contain vascular mesoderm cells expressing markers such as KDR, FLT1, and GATA2. Continued growth in Matrigel results in the formation of hemogenic endothelium expressing CD31, CD34. Between days 15 and 20, the cultures have endothelial tubes that generate hematopoietic progenitor / stem cells expressing RUNX1. By collecting the medium of HCO cultures, a wide range of differentiated hematopoietic cells were identified, including myeloid cells (basophils, neutrophils, eosinophils), monocytes, and macrophages. Using flow cytometry, cells expressing markers of immature B and T cells can also be seen. [Table 3]

[0076] Growth of cells harvested from the medium in Methocult™ H4434 Classic results in the formation of colonies composed of erythrocytes, granulocytes, and macrophages. Methocult™ H4434 Classic contains Iscove's MDM plus methylcellulose, fetal bovine serum, bovine serum albumin, 2-mercaptoethanol, recombinant human stem cell factor (SCF), recombinant human interleukin 3 (IL-3), recombinant human erythropoietin (EPO), and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0077] Generation of functional macrophages in HCO Continued culture with passaging of HCO into fresh Matrigel results in the generation of functional macrophages (from about day 20 to about day 34 onwards). Macrophages are functionally responsive to infectious stimuli such as lipopolysaccharide (LPS) or bacteria, phagocytosing bacteria and producing proinflammatory cytokines including IL6, IL8, CCL3, CCL4, and TNF-α spontaneously and in response to LPS. Macrophages also respond to IL10, resulting in decreased proinflammatory cytokine production. Macrophages also respond to M-CSF inhibition or addition, with decreased and increased macrophage numbers, respectively.

[0078] A method for generating human liver organoids with increased immune cell production Differentiation of hiPSCs into definitive endoderm is induced using a previously reported method with some modifications (Spence et al., 2011). Briefly, hiPSC colonies are dissociated with Accutase (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 150,000–300,000 cells are added to Matrigel- or laminin-coated tissue culture 24-well plates (Corning, Durham, NC, USA). When cells reached high density (>90% of the well was covered with cells), the medium was changed to RPMI 1640 medium (Life Technologies, Carlsbad, CA) containing 100 ng / mL activin A (R&D Systems, Minneapolis, MN) and 50 ng / mL bone morphogenetic protein 4 (BMP4, R&D Systems) on day 1, 100 ng / mL activin A and 0.2% fetal calf serum (FCS, Thermo Fisher Scientific Inc.) on day 2, and 100 ng / mL activin A and 2% FCS on day 3. On days 4–6, cells are differentiated into posterior midguts by culturing in Advanced DMEM / F12 (Thermo Fisher Scientific Inc.) supplemented with 2% B27 (Life Technologies), 1% N2 (Gibco, Rockville, MD), 2 mM L-glutamine (Gibco), and 1 mM HEPES (Gibco), 1% penicillin / streptomycin (Gibco), containing 500 ng / ml fibroblast growth factor (FGF4, R&D Systems) and 3 μM CHIR99021 (Stemgent, Cambridge, MA, USA). Cultures for cell differentiation are maintained at 37°C in an atmosphere of 5% CO2 / 95% air, and the medium is changed daily. Differentiated definitive endoderm showed budding on plates on day 7. If spheroids are not sufficient to embed in Matrigel, add the medium from days 4–6 again and incubate at 37°C overnight.

[0079] Differentiation into liver organoids. Four methods can be used to differentiate DE into liver organoids: "Matrigel drop method", "Matrigel sandwich method", "Matrigel-free method", and "spheroid-generating transwell method", each of which is described below.

[0080] Matrigel Drop Method: On days 7–8, definitive endoderm organoids with seeded cells are detached from the dish by gentle pipetting. The isolated spheroids are centrifuged at 800 rpm for 3 min, and after removing the supernatant, they are embedded in 100% Matrigel drop on the dish. 250 μL of Matrigel (Corning) is used per well of a 24-well plate for endoderm culture. One 80 μL Matrigel drop is made for each well of a 24-well plate (VWR Scientific Products, West Chester, PA). The plate is left at 37 °C in an atmosphere of 5% CO2 / 95% air for 5–15 min. After the Matrigel solidifies, Advanced DMEM / F12 is added with B27, N2, L-glutamine, HEPES, penicillin / streptomycin, and retinoic acid (RA, Sigma, St. Louis, MO) 2 μM for 1–5 days. The medium is changed every other day. After RA treatment, the organoids embedded in the Matrigel drops are cultured in hepatocyte culture medium (HCM Lonza, Walkersville, MD) supplemented with 10 ng / mL hepatocyte growth factor (HGF, PeproTech, Rocky Hill, NJ), 0.1 μM dexamethasone (Dex, Sigma), and 20 ng / mL oncostatin M (OSM, R&D Systems). Cultures for cell differentiation are maintained at 37 °C in an atmosphere of 5% CO2 / 95% air, and the medium is changed every 3 days. Around days 20-30, the organoids embedded in the Matrigel drops can be isolated by scraping and gently pipetting for any analysis.

[0081] Matrigel sandwich method: On days 7–8, gently pipette the definitive endoderm organoids with seeded cells to detach them from the dish. Centrifuge the isolated spheroids at 800 rpm for 3 min, remove the supernatant, and then mix them with 100% Matrigel. At the same time, mix the hepatocyte medium supplemented with all supplements with the same volume of 100% Matrigel. Place the mixture of HCM and Matrigel on the bottom of the dish to create a thick coating on the plate (0.3–0.5 cm) and leave it at 37 °C in an atmosphere of 5% CO2 / 95% air for 15–30 min. After the Matrigel has solidified, seed the spheroids mixed with Matrigel onto the thickly coated plate of Matrigel. Leave the plate at 37 °C in an atmosphere of 5% CO2 / 95% air for 5 min. Advanced DMEM / F12 is supplemented with B27, N2, L-glutamine, HEPES, penicillin / streptomycin, and 2 μM retinoic acid (RA, Sigma, St. Louis, MO) for 1–5 days. The medium is changed every other day. After RA treatment, organoids embedded in Matrigel drops are cultured in hepatocyte culture medium (HCM Lonza, Walkersville, MD) supplemented with 10 ng / mL hepatocyte growth factor (HGF, PeproTech, Rocky Hill, NJ), 0.1 μM dexamethasone (Dex, Sigma), and 20 ng / mL oncostatin M (OSM, R&D Systems). Cultures for cell differentiation are maintained at 37 °C in an atmosphere of 5% CO2 / 95% air, and the medium is changed every 3 days. Around days 20–30, organoids embedded in Matrigel drops are dissociated by scraping and gentle pipetting for optional analysis.

[0082] Matrigel-free method: On days 7–8, the definitive endoderm organoids with seeded cells are plated in Advanced DMEM / F12 (Thermo Fisher Scientific Inc.) supplemented with B27 (Life Technologies), N2 (Gibco, Walkersville, MD), L-glutamine, HEPES, penicillin / streptomycin, and retinoic acid (RA; Sigma, St. Louis, MD), 2 μM, for 4 days. The medium is changed every other day. After 4 days of plate culture, the organoids start to sprout, while the 2D cells differentiate into hepatocytes. Both organoids and hepatocytes can be maintained under hepatocyte culture medium (HCM Lonza, Walkersville, MD) supplemented with 10 ng / mL hepatocyte growth factor (HGF, PeproTech, Rocky Hill, NJ), 0.1 μM dexamethasone (Dex, Sigma), and 20 ng / mL oncostatin M (OSM, R&D Systems) for 10 days and 60 days. For organoid assays, floating organoids are collected into ultra-low attachment 6-well plates and can be used for subsequent assays, if necessary. Cultures for cell differentiation are maintained at 37 °C in an atmosphere of 5% CO2 / 95% air, and medium is changed every 3 days.

[0083] Spheroid generation transwell method: Posterior midgut spheroids are formed as described above. Anterior foregut spheroids are formed by slight modifications of day 4–6 differentiation. For anterior foregut spheroids, add Advanced DMEM / F12 (Thermo Fisher Scientific Inc.) supplemented with B27 (Life Technologies), N2 (Gibco, Walkersville, MD), L-glutamine, HEPES, penicillin / streptomycin, 500 ng / ml FGF4, 2 μM CHIR99021, and 200 ng / ml noggin, and change daily for 4–7 days. On day 8, cells from the anterior and posterior are dissociated into single cell suspensions by pipetting and trypsin digestion, and seeded into 96-well ultra-low attachment plates and incubated overnight. On day 9, cell aggregates are collected, and the anterior and posterior are mixed and incubated overnight. On day 10, the anterior and posterior Matrigel drops become attached to each other. Coat 12-well plates (Denville) with 50 μl Matrigel and incubate at 37°C for 2 min. Attached anterior and posterior aggregate spheroids are carefully picked up with a wide-mouth 10 μL pipette with minimal Matrigel and added to the Matrigel-coated 12-well plate. Add an additional 5 μL Matrigel to each spheroid. Add Advanced DMEM / F12 supplemented with B27, N2, L-glutamine, HEPES, and penicillin / streptomycin. On day 13, collect spheroids with a wide-mouth pipette, transfer to a transwell plate, and cover with an additional 5 μL Matrigel. Add Advanced DMEM / F12 supplemented with B27, N2, L-glutamine, HEPES, and penicillin / streptomycin to the bottom wells and change every 5 days.

[0084] Embedded liver organoid culture with hematopoietic cells: On days 7-8, definitive endoderm organoids with added cells are gently pipetted and detached from the dish. Cells are prepared using either the "Matrigel drop method", "Matrigel sandwich method", or the "spheroid generation transwell method" described above. Cultures are cultured in Advanced DMEM / F12 supplemented with B27, N2, L-glutamine, HEPES, and penicillin / streptomycin, but without retinoic acid. Cultures for cell differentiation are maintained at 37°C in an atmosphere of 5% CO2 / 95% air, and this medium is changed every 4 days. On days 13-15, red blood cells become visible in the iPS cell cultures. Addition of thrombopoietin (TPO) (10 ng / ml) and stem cell factor (SCF) (100 ng / ml) to the medium on day 7 increases the generation of hematopoietic cells (which can be maintained in the medium until the time of collection).

[0085] In vitro differentiation assay into multiple hematopoietic lineages in liver organoid cultures: On days 8–18 of culture, organoids are dissociated by mechanical force by pipetting and washed by PBS. Cells are then treated with 0.05% trypsin-EDTA (Life Technologies) to remove Matrigel and form a single cell suspension. Cells are seeded onto plates containing methylcellulose along with cytokines including transferrin, stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Stem Cell Technologies) and left in a humidified chamber in an incubator and maintained at 37 °C in an atmosphere of 5% CO2 / 95% air for 10 days, at which point colonies were observed and included erythroblast cells, macrophages, and basophils. Cells are identified by Wright-Giemsa staining.

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[0087] Unless otherwise specified, all percentages and ratios are calculated by weight.

[0088] All percentages and ratios are calculated based on the total composition unless otherwise specified.

[0089] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0090] The dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range that includes that value. For example, a dimension disclosed as "20 mm" is intended to mean "about 20 mm."

[0091] All documents cited herein, including cross-references or related patents or applications, are incorporated herein by reference in their entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in any combination with other references, teaches, suggests, or discloses such invention. Furthermore, to the extent that a meaning or definition of a term in this document conflicts with a meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall control.

[0092] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended that the appended claims cover all such changes and modifications that are within the scope of this invention.

Claims

1. 1. A method for producing hematopoietic stem cells (HSCs) or derivatives thereof, comprising: a. contacting definitive endoderm derived from progenitor cells with a wnt signaling pathway activator and an FGF signaling pathway activator until foregut cells are formed; b. Culturing the foregut cells in the absence of retinoic acid to form hepatic organoids that generate hematopoietic cells.

2. 2. The method of claim 1, wherein the progenitor cells are selected from one or both of embryonic stem cells and induced pluripotent stem cells (iPSCs).

3. The wnt signaling pathway activator may be Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, small molecule activators of the wnt signaling pathway (e.g., lithium chloride, 2-amino-4,6-disubstituted pyrimidine (hetero)arylpyrimidines, IQ1, QS11, NSC66803, 6, DCA β-catenin, 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl)pyrimidine), WAY-316606, SB-216763, or BIO (6-bromoindirubin-3'-oxime)), siRNA and / or shRNA activators of the Wnt signaling pathway, GSK3 inhibitors (e.g., Chiron / CHIR9902), and combinations thereof.

4. 4. The method of any of claims 1 to 3, wherein the FGF signaling pathway activator is selected from small molecule or protein FGF signaling pathway activators, FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, siRNA and / or shRNA activators of the FGF signaling pathway, and combinations thereof.

5. The method according to any one of claims 1 to 4, wherein the foregut cells form spheroids before forming the liver organoids.

6. The method according to any one of claims 1 to 5, wherein the foregut cells form spheroids before forming the liver organoids, and the method further comprises fragmenting the spheroids to form a plurality of cells.

7. The method of claim 6 , wherein the fragmentation is carried out via one or both of chemical and mechanical disruption.

8. 8. The method of claim 6 or 7, wherein the fragmentation comprises treatment with an enzyme, preferably the enzyme is an enzyme having one or both of proteolytic and collagenolytic enzyme activity, preferably one or more enzymes selected from Accutase, trypsin, collagenase, hyaluronidase, DNase, papain, Trypzean (Sigma), or combinations thereof.

9. said foregut being cultured in the presence of a cytokine selected from transferrin, stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), and combinations thereof, preferably said foregut being dissociated into single cells prior to said culturing, and said culturing with cytokines being for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days; 9. The method of any of claims 1-8, wherein the method is carried out for about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 9 weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or more than 12 weeks.

10. The method according to any of claims 1 to 9, further comprising contacting the human liver organoid with one or both of thrombopoietin (TPO) and stem cell factor (SCF), wherein the contacting with one or both of thrombopoietin (TPO) and stem cell factor (SCF) is performed for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 9 weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or more than 12 weeks.

11. The method according to any one of claims 1 to 10, wherein the human liver organoid is in a fetal state.

12. The method according to any one of claims 1 to 11, wherein the human liver organoid produces reduced albumin compared to human liver organoids treated with retinoic acid.

13. The method according to any one of claims 1 to 12, wherein the human liver organoid produces alpha-fetoprotein (AFP).

14. The method according to any one of claims 1 to 13, wherein the human liver organoids have increased endothelial markers CD34 and KDR compared to human liver organoids treated with retinoic acid.

15. The method according to any one of claims 1 to 14, wherein the human liver organoid has increased erythropoietin (EPO) and hemoglobin gamma (HBG) compared with the human liver organoid treated with retinoic acid.

16. The method according to any one of claims 1 to 15, wherein the foregut cells are suspended in a basement membrane matrix (Matrigel).

17. The method according to any one of claims 1 to 16, wherein the foregut cells are cultured on a stromal cell line derived from bone marrow.

18. 2. The method of claim 1, wherein the derivative cells are selected from myeloid cells (such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes up to platelets), lymphoid cells (such as T cells, B cells, and natural killer cells), and combinations thereof.

19. Culturing the colon organoids to form an organoid culture; and harvesting one or more immune cells from said colon organoid culture.

20. 20. The method of claim 19, wherein the colon organoids are cultured for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 9 weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or for more than 12 weeks, or until the colon organoids comprise one or more of hemogenic endothelium and endothelial ducts that generate hematopoietic progenitor / stem cells.

21. 21. The method of claim 19 or 20, further comprising isolating mesenchyme from said colon organoid culture and culturing said mesenchyme, preferably said mesenchyme culturing step is carried out for a period of about 4 days to 3 months, or about 5 days to 2 months, or about 6 days to about 1 month, or about 7 days to about 21 days, more preferably said mesenchymal culture is a suspension culture.

22. 22. The method of any of claims 19-21, wherein the colon organoids comprise mesenchyme, and the culturing step is carried out for a period of about 4 days to 3 months, or about 5 days to 2 months, about 6 days to about 1 month, or about 7 days to about 14 days, and optionally, the culturing step is carried out as a suspension culture for a period of about 1 week to 4 weeks, or about 1 week to allow proliferation of mesenchyme.

23. The method of claims 19-22, wherein the immune cells are selected from erythroid, myeloid, and mixed myeloid colonies.

24. The method of claims 19-23, wherein the immune cells are selected from one or more of macrophages, neutrophils, eosinophils, basophils, erythrocytes, leukocytes, and monocytes.

25. The method according to claims 19 to 24, wherein said colon organoids are derived from definitive endoderm derived from precursor cells, preferably embryonic stem cells or induced pluripotent stem cells.

26. The method of any one of claims 19 to 25, further comprising culturing with a T cell derived growth factor.

27. The method according to any of claims 19 to 26, further comprising disrupting the culture to disperse the colon organoids and disrupt the mesenchyme in the culture, followed by culturing in a basement membrane matrix for a period of about 1 week to about 4 weeks, or about 2 weeks to about 3 weeks.

28. A method for modeling a pathology selected from necrotizing enterocolitis, very early onset IBD30, infection from a bacterial pathogen (such as Clostridium difficile), infection from a viral pathogen (such as HIV, which readily infects fetal intestinal macrophages), comprising initiating said pathology in a colon organoid produced according to the method of any one of claims 19 to 27.

29. 28. A method for modeling innate immune mechanisms, comprising a colon organoid produced according to any of the methods of claims 19 to 27.

30. A human liver organoid (HLO), characterized in that said HLO comprises fetal liver tissue, said HLO generating hematopoietic cells.

31. Human colon organoids (HCOs) containing hemogenic endothelium.

32. The HCO of claim 30 or 31, wherein the hemogenic endothelium generates one or more of immune cells, preferably erythro-myeloid progenitor cells, lymphoid progenitor cells, and macrophages.

33. The HCO of claim 31 or 32, wherein the hemogenic endothelium produces macrophages that secrete pro-inflammatory cytokines.

34. 33. The HCO of claim 32, wherein the immune cells are bone marrow potential.

35. 31. The HCO of claim 30, wherein the HCO comprises hematopoietic progenitor cells having T cell potential.

36. The HCO of claim 30, wherein the HCO comprises hematopoietic progenitor cells, the progenitor cells are CD34+, the CD34 progenitor cells are within the organoid mesenchyme, and the hematopoietic progenitor cells are capable of forming T cells.

37. The method of any of claims 30-36, wherein the HCO comprises endothelial vessels, the ET is positive for CD34+, and the ET comprises RUNX1+ cells.

38. A method of making HCO / HIO capable of generating hematopoietic stem cells (HSCs), comprising contacting definitive endoderm derived from progenitor cells with one or more factors for a period of time sufficient to generate midgut / hindgut spheroids, optionally embedding said midgut / hindgut spheroids in a basement membrane matrix, and contacting said DE with a combination of factors including FGF, CHIR, Noggin, and a SMAD inhibitor in an amount and for a period of time sufficient to generate anterior foregut spheroids; The method, wherein the midgut / hindgut spheroids or anterior foregut spheroids generate HSCs.

39. 1. A method of treating an individual in need of immune cells, comprising: a. harvesting hematopoietic stem cells (HSCs) or derived cells from HCOs or HLOs according to any of the preceding claims; b. administering said HSC or derived cells to an individual in need thereof, said administering comprising transplanting said HSC into the bone marrow of said individual.

40. 40. The method of claim 39, wherein the treatment is treatment of anemia (including aplastic anemia, Fanconi anemia), immune deficiency, cancer (lymphoma, leukemia, carcinoma, solid tumors, etc.), genetic disorders of hematopoiesis, inherited storage diseases, thalassemia major, sickle cell disease, osteoporosis, or a combination thereof.

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