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Definitions

• bone marrow transplantation is the primary means of treatment.
• Stem and progenitor cells in donated bone marrow can multiply and replace the blood cells responsible for protective immunity, tissue repair, clotting, and other functions of the blood.
• the 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 increasing success to treat various diseases, including certain types of anemias such as aplastic anemia, Fanconi's anemia, immune deficiencies, cancers such as lymphomas or leukemias, carcinomas, various solid tumors, and genetic disorders of hematopoiesis. Bone marrow transplantation has also been applied to the treatment of inherited storage diseases, thalassemia major, sickle cell disease, and osteoporosis.
• HSCs hematopoietic stem cells
• the instant disclosure relates to compositions derived from precursor cells, and methods of using such compositions, for the manufacture of hematopoietic stem cells (HSCs) or derivative immune cells. More particularly, methods for obtaining hematopoietic stem cells from organoid tissue or cultures comprising organoids are disclosed, wherein the organoid tissue or cultures comprise liver or colonic tissue derived from precursor cells (such as embryonic stem cells or induced pluripotent stem cells), via directed differentiation.
• precursor cells such as embryonic stem cells or induced pluripotent stem cells
• FIG. Characterization of human liver organoid (HLO) gene expression at day 21 of culture.
• A. Albumin expression is modestly decreased while 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.
• EPO Erythropoietin
• HBG hemoglobin gamma
• FIG. Differentiation of myeloid lineages from HLO culture.
• C. CFC colony quantification comparing cells from HLO culture to umbilical cord blood (UCB) CD34 + cells and undifferentiated iPSCs (N.D. not detectable).
• FIG 3. Differentiation of B cells from HLO culture.
• FIG 4. Hemogenic endothelium co-develops in human colonic organoid cultures.
• B C
• E F
• Optical slices from (D). DA dorsal aorta.
• (H) Flow cytometry plot of gated CD34+ cells stained with CD45 and CD73. CD34+/CD45-/CD73- cells are boxed in an in black. CD34+/CD45+/CD73- cells are boxed in an in green.
• FIG. Erythro-myeloid and lymphoid progenitors are generated in HCO cultures.
• A Micrographs of cytospun cells from HCO cultures.
• B Examples of colonies formed in MethocultTM medium. Quantitation of colony formation in MethocultTM of cells from HCO derived from
• C Hl human embryonic stem cells and
• D IPSC 263-10.
• E Flow cytometry plots of CD45 gated cells stained with CD 3 and CD4 with and without treatment with T-cell differentiation inducing cytokines.
• FIG. HCOs contain co-developing macrophages.
• A Immunofluorescent staining of human colon biopsies for CD 163 (red) and CDH1 (green) counterstained with DAPI.
• A’ Inset of boxed region in (A).
• FIG 7. HCO have inflammatory macrophages which can secrete pro- inflammatory cytokines.
• A Heatmap of inflammation associated genes generated from RNAseq data of 35d HIOs and HCOs.
• B Immunofluorescent staining of 35 day old HCO for CD163 (green), iNOS (red) and CDH1 (white) counterstained with DAPI.
• B Inset of boxed area in B and excluding DAPI.
• C-D Luminex array data for IL-6, IL-8, MIP1A (CCL3) an MIP1B (CCL4). Each point represents Luminex values from an individual differentiation. Paired HIO and HCO samples (from same differentiation) are denoted with lines.
• FIG 8. HCO macrophage are functional.
• A Micrographs of a live imaging timecourse of HCOs treated with LPS.
• B-E Luminex array data for IL-6, IL-8, MIP1A (CCL3) an MIP1B (CCL4) of HCOs or HCOs treated with LPS.
• F Immunofluorescent staining of 35 day old HCO for -/+ pHRODO E. coli particles (green), and CD14 (red).
• FIG. 10 Experimental workflow.
• FIG. 12 Endothelial and hematopoietic cells co-develop within HCO cultures.
• FIG 13. Hemoglobin expression in HCO culture derived erythrocytes.
• FIG. Cytof analysis of immune cells present in HCOs.
• FIG 15. WELLS FIG S6. Macrophages persist within HCOs following transplantation into the mouse kidney capsule.
• FIG 16. WELLS FIG S7. Gene ontology analysis reveals parallel cytodifferentiation, macrophage maturation and inflammation in HCOs.
• FIG. Macrophages within HCOs can extend filipodia in response to E. coli particles.
• FIG. Imaging of placenta, liver, lung and colon. DETAILED DESCRIPTION
• the term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system.
• “about” can mean within 1 or more than 1 standard deviation, per the practice in the art.
• “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.
• the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
• totipotent stem cells are stem cells that can differentiate into embryonic and extra-embryonic cell types. Such cells can construct a complete, viable, organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.
• pluripotent stem cells also commonly known as PS cells, encompasses any cells that can differentiate into nearly all cells, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissues and nervous system).
• PSCs can be the descendants of totipotent cells, derived from embryonic stem cells (including embryonic germ cells) or obtained through induction of a non-pluripotent cell, such as an adult somatic cell, by forcing the expression of certain genes.
• iPSCs induced pluripotent stem cells
• iPS cells also commonly abbreviated as iPS cells, refers to a type of pluripotent stem cells artificially derived from a normally non-pluripotent cell, such as an adult somatic cell, by inducing a“forced” expression of certain genes.
• ESCs embryonic stem cells
• ES cells embryonic stem cells
• a precursor cell encompasses any cells that can be used in methods described herein, through which one or more precursor cells acquire the ability to renew itself or differentiate into one or more specialized cell types.
• a precursor cell is pluripotent or has the capacity to becoming pluripotent.
• the precursor cells are subjected to the treatment of external factors (e.g., growth factors) to acquire pluripotency.
• a precursor cell can be a totipotent (or omnipotent) stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; an oligopotent stem cells and a unipotent stem cell.
• a precursor cell can be from an embryo, an infant, a child, or an adult. In some aspects, a precursor cell can be a somatic cell subject to treatment such that pluripotency is conferred via genetic manipulation or protein/peptide treatment.
• the term“cellular constituents” are individual genes, proteins, mRNA expressing genes, and/or any other variable cellular component or protein activities such as the degree of protein modification (e.g., phosphorylation), for example, that is typically measured in biological experiments (e.g., by microarray or immunohistochemistry) by those skilled in the art.
• FGFs Fibroblast growth factors
• FGFs Fibroblast growth factors
• Exemplary FGF signaling pathway activators may 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 targeting cellular constituents associated with the FGF signaling pathway may be used to activate these pathways.
• siRNA and/or shRNA targeting cellular constituents associated with the FGF signaling pathway may be used to activate these pathways.
• Modulators/activators of the Wnt signaling pathway may include Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, and Wntl6.
• the modulation of the pathway may be through the use of small molecule modulators or protein modulators that activate the aforementioned pathways or proteins that activate the aforementioned pathways.
• Small molecule modulators of the Wnt pathway included, but is not limited to Lithium Chloride; 2-amino-4,6-disubstituted pyrimidine (hetero) arylpyrimidines; IQ1; QS11; NSC668036; DCA beta-catenin; 2-amino-4-[3,4- (methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl) pyrimidine.
• the extrinsic molecules may include small molecules such as WAY-316606; SB-216763; or BIO
• siRNA and/or shRNA targeting cellular constituents associated with the Wnt and/or FGF signaling pathways may be used to activate these pathways.
• target cellular constituents include but are not limited to SFRP proteins; GSK3, Dkkl, and FrzB.
• Additional modulators include molecules or proteins that inhibit GSK3, which activates the Wnt signaling pathway.
• Exemplary GSK3 inhibitors may include Chiron/CHIR9902l, for example, which inhibits GSK3 .
• the WNT signaling pathway activator may be administered in an amount sufficient to carry out the disclosed methods.
• One of ordinary skill in the art will readily appreciate the appropriate amount and duration.
• BMP Activators include may be selected from BMP2, BMP4, BMP7, BMP9, small molecules that activates the BMP pathway, proteins that activate the BMP pathway, and may include the following: Noggin, Dorsomorphin, LDN189, DMH-l, ventromophins, and combinations thereof.
• Organoid technology is a developing field.
• organoids are“organ like tissues,” or three-dimensional tissues having structural organization similar to that of the corresponding native organ.
• Organoids may be derived from precursor cells such as embryonic stem cells or induced pluripotent stem cells.
• Organoids are typically cultured in vitro using temporal series of growth factor manipulations that mimic embryonic development for the organ tissue of interest - a process referred to generally as directed differentiation of the precursor cell.
• organoids may contain differentiated cell types, which in many cases, are functional, for example, gastric parietal cells capable of secreting acid. That said, at present, organoids described in the literature are not identical in scope to that of naturally occurring organ tissue.
• organoids may lack a vasculature or one or more other features of the native organ that the organoid may be intended to mimic.
• organoids have not been recognized to possess a developed hematopoietic system or to produce significant amounts of immune cells. The instant disclosure seeks to address one or more such needs in the art.
• Blood cell production derives from a single type of cell, the hematopoietic stem cell, which through proliferation and differentiation, gives rise to the entire hematopoietic system.
• the hematopoietic stem cells are believed to be capable of self-renewal, expanding their own population of stem cells, and they are pluripotent (capable of differentiating into any cell in the hematopoietic system). From this rare cell population, the entire mature hematopoietic system, comprising lymphocytes (B and T cells of the immune system) and myeloid cells (erythrocytes, megakaryocytes, granulocytes and macrophages) is formed.
• lymphocytes B and T cells of the immune system
• myeloid cells erythrocytes, megakaryocytes, granulocytes and macrophages
• the lymphoid lineage comprising B cells and T cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like.
• the myeloid lineage which includes monocytes, granulocytes, megakaryocytes as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like.
• the erythroid lineage provides red blood cells, which act as oxygen carriers.
• compositions and methods may be used to produce hematopoietic cells, which may be used for treatment of any disease state in which the administration of hematopoietic cells is advantageous.
• the methods may further comprise isolating or harvesting hematopoietic cells from a disclosed organoid composition.
• Disease states which may be treated using the disclosed organoid-derived hematopoietic cells may include, for example, genetic diseases such as beta-thalassemia, sickle cell anemia, adenosine deaminase deficiency, recombinase deficiency, recombinase regulatory gene deficiency via introduction of a wild-type gene into the stem cells, for example, using CRISPR technology.
• the organoids and/or hematopoietic cells disclosed herein may be, in certain aspects, used to reconstitute an irradiated host or host subject to chemotherapy
• hematopoietic stem cell compositions for example, highly concentrated hematopoietic stem cell compositions, that are substantially free of differentiated or dedicated hematopoietic cells.
• substantially free of it may be meant that less than 10%, or less than 5% or less than 1 % of a cell exists in a population.
• the hematopoietic cells derived from the organoid compositions may be a substantially homogenous viable mammalian, or human, hematopoietic cell composition and may be produced for a variety of purposes, for example, bone marrow transplants, where the cells may be freed of neoplastic cells or other cells that are pathogenic, e.g., HIV-infected cells, transplantation in which avoidance of graft-versus-host disease is desired.
• substantially homogenous it is meant a 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, greater than 95% of the desired cell type, wherein the cell type may be hematopoietic stem cells.
• the hematopoietic cells may be modified by appropriate recombination, either homologous or non-homologous, to correct genetic defects or provide genetic capabilities naturally lacking in the stem cells, either as to the individual or as to stem cells generally.
• Such genetically modified cells i.e., using CRISPR methods well known in the art
• a method of making hematopoietic stem cells may comprise 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 culturing the foregut cells in the absence of retinoic acid to form a liver organoid producing hematopoietic cells.
• the precursor cell of any preceding paragraph may be selected from one or both of embryonic stem cells and induced pluripotent stem cells (iPSC).
• the wnt signaling pathway activator of any preceding paragraph may be selected from Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, small molecule activators of the wnt signaling pathway, (for example lithium chloride; 2-amino-4,6- disubstituted pyrimidine (hetero) arylpyrimidines; IQ1; QS11; NSC668036; DCA beta- catenin; 2-amino
• the FGF signaling pathway activator of any preceding paragraph may be selected from a small molecule or protein FGF signaling pathway activator, FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, an siRNA and/or shRNA activator of the FGF signaling pathway, and combinations thereof.
• any preceding paragraph may further comprise forming a spheroid from foregut cells prior to forming a liver organoid.
• the foregut cells may form a spheroid prior to forming a liver organoid
• the method may further comprise fragmenting the spheroid to form a plurality of cells derived from the spheroid. The fragmenting may be accomplished via one or both of chemical disruption and/or mechanical disruption.
• the fragmenting may comprise treatment with an enzyme, such as, for example, an enzyme 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 (manufactured by Sigma), or combinations thereof.
• an enzyme such as, for example, an enzyme 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 (manufactured by Sigma), or combinations thereof.
• cytokine may be any cytokine acceptable in the art, for example, 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.
• the foregut may be dissociated into single cells prior to the culturing.
• the culturing with cytokines may be carried out for a certain 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 three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or for about eight weeks, or for about nine weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or for greater than 12 weeks.
• the method of any preceding paragraph may further comprise contacting said a liver organoid, for example, a 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 carried out for a certain period of time, for example, a period of time selected from 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 three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or for about eight weeks, or
• the liver organoid of any preceding paragraph may be one that would be understood to be in a fetal state, for example, a human liver organoid derived from human precursor cells, such organoid comprising fetal liver tissue.
• the liver organoid may produce decreased albumin as compared to a human liver organoid that has been treated with retinoic acid.
• the liver organoid produces alpha- fetoprotein (AFP).
• the liver organoid has increased endothelial markers CD34 and KDR as compared to a liver organoid that has been treated with retinoic acid.
• the liver organoid has increased erythropoietin (EPO) and hemoglobin gamma (HBG) as compared to a liver organoid that has been treated with retinoic acid.
• EPO erythropoietin
• HBG hemoglobin gamma
• the method may comprise the steps of any preceding paragraph and further, suspending the foregut cells in a basement membrane matrix, for example, MatrigelTM.
• the foregut cells may be further cultured on a stromal cell line, for example, a stromal cell line derived from bone marrow.
• said derivative cell is selected from a myeloid cells (such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets), lymphoid cells (such as T cells, B cells, and natural killer cells) and combinations thereof.
• myeloid cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets
• lymphoid cells such as T cells, B cells, and natural killer cells
• human colonic organoids comprising a hemogenic endothelium, and methods of making same.
• the hemogenic endothelium of the HCOs as described herein produce immune cells, for example, one or more of erythro-myeloid progenitors, lymphoid progenitors, and macrophages.
• the hemogenic endothelium of the disclosed HCOs produces macrophages that secrete pro- inflammatory cytokines.
• the disclosed HCOs may further comprise a hematopoietic progenitor cell, wherein said progenitor cell is CD34+, wherein the CD34 progenitor cell is within organoid mesenchyme, wherein said hematopoietic progenitors are competent to form T-cells.
• the disclosed HCOs may comprise an endothelial tube, wherein the endothelial tube (ET) is positive for CD34+, and wherein the ET comprises RUNX1+ cells.
• the colonic organoids which may be derived from precursor cells, such as human precursor cells, may be used to obtain immune cells.
• a method comprising culturing a colonic organoid to form an organoid culture; and harvesting one or more immune cells from said colonic organoid culture is disclosed.
• the colonic organoid may 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 three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or for about eight weeks, or for about nine weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or for greater than 12 weeks, or until the colonic organoid comprises one or more of hemogenic endothelium and endothelial tubes producing hematopoietic progenitor/stem cells.
• the method may include separating a mesenchyme from said colonic organoid culture and culturing said mesenchyme.
• the mesenchyme culturing step may be carried out for a period of from about four days to three months, or about five days to two months, or from about 6 days to about one month, or from about seven days to about 21 days.
• the mesenchyme culture may be a suspension culture.
• the said colonic organoid may comprise a mesenchyme
• the culturing step may be carried out for a certain period of time, for example, from about four days to three months, or about five days to two months, or from about 6 days to about one month, or from about seven days to about 14 days.
• the culturing step may be carried out as a suspension culture for a period of time of from about one week to four weeks, or about one week, to allow for expansion of mesenchyme.
• immune cells of the disclosed methods may be selected from erythroid, myeloid, and mixed myeloid colonies.
• the immune cells may be one or more of macrophages, neutrophils, eosinophils, basophils, erythrocytes, leukocytes, and monocytes.
• the colonic organoid may be derived from a definitive endoderm derived from a precursor cell as described herein.
• the precursor cell is an embryonic stem cell or an induced pluripotent stem cell.
• the method may further comprise culturing the organoid culture with a T-cell inducing growth factor.
• the method may comprise disrupting the culture to disperse the colonic organoids into single colonic organoids and to disrupt the mesenchyme in culture. This step may be by culturing the resulting disrupted organoids and mesenchyme in a basement membrane matrix (e.g. Matrigel) for a period of time of from about one week to about four weeks, or about two weeks to about three weeks.
• a basement membrane matrix e.g. Matrigel
• the colonic organoids in particular human colonic organoids, may be used to model disease states.
• a method for modeling a disease state selected from necrotizing enterocolitis, Very Early Onset IBD30, infection from a bacterial pathogen, (such as Clostridium difficile), infection from a viral pathogens (such as HIV, which readily infects fetal intestinal macrophages), is disclosed.
• the method may comprise initiating a disease state in a colonic organoid, for example a human colonic organoid, made according to a method as disclosed herein.
• a method of making an HCO or HIO capable of producing hematopoietic stem cells comprising contacting definitive endoderm derived from a precursor cell with one or more factors for a period of time sufficient to produce mid/hindgut spheroids, optionally embedding said mid/hindgut spheroids in a basement membrane matrix, and contacting said DE with a combination of factors comprising FGF, CHIR, Noggin, and a SMAD inhibitor in an amount and for a period of time sufficient to produce anterior foregut spheroids; wherein the mid/hindgut spheroids or anterior foregut spheroids produce HSCs.
• a method of treating an individual in need of immune cells may comprise harvesting an hematopoietic stem cell (HSC) or derivative cell from an HCO or HLO according to any preceding paragraph; and administering said HSC or derivative cell to an individual in need thereof, wherein the administration comprises engrafting said HSC into bone marrow of the individual.
• HSC hematopoietic stem cell
• the treatment may be treatment of an anemia (including aplastic anemia, Fanconi’s anemia), an immune deficiency, a cancer (such as lymphoma, leukemia, carcinoma, a solid tumor), a genetic disorder of hematopoiesis, an inherited storage disease, thalassemia major, sickle cell disease, osteoporosis, or combinations thereof.
• anemia including aplastic anemia, Fanconi’s anemia
• an immune deficiency such as lymphoma, leukemia, carcinoma, a solid tumor
• a genetic disorder of hematopoiesis such as lymphoma, leukemia, carcinoma, a solid tumor
• a genetic disorder of hematopoiesis such as a genetic disorder of hematopoiesis, an inherited storage disease, thalassemia major, sickle cell disease, osteoporosis, or combinations thereof.
• pluripotent stem cells that are pluripotent or can be induced to become pluripotent may be used.
• pluripotent stem cells are derived from embryonic stem cells, which are in turn derived from totipotent cells of the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro.
• Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Methods for deriving embryonic stem cells from blastocytes are well known in the art. For example, three cell lines (Hl, H13, and H14) had a normal XY karyotype, and two cell lines
• H9-hESCs Human embryonic stem cells H9
• NSCB National Stem Cell Bank
• UCSF Human Embryonic Stem Cell Research Center
• WISC Wireless Cell Bank
• Wi Cell Research Institute the University of Wisconsin Stem Cell and Regenerative Medicine Center
• UW- SCRMC the University of Wisconsin Stem Cell and Regenerative Medicine Center
• Exemplary embryonic stem cells that can be used in aspects in accordance with the present invention include but are not limited to SA01 (SA001); SA02 (SA002); ES01 (HES-l); 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 (13); TE04 (14); TE06 (16); UC01 (HSF1); UC06 (HSF6); WA01 (Hl); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14).
• the stem cells are further modified to incorporate additional properties.
• Exemplary modified cell lines include but not limited to Hl OCT4-EGFP; H9 Cre-LoxP; H9 hNanog-pGZ; H9 hOct4-pGZ; H9 inGFPhES; and H9 Syn-GFP.
• embryonic stem cells More details on embryonic stem cells can be found in, 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,”.
• pluripotent stem cells can be derived from embryonic germ cells (EGCs), which are the cells that give rise to the gametes of organisms that reproduce sexually. EGCs are derived from primordial germ cells found in the gonadal ridge of a late embryo, have many of the properties of embryonic stem cells. The primordial germ cells in an embryo develop into stem cells that in an adult generate - li
• mice and humans it is possible to grow embryonic germ cells in tissue culture under appropriate conditions. Both EGCs and ESCs are pluripotent.
• iPSCs Induced Pluripotent Stem Cells
• iPSCs are derived by transfection of certain stem cell- associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection may be achieved through viral vectors, such as retroviruses. Transfected genes include the master transcriptional regulators Oct-3/4 (Pouf5l) and Sox2, although it is suggested that other genes enhance the efficiency of induction. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection.
• iPSCs include but are not limited to first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells.
• non-viral based technologies may be employed to generate iPSCs.
• an adenovirus can be used to transport the requisite four genes into the DNA of skin and liver cells of mice, resulting in cells identical to embryonic stem cells. Since the adenovirus does not combine any of its own genes with the targeted host, the danger of creating tumors is eliminated.
• reprogramming can be accomplished via plasmid without any virus transfection system at all, although at very low efficiencies.
• direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification.
• generation of mouse iPSCs is possible using a similar methodology: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency.
• the expression of pluripotency induction genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions.
• embryonic stem cells More details on embryonic stem cells can be found in, 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 el al.
• exemplary iPS cell lines include but not limited to iPS-DFl9-9; iPS-DFl9-9; iPS-DF4-3; iPS-DF6-9; iPS (Foreskin); iPS(IMR90); and iPS(IMR90).
• Definitive Endoderm The spheroids, organoids, and/or tissues described herein may be derived from a simple sheet of cells called the definitive endoderm (DE). Methods for deriving definitive endoderm from precursor cells are well known in the art, as taught by D’Armour et al. 2005 and Spence et al. Any methods for producing definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) are applicable to the methods described herein. In some aspects, pluripotent cells are derived from a morula. In some aspects, pluripotent stem cells are stem cells. Stem cells used in these methods can include, but are not limited to, embryonic stem cells.
• Embryonic stem cells can be derived from the embryonic inner cell mass or from the embryonic gonadal ridges. Embryonic stem cells or germ cells can originate from a variety of animal species including, but not limited to, various mammalian species including humans. In some aspects, human embryonic stem cells are used to produce definitive endoderm. In some aspects, human embryonic germ cells are used to produce definitive endoderm. In some aspects, iPSCs are used to produce definitive endoderm. In some aspects, 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.
• the one or more growth factors may comprise the Nodal/Activin and/or the BMP subgroups of the TGF-beta superfamily of growth factors.
• the one or more growth factors are selected from the group consisting of Nodal, Activin A, Activin B, BMP4, Wnt3a or combinations of any of these growth factors.
• the embryonic stem cells or germ cells and iPSCs are treated with the one or more growth factors for 6 or more hours; 12 or more hours; 18 or more hours; 24 or more hours; 36 or more hours; 48 or more hours; 60 or more hours; 72 or more hours; 84 or more hours; 96 or more hours; 120 or more hours; 150 or more hours; 180 or more hours; or 240 or more hours.
• the embryonic stem cells or germ cells and iPSCs are treated with the one or more growth factors at a concentration of 10 ng/ml or higher; 20 ng/ml or higher; 50 ng/ml or higher; 75 ng/ml or higher; 100 ng/ml or higher; 120 ng/ml or higher; 150 ng/ml or higher; 200 ng/ml or higher; 500 ng/ml or higher; 1,000 ng/ml or higher; 1,200 ng/ml or higher; 1,500 ng/ml or higher; 2,000 ng/ml or higher; 5,000 ng/ml or higher; 7,000 ng/ml or higher; 10,000 ng/ml or higher; or 15,000 ng/ml or higher.
• concentration of the growth factor is maintained at a constant level throughout the treatment. In other aspects, concentration of the growth factor is varied during the course of the treatment. In some aspects, the growth factor is suspended in media that include fetal bovine serine (FBS) with varying HyClone concentrations.
• FBS fetal bovine serine
• concentration of each growth factor may be varied independently.
• populations of cells enriched in definitive endoderm cells are used. In some aspects, the definitive endoderm cells are isolated or substantially purified.
• the isolated or substantially purified definitive endoderm cells express the SOX17, FOXA2, and/or the CXRC4 marker to a greater extent than the OCT4, AFP, TM, SPARC and/or SOX7 markers.
• Methods for enriching a cell population with definitive endoderm are also contemplated.
• 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 that is present on the surface of definitive endoderm cells but which is not present on the surface of other cells in the mixed cell population, and then isolating the cells bound to the reagent.
• the cellular constituent that is present on the surface of definitive endoderm cells is CXCR4.
• Additional methods for obtaining or creating DE cells include but are not limited to those described in United States Patent No. 7,510,876 to D’ Amour et al.-, United States Patent 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 hereby incorporated by reference herein in its entirety.
• soluble FGF and Wnt ligands are used to mimic early hindgut specification in culture to convert, through directed differentiation, DE developed from iPSCs or ESCs into hindgut epithelium that efficiently gives rise to all the major intestinal cell types.
• directed differentiation of DE is achieved through selective activating certain signaling pathways that are important to intestinal development. It will be understood by one of skill in the art that altering the expression of any Wnt signaling protein in combination with any FGF ligand can give rise to directed differentiation as described herein.
• DE culture is treated with the one or more modulators of a signaling pathway described herein for 6 or more hours; 12 or more hours; 18 or more hours; 24 or more hours; 36 or more hours; 48 or more hours; 60 or more hours; 72 or more hours; 84 or more hours; 96 or more hours; 120 or more hours; 150 or more hours; 180 or more hours; 200 or more hours, 240 or more hours; 270 or more hours; 300 or more hours; 350 or more hours; 400 or more hours; 500 or more hours; 600 or more hours; 700 or more hours; 800 or more hours; 900 or more hours; 1,000 or more hours; 1,200 or more hours; or 1,500 or more hours.
• DE culture may be treated with the one or more modulators of a signaling pathway described herein at a concentration of 10 ng/ml or higher; 20 ng/ml or higher; 50 ng/ml or higher; 75 ng/ml or higher; 100 ng/ml or higher; 120 ng/ml or higher; 150 ng/ml or higher; 200 ng/ml or higher; 500 ng/ml or higher; 1,000 ng/ml or higher; 1,200 ng/ml or higher; 1,500 ng/ml or higher; 2,000 ng/ml or higher; 5,000 ng/ml or higher; 7,000 ng/ml or higher; 10,000 ng/ml or higher; or 15,000 ng/ml or higher.
• concentration of signaling molecule is maintained at a constant throughout the treatment.
• concentration of the modulators of a signaling pathway is varied during the course of the treatment.
• a signaling molecule in accordance with the present invention is suspended in media comprising DMEM and fetal bovine serine (FBS).
• the FBS can be at a concentration of 2% and more; 5% and more; 10% or more; 15% or more; 20% or more; 30% or more; or 50% or more.
• concentration of signaling molecule in accordance with the present invention is suspended in media comprising DMEM and fetal bovine serine (FBS).
• the FBS can be at a concentration of 2% and more; 5% and more; 10% or more; 15% or more; 20% or more; 30% or more; or 50% or more.
• the regiment described herein is applicable to any known modulators of the signaling pathways described herein, alone or in combination, including but not limited to any molecules in the Wnt and FGF signaling pathways.
• the signaling molecules can be added simultaneously or separately.
• the concentration of each may be varied independently.
• Definitive hematopoietic progenitor cells arise from hemogenic endothelium that develops in close proximity to the embryonic colon.
• Applicant has engineered human pluripotent stem cell-derived colonic organoid cultures that co-develop hemogenic endothelium and hematopoietic progenitors competent to form myeloid and lymphoid derivatives.
• BMP signaling may be used for generating hindgut mesenchyme that is competent to form hemogenic endothelium, and that gives rise to RUNX1 -expressing hematopoietic progenitors.
• Macrophages may be maintained within the developing mesenchyme of HCOs following extended in vitro culture. Following transplantation of HCOs and 3 months of growth in vivo, PSC-derived human macrophages establish a close association with the colonic epithelium and were not displaced by the host-derived macrophages. HCO- associated macrophages were functional and responded to both LPS and pathogenic bacteria by production of inflammatory cytokines, undergoing transepithelial migration, and phagocytosing bacteria, all properties of tissue resident macrophages.
• Dogma has it that all immune cells of the gut derive from bone marrow-derived hematopoietic stem cells (HSCs).
• HSCs bone marrow-derived hematopoietic stem cells
• increasing evidence from animal studies supports the conclusion that some organs contain a population of tissue resident macrophages that co-develop during embryonic development 1 3 . It was recently shown that the colon contains a stable, self-maintaining population of macrophages that is derived both from embryonic progenitors and adult HSCs 4 .
• Hematopoietic cells develop from three sites. Primitive hematopoietic cells arise during gastrulation, migrate to the yolk sac and are short lived 5 . Definitive hematopoietic progenitors (HPCs) derive from hemogenic endothelium, either in the yolk sac or the aorta- gonado-mesonephros (AGM) region of the embryo, which is adjacent to the developing colon.
• HPCs Definitive hematopoietic progenitors
• AGM aorta- gonado-mesonephros
• the intraembryonic HPCs that derive from the AGM region express markers like Runxl and Tek, and emerge from the endothelium of the aorta and surrounding vessels adjacent to the hindgut 6 . Development of this posterior region of the embryo requires BMP signaling 7 10 . In addition, BMP signaling regulates expression of GATA2 a transcription factor which is required for hemogenic endothelial formation 11 .
• HCOs human colonic organoids
• Such HCOs contained both colonic epithelium and surrounding mesenchymal derivatives including fibroblasts, myofibroblasts and smooth muscle cells.
• Bioinformatic analyses of transcriptional changes that occurred during stages of HCO differentiation revealed a surprising enrichment in genes associated with hematopoietic development.
• Disclosed herein is a method of processing cultures to identify the extent of cell types present within HCOs as shown in FIG 10.
• Hematopoietic progenitors develop from caudal-lateral mesoderm.
• HOX genes which are expressed anteriorly and posteriorly. Consistent with previous findings, posterior hox genes were significantly upregulated by BMP treatment (FIG 11, panel D). Moreover, anterior hox factors including HOXA3 which inhibits EHT12, were downregulated in HCOs compared to HIOs.
• Hemogenic endothelium can further be distinguished from non-hemogenic endothelium by the lack of CD73 expression 14 .
• Analysis of 2l-day old HCO cultures by flow cytometry revealed the presence of CD34 + /CD73 endothelial cells, suggesting the presence of hemogenic endothelium (FIG 4 (H)).
• the transcriptional profile of HCOs as compared to HIOs, revealed pathway terms related to the immune cells and their function. These included neutrophil degranulation, innate immune system, platelet activation and leukocyte transendothelial migration.
• HCO cultures contain hemogenic endothelium capable of generating hematopoietic cells.
• HCO human immunodeficiency virus
• media was sampled and cytospins and giemsa staining was performed, which identified cells resembling macrophages, neutrophils, eosinophils, and basophils (FIG 5, A).
• No exogenous factors or mouse bone marrow stromal cells were added to the cultures, suggesting that mesenchymal cell types in HCOs were capable of supporting differentiation of myeloid cells types.
• MethocultTM assays were performed.
• HCOs from human embryonic and induced pluripotent stem cell lines were competent to generate erythromyeloid derivatives, demonstrating that this method is robust across PSC lines.
• Erythrocytes generated from HCOs expressed fetal (HBG1 and 2) and fetal/adult (HBA1, HBA2) hemoglobins, but not appreciable levels of embryonic hemoglobins (HBE1, HBZ), suggesting that HCO cultures contain definitive erythromyeloid progenitors.
• One of the hallmarks of intraembryonic, definitive hematopoietic cells is the ability to form lymphoid cell types like T cells 13 15 .
• lymphoid cell types like T cells 13 15 .
• Applicant posited that these progenitors would emerge after prolonged culture of HCO mesenchyme.
• Applicant therefore developed a culture method that permitted longer term maintenance of intact hemogenic endothelial tubes (FIG 10, C). HCO cultures were grown for an additional week allowing for expansion of mesenchyme, which was scraped off the plate and grown in suspension culture for up to an additional 3 weeks.
• T-cell inducing growth factors IL7 and FLT3 was added. Without 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 (FIG 5, E). The presence of T cell potential further supports the conclusion that hematopoietic cells formed from HCO cultures are definitive.
• tissue resident macrophages can colonize organs early in development and persist until birth.
• organs such as the lung and liver
• embryonic macrophages persist throughout life 5 .
• HSCs in the postnatal bone marrow give rise to macrophages that replace the embryonic macrophages.
• HSC-derived macrophages 16 17 some data suggest that embryonic macrophages are replaced by HSC-derived macrophages 16 17 .
• recent lineage tracing of embryonic macrophages suggest that they persist postnatally along with HSC-derived macrophages.
• HCOs were passaged at day 21 by trituration, which results in the disruption of mesenchyme and dispersal of individual HCOs. HCOs were then re-plated in Matrigel and cultured for another 14 days. When the transcriptional profile was examined by gene ontology analysis, an enrichment in GO terms associated with myeloid cell types including leukocytes, neutrophils, as well as defense and inflammatory response were observed.
• the human intestine is populated by multiple subtypes of macrophages.
• the expression of cell surface markers using CYTOF was examined.
• CYTOF analysis revealed the presence of at least 4 different monocyte populations including a CDl lbhi population, a CD14-/CD16+ population, a CD14+/CD16+ population and a CD14+/CD16-.
• HCOMacs HCO macrophages
• FIG 8 To determine if HCO macrophages (“HCOMacs”) can be maintained long term, the presence of human CD 163+ macrophages following transplantation of HCOs into the mouse kidney capsule (FIG 8) was examined. Applicant hypothesized that short-lived macrophages would be replaced by host-derived murine macrophages which express the mouse specific marker F/480.
• hCDl63+ cells Only a few hCDl63+ 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, hCDl63 macrophages were readily detectable in HCOs even after 12 weeks following transplantation. These macrophages were predominantly located in the lamina propia which lacked infiltration of F/480+ macrophages. In the muscularis layers, hCDl63+ cells were interspersed with F/480+ suggesting that host macrophages colonize these tissue layers.
• RNAseq data from 35-day HCOs revealed an inflammatory signature compared to HIOs.
• the secretion of pro-inflammatory cytokines into the media of HCOs was examined using Luminex multiplexed ELISAs (FIG 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 signature seen in HCOs.
• MIP1A macrophage inflammatory proteins 1A
• MIP1B macrophage inflammatory proteins
• HCOmacs HCO macrophages
• E. coli particles labeled with a pH sensitive fluorophore
• Live imaging revealed that HCOMacs continuously extend filopodia and survey the microenvironment.
• F-G pH sensitive fluorescence
• HCOMacs are functional, resident-like macrophages capable of responding to bacterial particles and live bacteria.
• BMP signaling has been shown to both activate a posterior HOX pattern the hindgut endoderm and mesenchyme but also to activate expression of the hemogenic endothelial transcription factor GATA2.
• BMP signaling also specifies hemogenic endothelium with definitive hematopoietic potential. This is consistent with normal human development in which the definite hematopoietic progenitors are formed from ventral posterior mesoderm.
• HCO cultures as described herein are believed to be closely mimicking a larger portion of the posterior embryo than initially thought.
• Hematopoietic progenitors from HCO cultures have erythroid-myeloid and lymphoid 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 develops 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 which express these factors is not dependent on BMP signaling. These cell types may be an alternative to using OP9-DLL4 cells which are of murine origin and thus likely to impart immunogenic properties on human hematopoietic progenitors 28,29 . In addition, these cell types also may be present in normal intestinal tissues since recent studies have shown that subsets of macrophages are self-maintained within the intestine 4 .
• HCOs The presence of co-developing macrophages within HCOs provide a new tool for examining the interaction between innate immune cells and the colonic epithelium.
• HCOs could be used to determine the niche factors that allow maintenance of tissue resident macrophages.
• HCOs should allow modeling of inflammatory diseases such as Necrotizing enterocolitis, Very Early Onset IBD30, bacterial pathogens such as Clostridium difficile and viral pathogens like HIV which readily infects fetal intestinal macrophages 31 .
• incorporation of other immune cell types could be used to study other innate immune mechanisms such as neutrophil driven inflammatory hypoxia 32 .
• Mid- hindgut spheroids were embedded in Matrigel (BD Biosciences) as previously described 10 12 and subsequently grown in Advanced DMEM/F12 (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen), L-glutamine, 10 mM HEPES, penicillin/streptomycin, and EGF (100 ng ml-l; R&D Systems).
• N2 Invitrogen
• B27 Invitrogen
• L-glutamine 10 mM HEPES
• penicillin/streptomycin penicillin/streptomycin
• EGF 100 ng ml-l; R&D Systems
• Noggin 100 ng ml-l; R&D Systems
• BMP2 100 ng ml-l; R&D Systems
• ActivinA lOOng/mL
• RPMI 1640 media Invitrogen
• dFBS HyClone defined fetal bovine serum
• DE cells are incubated in 2% dFBS-DMEM/Fl2 with 500ng/ml FGF4 and 500ng/ml Wnt3a (R&D Systems) for up to 4 days.
• 3- dimensional floating spheroids formed and are then transferred into three-dimensional cultures previously shown to promote intestinal growth and differentiation (Gracz AD, Ramalingam S, Magness ST.
• Human embryonic stem cells and induced pluripotent stem cells are grown in feeder-free conditions in six-well Nunclon surface plates (Nunc) coated with Matrigel (BD Biosciences) and maintained in mTESRl media (Stem Cell Technologies).
• mTESRl media Stem Cell Technologies.
• human ES or iPS cells are passaged with Accutase (Invitrogen) and plated at a density of 100,000 cells per well in a Matrigel-coated, Nunclon surface 24-well plate.
• Accutase split cells 10 mM Y27632 compound (Sigma) was added to the media for the first day. After the first day, media was changed to mTESRl and cells are grown for an additional 24 hours.
• Activin A Activin A for 3 days as previously described (Spence et al., 2011). Following DE induction, DE was then treated with hindgut induction medium (RPMI 1640, 2 mM L-glutamine, 2% decomplemented FBS, penicillin- streptomycin) for 4 d with 500 ng/mL FGF4 (R&D) and 3 pM Chiron 99021 (Tocris, WNT pathway activator; Inhibits GSK3) to induce formation of mid-hindgut spheroids.
• hindgut induction medium RPMI 1640, 2 mM L-glutamine, 2% decomplemented FBS, penicillin- streptomycin
• R&D hindgut induction medium
• 3 pM Chiron 99021 Tocris, WNT pathway activator; Inhibits GSK3
• Mid/hindgut spheroids are collected from 24 well plate and plated in Matrigel (BD) as previously described and subsequently grown in Advanced DMEM/F12 (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen), L-glutamine, 10 pM HEPES, penicillin/streptomycin, and EGF (100 ng ml-l; R&D Systems).
• HCOs Human Colonic Organoids
• spheroids are overlaid with 100 ng/mL EGF plus 100 ng/mL BMP (BMP2 or 4, R&D, or other BMP pathway activator may also be used) for at least 3 days. Media was changed after 3 days with only EGF being maintained in the media for all patterning conditions. Media was then changed twice weekly thereafter.
• HCOs After at least 3 days of BMP pathway activation (for example, Day 9), HCOs begin to contain hematovascular mesoderm cells expressing markers like KDR, FLT1 and GATA2. Continued growth in Matrigel results in formation of Hemogenic endothelium expressing CD31, CD34. Between day 15 and day 20, cultures have endothelial tubes that produce hematopoietic progenitor/stem cells that express RUNX1. Harvesting the media of HCO cultures identified a broad range of differentiated hematopoietic cells including myeloid cells (Basophil, Neutrophil, Eosinophil) and monocytes and macrophages. Using Flow cytometry, cells that express markers of immature B and T cells can also be observed.
• myeloid cells Basophil, Neutrophil, Eosinophil
• monocytes and macrophages Using Flow cytometry, cells that express markers of immature B and T cells can also be observed.
• MethoCultTM H4434 Classic contains methylcellulose in Iscove's MDM, fetal bovine serum, bovine serum albumin, 2-Mercaptoethanol, recombinant human stem cell factor (SCF), recombinant human interleukin 3 (IL-3), recombinant human erythropoietin (EPO), recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF)
• Macrophages are functionally responsive to infectious stimuli such as lipopoly saccharide (LPS) or bacteria, can phagocytose bacteria, and produce inflammatory cytokines including IL6, IL8, CCL3, CCL4, and TNF-alpha spontaneously and in response to LPS. Macrophages are also responsive to IL10 resulting in decreased inflammatory cytokine production. Macrophages are also responsive to M-CSF inhibition or addition with decreased and increased macrophage numbers respectively. [00110] Method of generating human liver organoid having increased immune cell production
• hiPSCs Differentiation of hiPSCs into definitive endoderm is induced using previously described methods with several modifications (Spence et al., 2011). In brief, colonies of hiPSCs are isolated in Accutase (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 150,000-300,000 cells are plated on Matrigel or laminin coated tissue culture 24 well plate (Coming, Durham, NC).
• 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) at Day 1, 100 ng/mL Activin A and 0.2 % fetal calf serum (FCS; Thermo Fisher Scientific Inc.) at Day 2, and 100 ng/mL Activin A and 2% FCS at Day 3.
• FCS fetal calf serum
• Matrigel Drop Method On Day 7-8, definitive endoderm organoids with plated cells are gently pipetted to delaminate from dishes. Isolated spheroids are centrifuged at 800 rpm for 3 minutes and, after removing supernatant, embedded in 100 % Matrigel drop on the dishes. 250pL Matrigel (Corning) is used per well of 24 well plate of endoderm culture. 80pL Matrigel drops are made, 1 per each well of 24 well plate (VWR Scientific Products, West Chester, PA). The plates are placed at 37°C in an atmosphere of 5% C0 2 /95 % air for 5- 15 minutes.
• Advanced DMEM/F12 is added with B27, N2, L- glutamine, HEPES, penicillin/streptomycin and retinoic acid (RA; Sigma, St. Louis, MO) 2 mM for 1-5 days. The media is replaced every other day.
• RA treatment organoids embedded in Matrigel drop are cultured in Hepatocyte culture medium (HCM Lonza, Walkersville, MD) with 10 ng/mL hepatocyte growth factor (HGF; PeproTech, Rocky Hill, NJ), 0.1 mM 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% C0 2 /95 % air and the medium is replaced every 3 days. Around Day 20-30, organoids embedded in Matrigel drop may be isolated by scratching and gentle pipetting for any analyses.
• Matrigel Sandwich Method On Day 7-8, definitive endoderm organoids with plated cells are gently pipetted to delaminate from dishes. Isolated spheroids are centrifuged at 800 rpm for 3 minutes, and after removing supernatant, mixed with 100% Matrigel. At the same time, hepatocyte culture medium with all supplements is mixed with the same volume of 100% Matrigel. HCM and Matrigel mix is plated to the bottom of the dish to make a thick coating on the plate (0.3-0.5 cm), and placed at 37°C in an atmosphere of 5% CO2/95 % air for 15-30 min. After the Matrigel was solidified, spheroids mixed with Matrigel is seeded on Matrigel thick coated plated.
• the plate is placed at 37°C in an atmosphere of 5% CO2/95 % air for 5 min.
• Advanced DMEM/F12 is added with B27, N2, L-glutamine, HEPES, penicillin/streptomycin and Retinoic acid (RA; Sigma, St. Louis, MO) 2 mM for 1-5 days. The media is replaced every other day.
• RA treatment organoids embedded in Matrigel drop are cultured in Hepatocyte culture medium (HCM Lonza, Walkersville, MD) with 10 ng/mL hepatocyte growth factor (HGF; PeproTech, Rocky Hill, NJ), 0.1 mM 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% C0 2 /95% air and the medium is replaced every 3 days. Around Day 20-30, organoids embedded in Matrigel drop are isolated by scratching and gentle pipetting for any analyses.
• Matrigel-Free Method On Day 7-8, definitive endoderm organoids with plated cells are continued in a planar culture in Advanced DMEM/F12 (Thermo Fisher Scientific Inc.) with B27 (Life Technologies), N2 (Gibco, Rockville, MD), L-glutamine, HEPES, penicillin/streptomycin, and retinoic acid (RA; Sigma, St. Louis, MO), 2 pM for 4 days. The media is replaced every other day. After the 4-day planar culture, the organoids begin to bud, whereas 2D cells differentiate into hepatocytes.
• Both organoids and hepatocytes can be maintained for over 60 days under hepatocyte culture medium (HCM Lonza, Walkersville, MD) with 10 ng/mL hepatocyte growth factor (HGF; PeproTech, Rocky Hill, NJ), 0.1 mM Dexamethasone (Dex; Sigma) and 20 ng/mL Oncostatin M (OSM; R&D Systems) for 10 days.
• HGF hepatocyte growth factor
• Dex 0.1 mM Dexamethasone
• OSM Oncostatin M
• floating organoids can be collected in Ultra-Low attachment 6 well plates and used for subsequent assays whenever appropriate. Cultures for cell differentiation are maintained at 37 °C in an atmosphere of 5% C0 2 /95 % air and the medium is replaced every 3 days.
• Spheroid Generation Transwell Method Posterior midgut spheroids are created as described above. Anterior foregut spheroids are created by slight modification of d4- 6 differentiation. For anterior foregut spheroids, Advanced DMEM/F12 (Thermo Fisher Scientific Inc.) with B27 (Life Technologies), N2 (Gibco, Rockville, MD), L-glutamine, HEPES, penicillin/streptomycin, 500 ng/ml FGF4, 2mM CHIR99021, and 200 ng/ml noggin is added and replaced each day for d 4-7.
• Advanced DMEM/F12 Thermo Fisher Scientific Inc.
• B27 Gibco, Rockville, MD
• L-glutamine L-glutamine
• HEPES penicillin/streptomycin
• 500 ng/ml FGF4 500 ng/ml FGF4, 2mM CHIR99021, and 200 ng/ml noggin is added and replaced each day for d
• Advanced DMEM/F12 with B27, N2, L-glutamine, HEPES, and penicillin/streptomycin was added.
• spheroids are collected by wide bore pipette and transferred to a transwell plate and covered with an additional 5pL Matrigel.
• Advanced DMEM/F12 with B27, N2, L-glutamine, HEPES, and penicillin/streptomycin is added to the bottom well and replaced every 5 days.
• TPO thrombopoietin
• SCF stem cell factor
• Cells are seeded onto plates containing methylcellulose with cytokines including 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) (Stem Cell Technologies) and kept in a humidified chamber inside the incubator and maintained at 37°C in an atmosphere of 5% C0 2 /95% air for 10 days, at which time colonies are observed including erythroid cells, macrophages, and basophiles. Cells are identified by Wright-Giemsa staining.
• Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function. Immunity 47, 183-198.el86 (2017).

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