Stem / precursor cells derived from duodenal brunner's glands, and method for isolating and using the same

Abstract

To provide stem / precursor cells derived from duodenal Brunner's glands, and a method for isolating and using the cells which are useful for supplying a stem / precursor cell for autologous cell therapy or heterologous cell therapy or gene therapy.SOLUTION: Provided are Brunner's glands stem / precursor cells (BGSCs) that have a phenotypic trait of an entodermal stem cell, in which a pluripotent marker is positive, and a method for isolating the cells from a human duodenum. Further also provided is that BGSCs are easily isolated from a human donor-derived duodenum, can be proliferated by culture or induced to be differentiated into liver and pancreatic systems, and can express a cell source for a clinical program of regenerative medicine.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 650,208, filed March 29, 2018. No. 6,313,935, filed on Oct. 23, 2003, which is hereby incorporated by reference in its entirety.

[0002] Multiple stem / progenitor cell microenvironments exist at specific anatomical sites within the fetal and postnatal human bile duct Stem / progenitor cell populations have long been recognized in fetal tissues, but few are known to exist in adult tissues. The persistence of peri-biliary glands, which are parallel to the intestinal crypts, is being newly recognized. PBG is located in the extrahepatic bile duct, the large intrahepatic bile duct, and the hepatopancreatic common bile duct. Late stage stem / progenitor cells and their derivatives are found in the gallbladder. The network is derived from stem / progenitor cells in the hepatopancreatic bile duct (PBG) and pancreatic ductal glands (PDG) in the pancreas. Stem / progenitor cells in all of these microenvironments continue to These are collectively called biliary stem / progenitor cells (BTSCs). BTSCs in the PBG have the following characteristics: proliferation, autonomy, and They have the characteristics of endodermal stem cells / progenitor cells, including regenerative ability and pluripotency, and they have the proliferation ability and They have the characteristics of progenitor cells in the PDG, including proliferation and pluripotency, but are more potent than stem / progenitor cells in the PBG. The self-regenerating ability is low.

[0003] BTSCs located at the level of the hepatopancreatic ampulla are primitive and express several multipotent markers. Co-expression of markers (e.g., OCT4, SOX2, NANOG) and self-renewal or can differentiate into functional hepatocytes, cholangiocytes, and pancreatic islets (they also give rise to acinar cells). Ongoing studies are investigating whether the BTSC-containing microenvironment can be expressed in the liver. Detailed anatomical studies in humans have revealed that the BTSC microenvironment The proximal to distal axis of the tissue is the hepatopancreatic ampulla, the proximal site where the most primitive stem cells are located. It has been shown that the cells progress from the endothelial cell to the liver or pancreas, distal sites where mature cells are found. This axis reconstructs the organogenesis of these organs and reflects their common embryonic origin. Indeed, from an embryological point of view, a common progenitor cell for the liver, biliary system, and pancreas originates from the foregut. It is present in the early stages of development of the definitive proximal endoderm that forms the primitive deuteron. The didenum harbours proximal endodermal stem / progenitor cells.

[0004] The most primitive stem / progenitor cells identified are those in the Brunner's gland, located in the submucosa of the duodenum. These cells are found in the stem / progenitor cell microenvironment that gives rise to the liver and pancreas. These cells may be the starting point for an entire network of endothelial cells. Isolation of these cells from the adult duodenum is difficult. which has not been achieved by any method disclosed in the art so far. The practical significance of these cells is manifold. For example, these cells can be used to Assessment of drug effects, generation of model systems (e.g., organoids), liver and pancreatic development, Analysis of functional maintenance and / or repair (although these include progenitor cells of both organs); Other clinical or analytical tests related to liver, pancreas and other endodermal tissues, liver, pancreas and Diagnosis or treatment of diseases or conditions involving or affecting endodermal and / or other endodermal tissues Cells from Brunner's gland can be used for treatment. These are rare sources of endodermal stem / progenitor cells present in the duodenum, a location where endodermal stem / progenitor cells can be easily accessed. Useful for providing stem / progenitor cells for autologous or heterologous cell or gene therapy Moreover, these Brunner's gland-derived tumors are logical targets for the treatment of various forms of cancer. Thus, they are known in the art as "Brunner's gland stem / progenitor cells." There remains a need to develop methods for isolating cells of interest. Summary of the Invention

[0005] In one aspect, the present disclosure provides Tra-1-60, Tra-1-81, OCT4, SOX2, Selected from the group consisting of NANOG, EpCAM, SOX9 and cytokeratin 7 (CK7). The cells express one or more markers of interest and express limited expression under culture conditions that support self-renewal. or stem cells isolated from the duodenum, further characterized as proliferative capacity with minimal differentiation. / progenitor cells (termed Brunner's gland stem / progenitor cells or BGSCs).

[0006] In another aspect, the present disclosure provides a method for the detection of an inflammatory cytokine, comprising administering to the patient a inflammatory cytokine comprising the group consisting of Lgr5, NIS, CD44 and CK19. The cells express one or more selected markers and are grown under culture conditions that support self-renewal. The duodenum-isolated leukocytes were further characterized as having a proliferative capacity with minimal or no differentiation. Concerning stem / progenitor cells (termed Brunner's gland stem / progenitor cells or BGSCs).

[0007] In another aspect, the present disclosure provides a method for the production of cells that express both SOX17 and PDX1 and support self-renewal. Further characterized as having the ability to proliferate under culture conditions with limited or minimal differentiation. Stem / progenitor cells isolated from the duodenum (termed Brunner's gland stem / progenitor cells or BGSCs) ) regarding.

[0008] In another aspect, the present disclosure provides Tra-1-60, Tra-1-81, OCT4, SOX2 , NANOG, EpCAM, SOX9, CK7, Lgr5, NIS, CD44 and CK 19, or expressing one or more markers selected from the group consisting of SOX1 7 and PDX1, and showed limited or minimal cell death under culture conditions that supported self-renewal. Stem / progenitor cells isolated from the duodenum (Bryomycin) were further characterized as their proliferation potential upon differentiation. (termed Lunar Gland Stem / Progenitor Cells or BGSCs).

[0009] In some embodiments, the BGSCs are pathogens and / or pathogenic and / or potent In some embodiments, the BGSCs are substantially free of beneficial microorganisms for at least one month. In some embodiments, the BGSCs can be expanded with limited or minimal differentiation. , and can proliferate with limited or minimal differentiation for at least two months. Morphologically, BGSCs were shown to proliferate with limited or minimal differentiation for at least 6 months. In some embodiments, the BGSC can be administered in a limited or minimal amount for at least 12 months. They can proliferate with limited differentiation.

[0010] In some embodiments, the culture conditions that support the self-renewal of BGSCs include serum-free medium, optionally In some embodiments, the culture conditions that support self-renewal include serum-free medium. The medium contains the

[0011] In one aspect, the present disclosure relates to a population of stem / progenitor cells isolated from the duodenum, At least a part, a substantial part, or a majority of the 81, a group consisting of OCT4, SOX2, NANOG, EpCAM, SOX9 and CK7 The gene expresses one or more markers selected from the group consisting of:

[0012] In one aspect, the present disclosure relates to a population of stem / progenitor cells isolated from the duodenum, At least a portion, or a substantial portion, or a majority of the and CK19.

[0013] In one aspect, the present disclosure relates to a population of stem / progenitor cells isolated from the duodenum, At least some, or a substantial portion, or most of the Manifest.

[0014] In one aspect, the present disclosure relates to a population of stem / progenitor cells isolated from the duodenum, At least a part, a substantial part, or a majority of the 81, OCT4, SOX2, NANOG, EpCAM, SOX9, CK7, Lgr5, N The present invention relates to a method for treating a patient with a inflammatory bowel disease, comprising administering to the patient a therapeutically effective amount of a marker selected from the group consisting of IS, CD44, and CK19. or at least a portion, or a substantial portion, or a majority of the cells express SOX 17 and PDX1.

[0015] In some embodiments, the population of stem / progenitor cells is a pathogen and / or a pathogenic and / or Or substantially free of beneficial microorganisms.

[0016] In some embodiments, the population of stem / progenitor cells is cultured in a limited or minimal amount for at least one month. In some embodiments, the population of stem / progenitor cells can be expanded to at least a limited degree of differentiation. Both are capable of proliferation for up to two months with limited or minimal differentiation. The stem / progenitor cell population is then expanded with limited or minimal differentiation for at least six months. In some embodiments, the population of stem / progenitor cells can be cultured for at least 12 months. These cells can be expanded efficiently or with minimal differentiation.

[0017] In some embodiments, the culture conditions that support the self-renewal of the stem / progenitor cell population are serum-free medium. , and optionally Kubota's medium. In some embodiments, the self-renewal of the stem / progenitor cell population is Supporting culture conditions include medium containing serum.

[0018] In one aspect, the present disclosure provides a method for the treatment of one or more BGSCs, or a population of BGSCs, by administering the BGSCs to a subject. 1. A method for isolating from the bidenum, a portion thereof, or a sample taken therefrom, comprising the steps of: (a) The mucosal layer of the duodenum, which is substantially free of intestinal mucus, is treated with an osmotic pressure characteristic outside the physiological range. The cells of the mucosal layer are contacted with a medium or solution having the above properties under conditions that induce an osmotic shock in the cells of the mucosal layer. To make; (b) removing at least one of the mucosal layer or cells thereof by mechanical, surgical and / or chemical methods; The method further comprises removing or dissolving a portion of the mucosa, leaving and / or exposing a remainder that may include the submucosa. To do so; (d) digesting or separating the remainder; (e) isolating one or more BGSCs or populations of BGSCs from the digested remainder. The present invention relates to a method comprising the steps of:

[0019] In some embodiments, the isolating step comprises isolating Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCAM, SOX9, CK7, Lgr5, NIS, CD44, and CK19. or at least a portion of the cells, or a substantial portion of the cells, or a majority of the cells, ra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCAM, S Selected from the group consisting of OX9, CK7, Lgr5, NIS, CD44, and CK19. The method includes isolating a population of BGSCs that express one or more markers that are associated with the proliferation and differentiation of the BGSCs.

[0020] In some embodiments, the isolating step comprises isolating BGSCs that express both SOX17 and PDX1; or at least a portion, or a substantial portion, or a majority of the cells express SOX17 and PD The study involves isolating a population of BGSCs that express both X1 and X2.

[0021] In one aspect, the present disclosure provides a method for treating one or more BGSCs, or a population of BGSCs, in a subject. 1. A method for isolating from the duodenum, a portion thereof, or a sample taken therefrom, comprising: Process (a) removing intestinal mucus; (b) A medium or solution having osmotic properties outside the physiological range is infiltrated into the cells of the mucosal layer. applying under conditions inducing osmotic shock; (c) Deletion of the mucosal layer or at least some of its cells by mechanical, surgical and / or chemical methods At least a portion of the tissue is removed or dissolved, leaving and / or exposing a remainder that may include the submucosa. The process of making; (d) the mucosal layer and / or the remainder are free of pathogens and / or pathogenic and / or A medium or solution for substantially killing, inactivating, or eliminating beneficial microorganisms. applying the liquid; (e) applying digestion or dissociation to the submucosa to produce digesta, dissociated cellular material, or cells; forming a suspension; (f) optionally, at least one of the cells from the digest, the separated cellular material, or the cell suspension; culturing a portion of the culture; (g)Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, Ep One or more of CAM, SOX9, CK7, Lgr5, NIS, CD44, and CK19 A cell or at least a portion, or a substantial portion, or a majority of the cells expressing Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCAM, Expresses one or more of SOX9, CK7, Lgr5, NIS, CD44, and CK19 Isolating a population of cells and / or cells that express both SOX17 and PDX1 and (b) substantially eliminating pathogens and / or pathogenic and / or beneficial microorganisms. The killing, inactivating or removing steps may be performed at any time or more than once. It is possible.

[0022] In some embodiments, removing intestinal mucus comprises compressing duodenal tissue.

[0023] In some embodiments, a medium or solution having osmolality properties outside the physiological range is a low These include tonic, hypotonic, hypertonic, or hyperosmotic solutions.

[0024] In some embodiments, the medium or solution having osmotic properties outside the physiological range is glucose solution, high salt solution, or distilled water.

[0025] In some embodiments, the removal is achieved by chemical destruction, including the use of emulsifiers and / or surfactants. This is done by destruction.

[0026] In some embodiments, the surfactant and / or emulsifier may be water, saline, and / or or in a buffer solution.

[0027] In some embodiments, the surfactant and / or emulsifier is applied for a short period of time (less than 15 minutes). will be done.

[0028] In some embodiments, the emulsifier is lecithin, polyoxyethylene sorbitan monolaurate, sorbitan monooleate (polysorbate 20), polyoxyethylene sorbitan monooleate (polysorbate Polysorbate 80), Polyoxyethylene sorbitan monopalmitate (Polysorbate 40) , Polyoxyethylene sorbitan monostearate (Polysorbate 60), Polyoxy Ethylene sorbitan tristearate (polysorbate 65), ammonium phosphatide sodium, potassium and calcium salts of fatty acids, magnesium salts of fatty acids, fats Mono- and diglycerides of acids, mono- and diglycerides of fatty acids Acetate esters, lactate esters of mono- and diglycerides of fatty acids, monoglycerides of fatty acids, Citric acid esters of mono- and diglycerides, mono- and diglycerides of fatty acids Monoacetyl tartaric acid esters and diacetyl tartaric acid esters of glycerides, monoacetyl tartaric acid esters of fatty acids, Mixed acetate and tartaric acid esters of monoglycerides and diglycerides, sucrose esters of fatty acids esters, sucrose glycerides, polyglycerol esters of fatty acids, polyglycerol polylysine Cinoleates, propane-1,2-diol esters of fatty acids, monoglycerides of fatty acids and diglycerides interacted with thermally oxidized soybean oil, sodium stearoyl-2-lactylate, and Calcium stearoyl-2-lactylate, sorbitan monostearate, sorbitol Sorbitan tristearate, sorbitan monolaurate, sorbitan monooleate, sorbitan tan monopalmitate and combinations thereof.

[0029] In some embodiments, the surfactant is 1-heptanesulfonic acid, N-lauryl sarcosine Lauryl sulfate, 1-octanesulfonic acid and taurocholic acid, benzalkonium chloride um, cetylpyridinium, methylbenzthonium chloride, decamethonium bromide, alkyl Betaine, alkylamidoalkylbetaine, N-dodecyl-N,N-dimethyl-3-a Mumonio-1-propanesulfonate, phosphatidylcholine, N-decyl AD-glucose Copyranoside, N-Decyl AD-Maltopyranoside, N-Dodecyl BD-Maltoside , N-Octyl BD-glucopyranoside, N-Tetradecyl BD-maltoside, Tri Ton (Triton X-100), Nonidet-P-40, Poloxamer 188, Lauryl Sulfate Sodium cholate, sodium deoxycholate, sodium dodecyl sulfate and combinations thereof The compound is selected from the group consisting of a combination of

[0030] In some embodiments, the remainder comprises the submucosa.

[0031] In some embodiments, the digestion or separation is performed enzymatically.

[0032] In some embodiments, pathogens and / or pathogenic and / or beneficial microorganisms are The medium or solution for substantially killing, inactivating or removing the bacteria is preferably hypochlorite. An aqueous solution of sodium chloride (NaClO) or any other suitable solution used for skin or surface disinfection. This includes any solution or drug.

[0033] In some embodiments, pathogens and / or pathogenic and / or beneficial microorganisms are Application of a medium or solution to qualitatively kill, inactivate or remove the bacteria is performed at the interface. This may be done before application of the activator and / or emulsifier or after digestion or separation. This is usually done before or after removal of mucus.

[0034] In some embodiments, the tissue sample is minced prior to the digestion or separation step.

[0035] In some embodiments, the digestion or separation step and / or isolation step comprises a low attachment plate. This will be carried out at the

[0036] In some embodiments, the isolation step comprises culturing in a serum-free medium, optionally comprising Kubota's medium. This is done using conditional culture selection.

[0037] In some embodiments, the isolating step comprises culturing cells using culture conditions comprising a medium containing serum. This is done using a collection.

[0038] In some embodiments, the isolated cells are spheroids, one or more organoids. The cells are cultured under conditions that support or result in the formation of cells, cell populations, or cell aggregates.

[0039] In one aspect, the present disclosure provides a method for culturing BGSCs, or a population of BGSCs, in low attachment plates. Regarding spheroids, organoids, cell aggregates or cell populations resulting from culturing do.

[0040] In one aspect, the present disclosure provides a method for culturing a BSGC, or a population of BSGCs, in suspension or 3D culture conditions. Spheroids, organoids, cell aggregates or cells obtained by culturing in Pertaining to groups.

[0041] In one aspect, the present disclosure relates to liver, pancreas, stomach, intestine, or other endodermal tissues, A method of treating a subject diagnosed with a disease or condition that affects the immune system, comprising administering to said subject a therapeutically effective amount of and administering to a subject an effective amount of a BGSC or a population of BGSCs. .

[0042] In one aspect, the present disclosure relates to or relates to liver, pancreas, stomach, intestine or other endodermal tissue. The present invention relates to a method for treating a subject diagnosed with a disease or condition that affects or affects the immune system, the method comprising administering an effective amount of BGS to a subject. The present invention relates to a method comprising administering

[0043] In one aspect, the present disclosure relates to or relates to liver, pancreas, stomach, intestine or other endodermal tissue. The present invention relates to a method for treating a subject diagnosed with a disease or condition that affects or affects the immune system, the method comprising administering an effective amount of BGS to a subject. The present invention relates to a method comprising administering a population of C.

[0044] In one aspect, the present disclosure provides a method for the treatment of autoimmune diseases comprising administering an effective number of BGSCs, or a population of BGSCs. Related to cell or gene therapy.

[0045] In one aspect, the present disclosure provides an allogeneic method comprising administering an effective number of BGSCs, or a population of BGSCs. Related to cell or gene therapy.

[0046] In one aspect, the present disclosure relates to liver, pancreas, stomach, intestine, or other endodermal tissues, A method of treating a subject diagnosed with a disease or condition that affects the immune system, comprising administering to said subject a therapeutically effective amount of The method comprises administering to a subject a therapeutically effective amount of a BGSC or population of BGSCs, the cells comprising administering to said subject an effective amount of a BGSC or population of BGSCs, the cells The present invention relates to methods in which the cells are genetically engineered or modified.

[0047] In one aspect, the present disclosure relates to liver, pancreas, stomach, intestine, or other endodermal tissues, A method for treating a subject diagnosed with a disease or condition that affects or is associated with a pulmonary circulation disorder, comprising administering to a subject an effective amount of BG The method includes administration of SC or BGSC, wherein the cells are genetically engineered or modified cells. Regarding the law.

[0048] In one aspect, the present disclosure relates to liver, pancreas, stomach, intestine, or other endodermal tissues, A method for treating a subject diagnosed with a disease or condition that affects or is associated with a pulmonary circulation disorder, comprising administering to a subject an effective amount of BG The method includes administering a population of SCs, wherein the cells are genetically engineered or modified cells. do.

[0049] In one aspect, the present disclosure provides a method for the treatment of autoimmune diseases comprising administering an effective number of BGSCs, or a population of BGSCs. A method of cell or gene therapy, wherein the cells are genetically engineered or modified cells. Regarding.

[0050] In one aspect, the present disclosure provides an allogeneic method comprising administering an effective number of BGSCs, or a population of BGSCs. A method of cell or gene therapy, wherein the cells are genetically engineered or modified cells. Regarding.

[0051] In one aspect, the present disclosure provides autologous or allogeneic cells or may involve or be directed to liver, pancreas, stomach, intestine, or other endodermal tissues for gene therapy. The present invention relates to the use of cells of a BGSC for the treatment of a disease or condition affecting or affecting the immune system.

[0052] In one aspect, the present disclosure provides autologous or allogeneic cells or may involve or be directed to liver, pancreas, stomach, intestine, or other endodermal tissues for gene therapy. Use of cells of a BGSC for the treatment of a disease or condition affecting or affecting the immune system, the cells comprising: genetically engineered or modified, relating to use;

[0053] In one aspect, the present disclosure provides autologous or allogeneic cells or may involve or be directed to liver, pancreas, stomach, intestine, or other endodermal tissues for gene therapy. The present invention relates to the use of a population of BGSCs for the treatment of a disease or condition affecting or affecting the immune system.

[0054] In one aspect, the present disclosure provides autologous or allogeneic cells or may involve or be directed to liver, pancreas, stomach, intestine, or other endodermal tissues for gene therapy. Use of a population of BGSCs for the treatment of a disease or condition affecting or affecting a cell The cells are genetically engineered or modified for use.

[0055] In some embodiments, the spheroids, organoids, cell aggregates or populations of cells are The ability to differentiate BGSCs, or populations of BGSCs, into cells of later lineage stages, including mature cells The present invention further includes potent culture conditions.

[0056] In one aspect, the present disclosure provides a method for the production of Brunner's gland stem / progenitor cells (BGSCs), or a collection of BGSCs. The method comprises isolating a polypeptide from the duodenum of a subject, a portion thereof, or a sample taken therefrom. There was, (a) digesting or dissociating the duodenum, a portion thereof, or a sample taken therefrom; producing digests or separated cellular material; (b) From digested or isolated cellular material: (i) Tra-1-60, Tra-1-81 , OCT4, SOX2, NANOG, EpCAM, SOX9, CK7, CK7, Lgr5 These cells express one or more of: NIS, CD44 and CK19; or At least a portion, a substantial portion, or a majority of the cells are Tra-1-60, Tra-1 -81, OCT4, SOX2, NANOG, EpCAM, SOX9, CK7, CK7, L A population of cells expressing gr5, NIS, CD44, and CK19; and / or i) at least one of these cells, or cells, that express both SOX17 and PDX1; A population of cells in which a portion, a substantial portion, or the majority express both SOX17 and PDX1 The present invention relates to a method for producing a medicament for use in a medical device, comprising:

[0057] In some embodiments, the duodenum, a portion thereof, a sample taken therefrom, a digesta, a separation The cellular material, or combinations thereof, may be pathogens and / or pathogenic and / or or a medium or a method for the preparation of ... or a solution.

[0058] In one aspect, the present disclosure provides a method for the detection of one or more desired biomarkers. A plurality of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells a population of cells expressing one or more desired biomarkers in the mucosal layer and submucosa; 1. A method for isolating a tissue (or a part or sample thereof) having a layer, comprising the steps of: or in other embodiments, the following steps may occur in a different order: (a) The mucosal layer of a tissue having a mucosal layer and a submucosa is treated with an osmotic pressure characteristic that is outside the physiological range. The cells of the mucosal layer are contacted with a medium or solution having the above properties under conditions that induce an osmotic shock in the cells of the mucosal layer. The process of making; (b) the mucosal layer or at least some of its cells are destroyed by mechanical, surgical and / or chemical methods; At least a portion of the tissue is removed or dissolved, leaving and / or exposing a remainder that may include the submucosa. The process of making; (c) removing substantially all pathogens and / or pathogenic and / or beneficial microorganisms from the remainder. Contacting with a medium or solution to remove, inactivate, or remove ; (d) digesting or separating the remainder; (e) one or more pluripotent cells, or at least a portion, or a substantial portion, of a cell. Isolate a population of cells that expresses one or more desired biomarkers, in part or in large part. and

[0059] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. The method of isolating from tissue (or a portion or sample thereof) having the mucus further comprises removing the surface mucus. Including etc.

[0060] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a portion or sample thereof) having a physiological range Media or solutions with osmotic properties outside the Includes solutions.

[0061] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a portion or sample thereof) having a physiological range Media or solutions with external osmotic properties include glucose solutions, high salt solutions, or distilled water. Contains water.

[0062] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. 20. A method for isolating from a tissue (or a portion or sample thereof) having This is accomplished by chemical destruction, including the use of agents and / or surfactants.

[0063] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. The method for isolating a tissue (or a part or sample thereof) having and / or the emulsifier is in water, saline and / or a buffer.

[0064] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. The method for isolating a tissue (or a part or sample thereof) having and / or the emulsifier is applied for a short period of time (less than 15 minutes).

[0065] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating a tissue (or a portion or sample thereof) having Cytin, polyoxyethylene sorbitan monolaurate (polysorbate 20), polio Polyoxyethylene sorbitan monooleate (Polysorbate 80), Polyoxyethylene so Rubitan monopalmitate (polysorbate 40), polyoxyethylene sorbitan mono Stearate (Polysorbate 60), Polyoxyethylene sorbitan tristearate (Polysorbate 65), Ammonium Phosphatide, Sodium, Potassium and and calcium salts, magnesium salts of fatty acids, mono- and diglycerides of fatty acids esters of mono- and diglycerides of fatty acids, acetate esters of monoglycerides of fatty acids Lactic acid esters of mono- and diglycerides, chlo- and diglycerides of fatty acids Enoic acid esters, monoacetyl tartaric acid esters of mono- and diglycerides of fatty acids esters of tartaric acid and diacetyl tartaric acid, and mixtures of mono- and diglycerides of fatty acids. Synthetic acetic and tartaric acid esters, sucrose esters of fatty acids, sucroglycerides, polysaccharides of fatty acids Ligylcerol Esters, Polyglycerol Polyricinoleate, Propane-1 of Fatty Acids ,2-diol esters, and heat interacted with mono- and diglycerides of fatty acids. Oxidized soybean oil, sodium stearoyl-2-lactylate, stearoyl-2-lactylic acid Calcium, Sorbitan Monostearate, Sorbitan Tristearate, Sorbitan Mo from the group consisting of sorbitan monolaurate, sorbitan monooleate, and sorbitan monopalmitate Be selected.

[0066] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a portion or sample thereof) having , 1-heptanesulfonic acid, N-lauryl sarcosine, lauryl sulfate, 1-octane Sulfonic and taurocholic acids, benzalkonium chloride, cetylpyridinium chloride, methyl chloride Rubensthonium, decamethonium bromide, alkylbetaine, alkylamidoalkylbetaine Tyne, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate , phosphatidylcholine, N-decyl AD-glucopyranoside, N-decyl AD-macro BD-maltoside, N-dodecyl BD-maltoside, N-octyl BD-glucopyranoside N-tetradecyl BD-maltoside, Triton (Triton X-100), Nonide P-40, Poloxamer 188, Sodium Lauryl Sulfate, Sodium Deoxycholate sodium dodecyl sulfate, and sodium dodecyl sulfate.

[0067] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating a pathogen and a tissue (or a part or sample thereof) having the pathogen and a sample comprising the steps of: and / or substantially kills or inactivates pathogenic and / or beneficial microorganisms; The medium or solution for removal is an aqueous solution of sodium hypochlorite (NaClO). or any solution or agent used for disinfecting the skin or a surface.

[0068] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating a pathogen and a tissue (or a part or sample thereof) having the pathogen and a sample comprising the steps of: and / or substantially kills or inactivates pathogenic and / or beneficial microorganisms; or the medium or solution to be removed prior to application of the surfactant and / or emulsifier. It may be performed before or after digestion or separation, or after removal of mucus.

[0069] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating a tissue (or a part or sample thereof) having Includes the lower layer.

[0070] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a part or sample thereof) comprising the steps of: The separation is carried out enzymatically.

[0071] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a portion or sample thereof) having Minced prior to digestion or separation steps.

[0072] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a part or sample thereof) comprising the steps of: The isolation may involve separating the submucosal tissue into a cell suspension, a mixture, a population, clumps or aggregates of cells, and / or or disintegrate into tissue fragments.

[0073] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a part or sample thereof) having Use culture selection using culture conditions containing serum-free medium and, optionally, Kubota's medium This is carried out.

[0074] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a part or sample thereof) having This is accomplished using culture selection using culture conditions that include medium containing serum.

[0075] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a part or sample thereof) having Performed in low attachment plates.

[0076] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a portion or sample thereof) having the The cell or cell population may be a spheroid, one or more organoids, cell populations, or The cells are cultured under conditions that support or result in cell aggregates.

[0077] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating a physiologically acceptable The method further comprises one or more washing steps with a culture medium to be cultured.

[0078] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a portion or sample thereof) having It is a leaf tissue.

[0079] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating from a tissue (or a portion or sample thereof) having , main bronchi, esophagus, stomach, duodenum, small intestine, large intestine and rectum.

[0080] In some embodiments, one or more of the desired biomarkers are expressed in a single or multiple a number of pluripotent cells, or at least a portion, or a substantial portion, or a majority of the cells, A population of cells expressing one or more desired biomarkers is isolated from the mucosal and submucosal layers. A method for isolating a tissue (or a portion or sample thereof) having , pancreas, gallbladder and bile duct, the bile duct including the common bile duct and the cystic duct. . [Brief description of the drawings]

[0081] [Figure 1] Figure 1 shows A) human duodenum stained with Periodic Acid Schiff (PAS). The duodenal mucosa is elevated up to the intestinal villi and folded at the intestinal crypts (arrowheads). The submucosa (SM) contains many PAS+ glandular elements (Brunner's glands: BG, dotted lines). The BG is anatomically continuous with the intestinal crypts via the muscularis mucosa (MM). Few BG acini are located within the lamina propria of the mucosa and are continuous with the intestinal crypts (arrows in the right image). B) Immunohistochemical staining of cytokeratin 7 (CK7) in the human duodenum. CK7 is specifically expressed by BG but not by intestinal crypts. C) Immunohistochemical staining of SOX9 in the human duodenum. Both intestinal crypts and BG contain cells expressing SOX9 (arrows). D) Immunofluorescent staining of SOX9 (red) and CK7 (green), nuclei are represented in blue. In BG, SOX9 is co-expressed with CK7 in the same cells (arrows). [Diagram 2] Figure 2 shows A) immunohistochemistry of proliferating cell nuclear antigen (PCNA), CD44, epithelial cell adhesion molecule (EpCAM), leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), Tra-1-60, and Tra-1-81 in human duodenum. PCNA+, CD44+, EpCAM+, and Lgr5+ cells are found in both intestinal crypts (arrows) and Brunner's glands (arrowheads). Tra-1-60+ and Tra-1-81+ cells are present in Brunner's glands (arrows) but not in intestinal crypts. MM = muscularis mucosae. B) Immunohistochemical and immunofluorescence staining of transcription factors associated with pluripotency. Oct4A and SOX2 are positive in cells in Brunner's glands and are co-expressed in Tra-1-60+ cells (arrows). [Diagram 3]Figure 3 shows A) hematoxylin and eosin (H&E) stained sections of human duodenum before and after chemical and mechanical removal of mucosa. Except for rare intestinal crypts (dotted circle in right image), the surface epithelium (villi) and almost all epithelial cells within the intestinal crypts (arrowheads) have been removed. The Brunner's glands in the submucosa are preserved (asterisk). B) Immunohistochemical staining for cytokeratin 7 (CK7) in human duodenum after mechanical and chemical removal of mucosa. CK7+ cells in Brunner's glands are retained. C) Immunohistochemical staining for Tra-1-60 in human duodenum after mechanical and chemical removal of mucosa. Tra-1-60+ cells in Brunner's glands are preserved. MM=muscularis mucosa. [Figure 4] Figure 4 shows A-B) flow cytometry of epithelial cell adhesion molecule (EpCAM), leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and Tra-1-60 in cells isolated from duodenal submucosa. Immunoselection of EpCAM and Tra-1-60 resulted in partial enrichment of EpCAM+ (A) and Tra-1-60+ (B) populations, respectively. C-D) Tra-1-60+ cells were further selected by culture selection strategy. In C, cells were seeded at uniform density on plastic in Kubota's medium. Cells began to proliferate after a lag phase of 1-2 days and formed small clusters of 10-15 cells after 6-8 days. After 14 days, large colonies were observed. Each colony was formed by small, dense, uniform cells of Tra-1-60+. In D, cells were cultured in conditions adjusted for organoid formation, and single Brunner's gland stem cells (BGSCs) began to self-organize into spherical structures and further expanded in size and number. Organoid formation determined the enrichment of Tra-1-60+ cells, which represent the main phenotype that forms organoids. Ph-C: Phase contrast. Scale bar = 200 μm. [Diagram 5]Figure 5 shows A-B) Phase contrast (Ph-C), hematoxylin-eosin (H&E), and periodic acid-Schiff (PAS) staining, immunohistochemistry, and immunofluorescence of endoderm and pluripotency markers. In panel A, Brunner's gland stem cells (BGSCs) were cultured under self-renewal conditions (i.e., serum-free Kubota's medium). In panel B, BGSCs were cultured under conditions adjusted for organoid formation. In both conditions, the cultured cells exhibited a typical phenotype similar to that observed in situ by cells forming Brunner's gland. Nuclei are shown in blue. C-D) RT-PCR analysis confirmed the expression of endoderm (C) and pluripotency (D) genes. Biliary stem / progenitor cells (BTSCs) and Ntera cell lines were used as positive controls for endoderm and pluripotency genes, respectively. *=p<0.05 vs. other groups. GOI=gene of interest. [Figure 6]Figure 6 shows A) in vitro hepatocyte differentiation. Phase contrast (Ph-C), Periodic Acid Schiff (PAS) staining and immunofluorescence staining of albumin in human Brunner's gland stem / progenitor cells (hBGSC) cultured in hormonally defined medium for hepatocyte differentiation (HDM-H). After 14 days in HDM-H, the morphology of most cells changed significantly to polygonally shaped cells. These cells aggregated to form multicellular zones, were PAS positive (glycogen storage) and expressed albumin. Nuclei are shown in blue. Real-time PCR of hepatocyte markers in cells cultured for 14 days in HDM-Liver. Hepatocyte-specific genes, including albumin (ALB), transferrin (TF), and cytochrome P450 3A4 (CYP3A4), were increased compared to cells under self-renewal conditions (i.e., serum-free Kubota's medium: KM). Primary human hepatocytes (hHep) are used as a positive control. *=p<0.05 vs. other groups. GOI=gene of interest. B) Endocrine pancreatic differentiation in vitro. Ph-C, hematoxylin and eosin (H&E) staining and immunofluorescence for neurogenin 3 (NGN3) and insulin in human Brunner's gland stem / progenitor cells (hBGSCs) cultured in hormonally defined medium for islet cell differentiation (HDM-P). After 14 days in HDM-P, islet-like structures appeared in the cultures and these aggregates were positive for NGN3 and insulin. Nuclei are shown in blue. Real-time PCR of pancreatic endocrine markers in cells cultured for 14 days in HDM-P. PDX1, insulin and glucagon showed a high increase compared to cells in self-renewal conditions (i.e. Kubota's medium: KM). Normal islet cells were used as a positive control. *=p<0.05 vs. hBGSCs in KM. GOI=gene of interest. [Figure 7]Figure 7 shows in vivo transplantation of human Brunner's gland stem cells (hBGSCs) into the liver of SCID mice by intrasplenic injection. A) After 30 days, cells expressing human mitochondrial antigen (hMito) were present in the mouse liver, mainly located around the portal triad space (arrows). There were no positive cells in mice injected with saline (Veh). B) hMito-positive cells accounted for nearly 5% of hepatocytes in the mouse liver. C) Expression of human albumin (hALB) gene was detected by RT-qPCR in the liver of mice injected with hBGSCs, but not in mice injected with Veh (ND: not detected). Data are expressed as the mean ± SD of three experiments. D-E) Expression of markers of mature human hepatocytes, such as human albumin (hAlb), Hep-Par1, and hMito, was confirmed in the same cells by double immunofluorescence staining. Nuclei (Nu) are shown in blue (DAPI). [Figure 8] Figure 8 shows selective solubilization of duodenal mucosa: A) washing method; B) clamping of the duodenal limb; C) tissue cutting and mucus removal; D) mechanical filtration through a strainer. [Figure 9] Figure 9 shows immunohistochemistry of cytokeratin 7 (CK7) in human duodenum. The duodenal wall contains mucus, submucosa and mucosa layers. Brunner's glands (BG) are located within the submucosa. CK7 is specifically expressed by BG but not by cells in the intestinal crypts. The area within the box is enlarged in the image on the right. [Figure 10] FIG. 10 shows A) immunofluorescence staining of SOX9 (red) and Lgr5 (green) in human duodenum. Nuclei are shown in blue. In Brunner's glands (included in dotted line), Lgr5 colocalized with SOX9 (arrows) and its expression was higher in acini located inside the muscularis mucosae and in continuity with intestinal crypts (arrowheads) than in acini located in deeper layers within the submucosa. MM = muscularis mucosae. B-D) Immunohistochemical staining of sodium / iodine symporter (NIS). NIS is expressed at the base of intestinal crypts (arrowheads in panels A and C) and in Brunner's glands (arrows in panels A and B). [Figure 11]Figure 11 shows failed protocols and techniques for the removal of mucosal epithelial cells with preservation of submucosal viability. Technique 1: surgical incision, 2: mucosectomy with prior injection of submucosal saline, 3: mucosal scraping. These strategies resulted in partial removal of the mucosal layer and preservation of intestinal villi (arrowheads) and crypts (arrows) as shown in hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining. [Figure 12] Figure 12 shows real-time PCR in human Brunner's gland stem / progenitor cells (hBGSCs) cultured for 7 days in hormonally defined medium (HDM-P) for islet cell differentiation. PDX1 and glucagon genes, but not insulin genes, are significantly increased after 7 days when compared to cells under self-renewal conditions (i.e., Kubota's medium: KM). Normal islet cells were used as a positive control. *=p<0.05 vs. hBGSCs in KM. GOI=gene of interest. [Figure 13]Figure 13 shows that the duodenal submucosal glands of mice represent a distinct compartment relative to the intestinal crypts and display proliferative characteristics. A) Hematoxylin and eosin (H&E) staining and immunofluorescence staining for cytokeratin 19 (Ck19), SOX9 and proliferating cell nuclear antigen (PCNA) in mouse (m) duodenum. Dotted lines individualize the border between intestinal crypts and submucosal glands (SGs: asterisks). Duodenal SGs are distinguishable due to their clearer cytoplasm and mucus content than crypts. In rodent duodenum, intestinal crypts and j-intestinal crypts are Ck19 positive, whereas SGs (white asterisks) are mostly negative. SOX9+ cells are mainly located within SGs (green cells), whereas PCNA+ cells are mainly located within intestinal crypts (red cells). Nuclei are shown in blue. Scale bar = 200 μm (H&E and Ck19) or 100 μm (SOX9 / PCNA). B) Hematoxylin-eosin and immunofluorescence staining for Ck19, SOX9 and PCNA in mouse (m) jejunum. In rodent jejunum, intestinal crypts and villi are positive for Ck19. The distinction with respect to the duodenum is that SOX9+ cells are located in the crypts and co-express PCNA (yellow arrows). Nuclei are shown in blue. Scale bar = 200 μm (H&E and Ck19) or 100 μm (SOX9 / PCNA). C) Immunofluorescence staining for Ck19 and TdTomato (Td-Tom) in Krt19CreTdTomatoLSL mice 14 days after tamoxifen injection. In the mouse (m) jejunum, most intestinal crypts are Td-Tom+ (red arrows), with negative crypts (green arrows) located in contact with the positive ones. Villi located above Td-Tom+ crypts are entirely Td-Tom positive. In the mouse duodenum, SGs are mostly td-Tom- and Ck19-, thus excluding their origin from td-Tom+ crypts (red arrows). White asterisks indicate SGs. Dotted lines individualize the border between intestinal crypts and SGs. Nuclei are shown in blue. Scale bar = 200 μm. D) In ​​the mouse duodenum, PCNA+505 and SOX9+ cells are always Td-Tom negative (red arrows). Yellow and green arrows point to Td-Tom+ intestinal crypts, which are PCNA positive and SOX9 negative in the duodenum. Nuclei are shown in blue.The dotted line marks the border between the intestinal crypts and the SGs. The images in Figure 13 represent n=5 animals. [Figure 14]Figure 14 shows that Tra-1-60+ cells isolated from duodenal submucosa are restricted to the endocrine pancreas in vitro and exhibit the ability to differentiate into insulin+ cells in vivo. A-C) Endocrine pancreatic differentiation in vitro. Panel A) shows phase contrast (Ph-C) and hematoxylin and eosin (H&E) staining of cells isolated from human duodenal submucosal glands and cultured in defined medium for pancreatic differentiation (PM) or in self-renewing conditions (Kubota's medium: KM). In PM, islet-like structures appear after 7 days (PM7) and increase in number after 14 days (PM14); *p<0.001 vs. other groups. Scale bar=100 μm. n=5 biological replicates. B) Real-time (RT)-PCR shows increased PDX1 gene expression at PM7-14, with 583 nuclear Pdx1 expression confirmed by immunofluorescence staining. n=4 biological replicates. C) Insulin (INS) and glucagon (GLU) gene expression is increased after PM14 (n=4 biological replicates). Human islet cells (ISL) are used as a control (n=3 biological replicates). At PM14 days, islet-like structures show insulin and glucagon expression by immunofluorescence staining. Scale bar=100 μm. D-G) Experimentally induced diabetes in mice can induce proliferation and pancreatic traits in dSG in vivo (n=5 animals per group). D) Mice treated with streptozotocin (STZ) show increased extent of the areal fraction (asterisk) of dSG compared to controls (CTR). Dotted lines individualize intestinal crypts from dSG. Scale bar=200 μm. E) dSG from STZ mice show increased expression of proliferating cell nuclear antigen (PCNA), Pdx-1, neurogenin 3 (Ngn3), and insulin compared to controls as determined by immunofluorescence (IF). Scale bar = 100 μm. F) Shows a heatmap of IF scores. G) Shows that specimens from rodent duodenums show slightly increased expression of NGN3 and INS genes in STZ mice compared to controls as determined by RT-PCR analysis. Pancreatic tissue from the same mice is used as a reference. H) Shows a study of insulin expression in human duodenum obtained from a patient suffering from type 2 diabetes (T2D).In these organs (n=5 duodenum), rare insulin+ cells are present within the SG (arrows). Pancreatic tissue is shown on the right. Scale bar=50 μm. In immunofluorescence staining, nuclei appear in blue. For A-D and G, error bars indicate mean ± sd. For A-D and G, p values ​​were determined by two-tailed t-test. [Figure 15] Figure 15 shows the acquisition of self-renewing Brunner's gland cells by endoscopic biopsy of the human duodenal bulb. A) The biopsy shows the removal of the submucosa containing the Brunner's gland (asterisk) (left panel). SOX9 expressing cells in the Brunner's gland are indicated by arrows (right panel). B) In vitro cell colonies (dotted lines) obtained by culturing Brunner's gland cells isolated from fetal duodenum under self-renewing conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] definition As used in the description of this invention and the appended claims, the singular forms "a," " "An" and "the" include the plural unless the context clearly indicates otherwise. It is intended.

[0083] As used herein, the term "about" refers to a measurable value, such as an amount or concentration. In some cases, this may include variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of a particular amount. It means to include.

[0084] Used to describe the selection of any ingredient, range, dosage form, etc. disclosed herein The terms "acceptable," "effective," or "sufficient" are used to describe the components, ranges, dosage forms, etc. is intended to be suitable for its intended purpose.

[0085] Also, as used herein, "and / or" refers to one or more associated Any and all possible combinations and alternatives of the listed items. ("or") means and includes the absence of a combination.

[0086] As used herein, the term "comprising" refers to compositions and methods that include recited elements. As used herein, the term "substance" is intended to mean inclusive of, but not exclusive of, others. In this case, the transitional phrase "consisting essentially of" (and grammatical variations) refers to the listed materials or or steps and "that do not materially affect the basic and novel characteristics" of the recited embodiment. In re Herz, 537 F. 2d 549,551-52,190 USPQ461,463(CCPA 1 976)(emphasis in the original);MPEP § 21 See 11.03. Accordingly, as used herein, "consisting essentially of" The term "comprises" should not be construed as equivalent to "comprises." "Consists" means that other components and a method for administering the compositions disclosed herein. Each of these transition terms means to exclude a method step that is Embodiments as defined above are within the scope of this disclosure.

[0087] The terms "equivalent" or "bioequivalent" refer to a specific molecular, biological, or cellular Used interchangeably when referring to materials, the materials that are Intended to be of the same sex.

[0088] As used herein, the term spheroid refers to cells that are cultured together in a suspension culture environment. Organized into a three-dimensional (3D) structure that allows the The term is used to refer to an aggregate of cells of the same type.

[0089] As used herein, the term organoid refers to a culture medium in which cells interact with each other in a suspension culture environment. One or more types of cells organized into a three-dimensional (3D) structure that allows In some cases, organoids are aggregates of cells from [human or The invention may mimic aspects of the structure and function of an animal organ or tissue.

[0090] As used herein, the term microorganism refers to a method for obtaining a desired cell or cell population. Pathogenic or disease-causing agents present inside or outside the tissue being treated may or may not cause, or may or may not be beneficial; This refers to microorganisms that are

[0091] As used herein, the term "expression" refers to the process by which a DNA or polynucleotide The process of transcription into mRNA and / or the subsequent synthesis of peptides, polypeptides, and The term refers to the process by which a polynucleotide is translated into a polypeptide or protein. If derived from endoplasmic DNA, expression may include splicing of the mRNA in eukaryotic cells. The expression level of a gene can be determined, for example, by measuring the level of mRNA or protein in a cell or tissue sample. and further, the expression level of the plurality of genes may be determined by measuring the amount of This may be determined to establish the expression profile of a particular sample.

[0092] As used herein, the term "functional" means to achieve a particular effect. It can be used to modify any molecular, biological, or cellular material to do.

[0093] As used herein, the term "gene" or "genetic" refers to DNA or is the RNA coding (e.g., mRNA) or non-coding (e.g., ncRNA) Any nucleic acid sequence, whether or not capable of being transcribed into an RNA molecule, is The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are intended to broadly include "nucleic acid," "polynucleotide," and "oligonucleotide." The terms " deoxyribonucleotides" and " ribonucleotides" are used interchangeably and are used interchangeably with each other. Polynucleotides refer to polymeric forms of nucleotides of any length, either nucleotides or analogs thereof. The peptides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments. (e.g., probes, primers, EST or SAGE tags), exons, intros RNA, messenger RNA (mRNA), microRNA, transfer RNA, ribosomal RNA , RNAi, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide , plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid promoters probes and primers.

[0094] The term "genetically engineered or modified" as used herein refers to a gene or deletion, addition or modification of genetic material, recombinant DNA techniques, viral vectors or electroporation Troporation-mediated gene modification, CRISPER (Clustered Regularly Interspaced Recombinant Proteins) gene targeting or editing via short palindromic repeats (or other short palindromic repeats); The present invention relates to a method for the preparation of a cell or its genome, including, but not limited to, deletion or addition of a NA fragment, correction of a genetic mutation, etc. The term "modification" is meant to broadly include any form of modification of a genetic material.

[0095] Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, it may be used prior to or after assembly of the polynucleotide. Modifications to the nucleotide structure can be added later. The sequence of nucleotides is The polynucleotide may be interrupted by non-nucleotide components. The term can also refer to a double-stranded molecule, which can be further modified after polymerization, such as by attachment of Unless otherwise specified or required, the term "polynucleotide" refers to both single-stranded and non-polynucleotide molecules. Any embodiment of this technology may comprise a double-stranded form and a double-stranded form known or predicted to be composed of The term encompasses both the two complementary single-stranded forms of each of which is shown in FIG.

[0096] The terms "protein," "peptide," and "polypeptide" refer to two or more subunits. To mean a compound of the unit amino acid, amino acid analog, or peptidomimetic, Used interchangeably in the broadest sense, the subunits are linked by peptide bonds. In another embodiment, the subunits may have other linkages, e.g., esters, ethers, etc. A protein or peptide may be linked by a linker. This limits the maximum number of amino acids that may make up a protein or peptide sequence. As used herein, the term "amino acid" includes glycine and D and Natural and / or synthetic derivatives, including both optical isomers, amino acid analogs, and peptidomimetics. The term refers to either unnatural or synthetic amino acids.

[0097] As used herein, the terms "subject" and "patient" are used interchangeably. is intended to mean any human or animal. The elephant may be a mammal. In further embodiments, the subject is a human or non-human animal (e.g. For example, a mouse or a rat.

[0098] As used herein, the term "tissue" refers to any tissue from a living or dead organism. Derived from or imitating an embodiment of body tissue or part of a living or dead organism The term refers to any tissue that is designed to mimic. Tissues may be healthy, diseased, and "Native tissue" or "living tissue" means a tissue that is naturally occurring and / or has genetic mutations or modifications. Such terms and variations as used herein refer to organisms in their natural state or derived from living organisms. "Micro-organs" refer to living tissue that exists in an unmodified state from the time it is created. Segments of "bioengineered tissue" that are modeled or mimic "natural tissue" Refers to...

[0099] A biological tissue is any single tissue (e.g., a collection of cells that may be interconnected) or a part of an organism. It may include a group of tissues that make up an organ or part or region of the body. It may contain heterogeneous cellular material or may be, for example, lung tissue, skeletal tissue, and / or muscle tissue. It may also be a complex structure such as found in areas of the body including the breast which may contain tissue. Exemplary tissues include liver, lung, thymus, skin, pancreas, blood vessels, bladder, kidney, brain, bile duct, and duodenal tract. the abdominal aorta, iliac vein, heart and intestine, any combination thereof, Not limited.

[0100] The term "isolated" as used herein means substantially free of other materials. The term "isolated" also refers to a molecule or biological or cellular material that is not isolated from the Material that has been removed from its natural environment (e.g., from in vivo to ex vivo or in vitro). The term "sterile" as used herein refers to a material that is Refers to material that is free of bacteria or other living microorganisms (i.e., sterile, aseptic, antiseptic). (e.g., disinfection, etc.).

[0101] Isolated Brunner's gland stem / progenitor cells and cell culture medium As used herein, the term "cell" refers to a eukaryotic cell. The cells may be of human or animal origin and may be stem or progenitor cells or somatic cells. The term "population of cells" refers to at least a portion, or a substantial portion, or The majority are of the same or different origin and / or lineage stage It refers to a group of one or more cells of a cell type. In some embodiments, the population of cells is In some embodiments, the population of cells may be derived from a cell line, and in some embodiments, the population of cells may be derived from the organ or or may be derived from a tissue, part thereof, or a sample thereof.

[0102] The term "stem cell" refers to a cell that is capable of self-renewal (producing daughter cells identical to the parent cell) and A cell population that is pluripotent, i.e., capable of giving rise to multiple types of adult cells, As used herein, the term "progenitor cell" or "precursor" refers to a variety of Although it is possible that progenitor or precursor cells may be multipotent, the extent or range of pluripotency of progenitor cells is greater than that of stem cells. The term "progenitor cell" or "precursor" may also refer to a stem cell. A progeny is broadly defined to include a progeny and its descendants. A progenitor may be pluripotent, bipotent, or Although they may be a unipotent cell population, they may have a more limited self-renewal capacity than stem cells. The selected progenitors are those that can differentiate into specific lineages. Examples include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, endometrial stem cells, and determined stem cells derived from human stem cells, adult stem cells, perinatal stem cells, amniotic fluid-derived stem cells, mesenchymal stem cells (MSCs), and These include, but are not limited to, stem cells and committed progenitors. Intermediates between these include cell populations such as hepatoblasts and pancreatic ductal precursors, and those that may be multipotent. However, this includes other forms of transiently proliferating cells that may have limited replicative capacity.

[0103] The cells of interest in this disclosure are herein referred to as Brunner's gland stem / progenitor cells (BGSCs). and can be derived from the duodenum (but not elsewhere in the intestine) of humans or animals These BGSCs are distinguishable from intestinal stem cells based on at least the following characteristics: [Table 1]

[0104] Applicant has identified the duodenum and its Brunner's gland as a separate organelle from the liver, pancreas, and other endodermal cells and The duodenal submucosa is a part of the glandular stem / progenitor cell microenvironment that gives rise to the duodenal ductal and endothelial tissues. BGSCs are essential stem / progenitor cells in the Brunner's gland (duodenal submucosal gland). A small subpopulation (e.g., about 5%) of cells in the TRA-1-6 0, Tra-1-81, OCT4, SOX2, and NANOG. BGSCs are recognized as a part of the liver, bile duct, pancreas, intestine, and Lgr 5, NIS, CD44, CK19, SOX9, and EpCAM. or other endodermal tissues with biomarkers that may include both SOX17 and PDX1. Related.

[0105] BGSCs are distinct from intestinal stem cells as mentioned above. BGSCs are characterized by the following: Tra-1-60, Tr a-1-81, express OCT4 and CK7, whereas intestinal stem cells do not. BGSC have also demonstrated that BGSCs resemble hepatic stem cells in that they express both SOX17 and PDX1. Hepatic stem cells, which are also distinct from pancreatic stem cells, express SOX17 but not PDX1. Pancreatic stem cells express PDX1 but not SOX17.

[0106] Thus, in one aspect, the present disclosure provides Tra-1-60, Tra-1-81, OCT4 , SOX2, NANOG, EpCAM, SOX9 and CK7 BGSCs isolated from the duodenum expressing one or more markers, and the ability to proliferate with limited or minimal differentiation under culture conditions that support self-renewal. In another embodiment, the BGSCs isolated from the duodenum are characterized by the expression of Lgr5, N The present invention relates to a method for treating a patient with a inflammatory bowel disease, comprising administering to the patient a therapeutically effective amount of a marker selected from the group consisting of IS, CD44, and CK19. We have shown that BGSCs proliferate with limited or minimal differentiation under culture conditions that support self-renewal. In another embodiment, the BGSCs isolated from the duodenum are further characterized as having the ability to express both SOX17 and PDX1, and BGSCs grow in culture conditions that support self-renewal. These are further characterized as the ability to proliferate with limited or minimal differentiation. In terms of morphology, the isolated BGSCs were characterized as Tra1-60, Tra-1-81, OCT4, SO X2, NANOG, EpCAM, SOX9, CK7, Lgr5, NIS, CD44, CK 19, and SOX17 and PDX1 In some embodiments, the isolated BGSCs may express both the pathogen and and / or substantially free of pathogenic and / or beneficial microorganisms.

[0107] In some embodiments, the isolated BGSCs are cultured in a limited or minimal amount for at least one month. In some embodiments, the isolated BGSCs can be expanded to a limited extent. Both are capable of proliferation for up to two months with limited or minimal differentiation. The isolated BGSCs can be cultured for at least 6 months or at least 12 months in a limited or are capable of proliferation with minimal differentiation.

[0108] In some embodiments, the culture conditions that support self-renewal of BGSCs comprise serum-free medium. In some embodiments, the serum-free medium comprises Kubota's medium.

[0109] The term "culture" or "cell culture" refers to the maintenance of cells in an in vitro environment. "Cell culture system" as used herein refers to a system in which a population of cells is cultured ex vivo. Cultures refer to culture conditions in which cells of a single type or different cell types can grow. The mixture may include:

[0110] The cell culture may include a method for culturing cells in which the cells have a coating, such as a coating of extracellular matrix components. or on a surface that does not have biological fluids (e.g., serum or lymph) and / or Defined mixture of hormones, growth factors and cytokines (hormonally defined medium or HDM) supplemented with nutrient medium (minerals, vitamins, amino acids, lipids). HDM is a single layer of cells that are formed at specific mature lineage stages. The usefulness of a cell or population of cells is empirically defined.

[0111] Cell cultures are also floating populations or aggregates of cells plated on low-attachment dishes. The cells may be floating populations or aggregates, and / or suspension cultures. The medium supporting the suspension culture can be the same medium used for the monolayer culture. The medium may be serum-free or may contain serum. Serum-free medium allows the formation of a monolayer of floating aggregates. The floating aggregates are essentially If they contain primarily or predominantly one cell type, they are called spheroids. If they involve multiple cell types (e.g., epithelial and mesenchymal cell partners), b Cell clusters or aggregates floating in suspension culture, called spheroids Ids and organoids are considered to be in a 3D microenvironment, where cells interact in three dimensions. It is possible.

[0112] As used herein, "culture medium" refers to a nutrient solution for the cultivation, growth, or proliferation of cells. Culture media refers to, but is not limited to, the medium that induces cells into a particular state (e.g., pluripotent state, quiescent state, etc.). the ability to maintain pluripotent cells in a stable state (e.g., pluripotent stem cells), promote cell maturation, and in some cases, the ability to specifically target pluripotent cells They may be characterized by functional properties, such as the ability to promote differentiation into cells of a lineage.

[0113] Non-limiting examples of media include serum (commercial and agricultural) typically at a level of about 10%. Serum-supplemented medium, any basal medium supplemented with serum derived from animals routinely slaughtered for (SSM).

[0114] The term "isolated" as used herein means that the tissue is free from other materials (such as culture medium and / or is substantially free of the extracellular matrix and its respective components The term "isolated" also refers to a substance or biological material or cellular material. Describes materials that have been removed from their natural environment (e.g., from in vivo to ex vivo or in vitro). It is also used to clarify.

[0115] The terms "sterilized, disinfected or sanitized" as used herein refer to a pathogen-free and / or pathogenic and / or beneficial microorganisms (i.e., germ-free, sterilized, aseptic, antimicrobial, This refers to materials that do not contain any harmful substances (corrosive, disinfectant, etc.).

[0116] Isolated populations of BGSCs In another aspect, the present disclosure provides a method for the preparation of a method for the preparation of a cell comprising the steps of: However, Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCA M, SOX9 and CK7. These characteristics are characterized as the ability to proliferate with limited or minimal differentiation under culture conditions that support self-renewal. In another aspect, the present invention relates to a population of stem / progenitor cells isolated from the duodenum, which are characterized. The disclosure relates to a population of stem / progenitor cells isolated from the duodenum, wherein at least a portion of the cells, or or a substantial part or majority of the In another aspect, the present disclosure provides a method for the preparation of a medicament for the treatment of a cancer, comprising: Regarding the population of stem / progenitor cells isolated from the diodenum, at least a portion of the cells, or substantially In some embodiments, a large proportion of the tumor cells express both SOX17 and PDX1. , at least a portion of the cells in a stem / progenitor cell population isolated from the duodenum, or parenchyma. A large part or the majority of the genes are Tra 1-60, Tra-1-81, OCT4, and SOX2. , NANOG, EpCAM, SOX9, CK7, Lgr5, NIS, CD44, CK19 and one or more markers selected from the group consisting of SOX17 and PDX1. This method is expressed as follows.

[0117] In some embodiments, the isolated population of stem / progenitor cells described in the preceding paragraph is a pathogen and / or the presence of pathogenic and / or beneficial microorganisms. do not have.

[0118] In some embodiments, the present disclosure provides Tra-1-60, Tra-1-81, OCT4, S OX2, NANOG, EpCAM, SOX9, CK7, Lgr5, NIS, CD44, C One or more markers selected from the group consisting of K19, CD44, and CK19; and and a composition comprising an isolated BGSC population expressing both SOX17 and PDX1, In some embodiments, the BGSC population is sterilized, disinfected, or sanitized.

[0119] In another aspect, the present disclosure provides a method for the preparation of a method for the preparation of a cell comprising the steps of: However, Tra 1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCA from the group consisting of M, SOX9 and CK7, Lgr5, NIS, CD44 and CK19 The present invention relates to a method for identifying a tumor that expresses one or more selected markers, as well as both SOX17 and PDX1, and that is autologous. Under culture conditions that support self-renewal, optionally on low-attachment plates or in suspension, including but not limited to The isolated BGSCs are further characterized as their ability to proliferate with limited or minimal differentiation. In some embodiments, the present invention relates to organoids produced by culturing a population of The luganoids further comprise a medium, the medium being capable of transforming the BGSCs into a later lineage stage, including mature cells. They have the ability to differentiate into cells.

[0120] In some embodiments, the culture conditions that support self-renewal include serum-free medium. In particular, serum-free media include Kubota's medium.

[0121] In some embodiments, serum-free culture conditions are suitable for cells, whether epithelial or mesenchymal cells. In addition to SSM, it is a hormone-defined medium (HDM) designed for specific mature lineage stages of In addition, we developed a novel immunotherapeutic agent designed for a specific mature lineage stage of cells, whether epithelial or mesenchymal. This example is a serum-free medium designed for endodermal stem / progenitor cells. The Kubota medium is copper and calcium free, and contains insulin, transferrin / Fe and various nutrients, but no cytokines or growth factors were added to the basal medium. It contains a culture medium (a nutrient medium containing minerals, amino acids, sugars, salts, vitamins, and lipids). can support endodermal stem / progenitor cells from the liver, pancreas, lung, and intestine.

[0122] As used herein, "Kubota's medium" is copper-free but calcium-free (<0.01%). 0.5mM), selenium, zinc, insulin, transferrin / Fe, bound to purified albumin A mixture of combined free fatty acids and, optionally, high density lipoprotein (HDL) In some embodiments, Kubota's medium is low in copper, calcium (e.g., For example, 0.3 mM), about 10-9 M selenium, about 0.1% bovine serum albumin or human blood albumin (highly purified and fatty acid free), 4.5 mM nicotinamide, 0.1 mM M zinc sulfate heptahydrate, and about 10-8 M hydrocortisone (used for hepatic precursors, but (optional components not used for pancreatic progenitors), approximately 5 μg / ml transferrin / Fe; Approximately 5 μg / ml insulin, approximately 10 μg / ml high density lipoprotein, and purified blood Any medium that does not contain a mixture of purified free fatty acids added after binding to serum albumin (e.g., RPMI 1640 or DMEM-F12). The free fatty acid mixture includes Approximately 100 mM of palmitic acid, palmitoleic acid, oleic acid, linoleic acid, and linoleic acid were A non-limiting exemplary method for the preparation of this medium is For example, Kubota H, Reid LM, Proc. Nat. A cad.Scien.(USA)2000;97:12132~12137, and The disclosure of which is incorporated herein by reference.

[0123] Stem cells / progenitors were cultured to specific cell types, such as hepatocytes (HDM-H) or cholangiocytes (HDM-C). There are other serum-free HDMs that can be engineered for adult fate. Some of these HDMs In some embodiments, the medium is a medium for inducing cells to grow in a given environment. In another embodiment, the medium may be a "seeding medium" used to present or introduce the plant to the environment. Alternatively, the medium may be a "differentiation medium" used to promote differentiation of cells.

[0124] Such media are mixtures of nutrients, minerals, amino acids, sugars, lipids, and trace elements. (e.g., Dulbecco's Modified Eagle's Medium or DME, as well as H am's F10 or F12, and RPMI 1640, Roswell Park (including the defined basal medium established by the University of Maryland Memorial Institute). These basal media contain serum (serum-supplemented medium or SSM) or purified hormones, Hormonally defined media supplemented with either a defined mixture of growth factors and nutrients The HDM is a medium containing 20% ​​soluble cellulose and can be used for the maintenance of cells ex vivo. When used in, "HDM-H" is a novel method for the development of organoids or endoderm cells into mature hepatocytes. To induce mesenchymal stem / progenitor differentiation, the cells were seeded on a substrate of type IV collagen and laminin. HDM-C is used for organoids. or by using type I collagen and fibronectin to induce the cells into mature cholangiocytes. HDM used for monolayers seeded on Chin's substrate.

[0125] Specific hormone-defined medium (HDM) compositions are described in further detail below: Modified KM (MKM): All three of the following HDMs were at 0.6 mM concentration, 10 -12 M KM supplemented with copper and calcium to reach 20ng / ml FGF did. Hepatocyte differentiation (HDM-L): MKM with 7ug / L glucagon, 2g / L galactose, 1nM triiodothyroxine 3 (T3), 10ng / ml oncostatin M (OSM), 10ng / ml epidermal growth factor (EGF), 20ng / ml hepatocyte growth factor (HGF), and 1 μM dexamethasone. ·Cholangiocyte differentiation (HDM-C): 20ng / ml vascular endothelial growth factor (VEGF) 16 MKM was prepared by adding 5 and 10 ng / ml HGF. Defined medium for pancreatic differentiation (PM or HDM-P): does not contain hydrocortisone and In addition, 2% B27, 0.1 mM ascorbic acid, 0.25 μM cyclopamine, 1 μM retinol MKM supplemented with nonic acid was used for the first 4 days, and bFGF was used for the remaining was replaced with 50 ng / ml exendin-4 and 20 ng / ml HGF for a period of .

[0126] Basal media is a buffer used in cell culture that contains amino acids, sugars, lipids, vitamins, minerals, and Contains various nutrients, salts, trace elements, and other compounds in a composition that mimics the chemical components of the interstitial fluid surrounding cells. In addition, cell culture media typically contain nutrients that support biological processes (e.g., proliferation, differentiation, etc.). Provides essential signaling molecules (hormones, growth factors) required to bring about Serum consists of a basal medium supplemented with a low percentage (typically 2-10%) of serum to promote cell proliferation. The cells can be autologous to the cell type used in culture, but are most commonly derived from bovine, human, or Blood from animals routinely slaughtered for agricultural or food purposes, such as serum from horses, goats, etc. Serum is also used to inactivate enzymes that are part of the tissue dissociation process. can be.

[0127] Method for isolating pluripotent cells from tissue having a mucosal layer and a submucosa - Patent Application 20070229633 In another aspect, the present disclosure provides a method for the detection of one or more desired biomarkers. a plurality of pluripotent stem / progenitor cells, or at least a portion, or a substantial portion, of the cells; The method further comprises the steps of: extracting a population that predominantly expresses a desired biomarker from tissues having a mucosal layer and a submucosal layer; 1. A method for isolating a compound comprising the steps of: (a) The mucosal layer of the duodenum, which is substantially free of intestinal mucus, is treated with an osmotic pressure characteristic outside the physiological range. The cells of the mucosal layer are contacted with a medium or solution having the above properties under conditions that induce an osmotic shock in the cells of the mucosal layer. To make; (b) the mucosal layer or at least some of its cells are destroyed by mechanical, surgical and / or chemical methods; At least a portion of the tissue is removed or dissolved, leaving and / or exposing a remainder that may include the submucosa. To make; (c) digesting or separating the remainder; (d) isolating one or more BGSCs or populations of cells from the digested remainder. The present invention relates to a method comprising the steps of:

[0128] As used herein, the term "osmotic shock" refers to a process that causes damage to a cell. In some embodiments, the osmotic pressure change refers to the change in osmotic pressure relative to the physiological osmotic pressure in a cell. A medium or medium having osmotic properties outside the physiological range under conditions that induce shock in cells. By applying the solution, the cells are damaged by osmotic shock. A medium or solution with different osmotic properties is called hypotonic, which has an osmotic pressure lower than physiological osmotic pressure. Alternatively, the solution may be hypotonic or low osmolarity. The medium or solution is hypertonic or hyperosmolar, which has an osmolality higher than physiological osmolality. A medium or solution having osmotic properties outside the physiological range may be used. The solution may be any type of solution. In some embodiments, the solution is outside the physiological range. Media or solutions with osmotic properties include water, ultrapure water, distilled water, 5% glucose solution, high salt It may be a solution or the like.

[0129] Osmotic shock occurs when a medium or solution with osmotic properties outside the physiological range is introduced into the lumen. or by applying such a medium or in an amount that induces duodenal distension. In some embodiments, the biosensor may be induced by applying a solution to the mucosal layer of the tissue. Media or solutions with osmotic properties outside the physical range are incubated for about 0.5 minutes, 1 minute, about 2 minutes, Contact with the lumen or mucosal layer may be for about 5 minutes, about 10 minutes, or about 15 minutes.

[0130] In some embodiments, one or more desired proteins from a tissue having a mucosal layer and a submucosa layer are or one or more pluripotent cells expressing a biomarker for The method of isolating a population containing the mixture comprises exposing the mixture to a cell suspension prior to the sorting or isolation step. In some embodiments, the method comprises the steps of: one or more pluripotent cells expressing a desired biomarker or biomarkers, or The method for isolating a population containing such cells involves a single incubation in a physiologically acceptable medium. or multiple washing steps.

[0131] The present invention provides a method for producing one or more desired biomarkers derived from tissues having a mucosal layer and a submucosa. and isolating one or more pluripotent cells, or a population containing such cells, that express the pluripotent The method can be used to isolate pluripotent stem cells from any suitable tissue having a mucosal layer and a submucosal layer. In some embodiments, the tissue is an endodermal tissue. In some embodiments, the tissue is selected from the group including the small intestine, the large intestine, and the rectum. It is selected from the group including the main bronchus, the esophagus, the stomach and the duodenum.

[0132] In certain aspects, the present disclosure provides a method for treating one or more BGSCs or a population comprising BGSCs, comprising: Method for isolating from a tissue, a part of such tissue, or a sample thereof taken from the duodenum of a subject It is a law, (a) Removing intestinal mucus; (b) A medium or solution having osmotic properties outside the physiological range is infiltrated into the cells of the mucosal layer. application under conditions inducing osmotic shock; (c) Deletion of the mucosal layer or at least some of its cells by mechanical, surgical and / or chemical methods At least a portion of the tissue is removed or dissolved, leaving and / or exposing a remainder that may include the submucosa. To make; (d) the mucosal layer and / or the remainder are free of pathogens and / or pathogenic and / or A medium or solution for substantially killing, inactivating, or eliminating beneficial microorganisms. Applying the liquid; (e) applying digestion or separation to the remainder, including the submucosa, to produce a digest, separated cellular material; or producing a cell suspension; (f) optionally, at least one of the cells from the digest, the separated cellular material, or the cell suspension; Also, some of them can be cultivated; (g)Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, Ep One or more of CAM, SOX9, CK7, Lgr5, NIS, CD44, and CK19 A cell or at least a portion, or a substantial portion, or a majority of the cells expressing Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCAM, Expresses one or more of SOX9, CK7, Lgr5, NIS, CD44, and CK19 Isolating a population of cells and / or cells that express both SOX17 and PDX1 The present invention relates to a method, comprising:

[0133] As used herein, the term "remnant" refers to a portion of the mucosal layer that has been removed such that the submucosal layer is exposed. Partially or completely destroyed and / or removed tissue, parts thereof, or samples thereof The desired multipotent cells, such as BGSCs, are contained within the remaining submucosa.

[0134] One or more BGSCs, or a population containing BGSCs, are then cultured in the duodenum, or a portion thereof, of a subject. In some embodiments of the method for isolating or isolating from a sample thereof, the removing step comprises removing an emulsifier and / or or by chemical destruction, including the use of surfactants. Alternatively, sterilization is performed in step (d) using a sodium hypochlorite solution. In some cases, digestion is performed enzymatically. In some embodiments of the method for isolating the mucosa, the remainder after the removal or dissolution step includes the submucosa. The digested remainder comprises tissue fragments. In some embodiments of the method for isolating a population, the tissue or tissue portion or sample is subjected to a digestion process. The method is performed before step (e). In some embodiments of the method, the isolating step (f) is performed in a serum-free medium, optionally in Kubota's medium. Selection is performed using culture conditions that include one or more BGSCs. In some embodiments of the method for isolating a population comprising BGSCs, the isolating step (f) comprises isolating a population comprising BGSCs from blood. Selection is performed using culture conditions containing one or more BGS. In some embodiments of the method for isolating a population containing C or BGSCs, the digestion step (e) and and / or the isolation step (f) is carried out on a low attachment plate. In some embodiments of the method for isolating cells, the isolated cells are spheroids, single or multiple. Suspension or 3D culture media that support or produce multiple organoids, cell populations, or cell aggregates The cells are cultured under these conditions.

[0135] Mechanical disruption / mucosectomy involves the removal of the mucosal layer by a variety of possible techniques and tools. In some embodiments, the cell surface layer is detached. Small beads are used. shearing the cell walls, using ultrasonic treatment to break down the cell walls, mortar and pestle Using a pestle grinder, a blender, or freeze and thaw cycles using microwaves to break the bonds within the cell walls, and proteins Many of the methods involve mechanical disruption of the cell layer, such as denaturing the cells or using high pressure. Different methods are known.

[0136] Some embodiments of the method for isolating one or more BGSCs or a population of cells containing BGSCs are described below. In an embodiment, the removal or decomposition step comprises removing or decomposing lecithin, polyoxyethylene sorbitan monolaurate, Polysorbate 20, Polyoxyethylene Sorbitan Monooleate (Polysorbate Polysorbate 80), Polyoxyethylene sorbitan monopalmitate (Polysorbate 40) ), polyoxyethylene sorbitan monostearate (polysorbate 60), polyoxyethylene Diethylene sorbitan tristearate (Polysorbate 65), Ammonium phosphatase fatty acids, sodium, potassium and calcium salts, magnesium salts of fatty acids, Mono- and diglycerides of fatty acids, mono- and diglycerides of fatty acids acetate esters of fatty acids, lactate esters of mono- and diglycerides of fatty acids, Citric acid esters of mono- and diglycerides, monoglycerides of fatty acids and Mono- and diacetyl tartaric acid esters of diglycerides, fatty acids Mixed acetate and tartaric acid esters of mono- and diglycerides, sucrose esters of fatty acids esters, sucrose glycerides, polyglycerol esters of fatty acids, polyglycerol poly Ricinoleate, propane-1,2-diol esters of fatty acids, monoglycerides of fatty acids Interaction of thermooxidized soybean oil, sodium stearoyl-2-lactylate, and diglycerides with glycerol and diglycerides Sodium, Calcium Stearoyl-2-Lactylate, Sorbitan Monostearate, Sorbitan Sorbitan tristearate, sorbitan monolaurate, sorbitan monooleate, sorbitan By chemical destruction, including the use of emulsifiers selected from vitamin monopalmitate. do.

[0137] In some embodiments of the method for isolating one or more BGSCs, a removal or decomposition step 1-heptanesulfonic acid, N-lauryl sarcosine, lauryl sulfate, 1-octane Sulfonic and taurocholic acids, benzalkonium chloride, cetylpyridinium chloride, methyl chloride Chilbenzthonium, decamethonium bromide, alkyl betaine, alkyl amido alkyl Betaine, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate , Phosphatidylcholine, N-Decyl AD-glucopyranoside, N-Decyl AD- Maltopyranoside, N-dodecyl BD-maltoside, N-octyl BD-glucopyranoside Noniside, N-Tetradecyl BD-Maltoside, Triton (Triton X-100), Noni Det-P-40, Poloxamer 188, Sodium Lauryl Sulfate, Sodium Deoxycholate and sodium dodecyl sulfate. This is done by chemical destruction.

[0138] In one aspect, the present disclosure provides a method for the treatment of one or more BGSCs or cell populations comprising BGSCs. A method for isolating from a tissue sample taken from the duodenum of an elephant, comprising the steps of: (a) A solution having osmotic pressure characteristics outside the physiological range is passed through the mucosal layer to penetrate the cells of the mucosal layer. Applying under conditions that induce pressure shock; (b) the mucosal layer or at least some of its cells are destroyed by mechanical, surgical and / or chemical methods; At least a portion of the tissue is removed or dissolved, leaving and / or exposing a remainder that may include the submucosa. To make; (c) removing substantially all pathogens and / or pathogenic and / or beneficial microorganisms from the remainder. Contacting with a medium or solution to remove, inactivate, or remove ; (d) digesting or dissociating the remainder to result in a cell suspension; (e) optionally, digesting or isolating at least some of the cells from the separated cellular or tissue material; Also some can be cultivated; (f) one or more pluripotent cells expressing one or more desired biomarkers. or at least a portion, or a substantial portion, or a majority of the cells are one or more and isolating a population of cells expressing a desired biomarker of the present invention. In some embodiments, mucus is a barrier that protects the mucosal layer from a solution having osmotic properties that are outside the physiological range. The tissue or tissue section is then pretreated with osmotic shock-inducing conditions prior to exposure of the cells of the mucosal layer. minutes or removed from the sample.

[0139] In another aspect, the present disclosure provides a method for treating BGSCs in the duodenum, a portion thereof, or a sample obtained therefrom. 1. A method for isolating from a sample, comprising the steps of: (a) Digesting or separating the duodenum, or a portion thereof, or a sample taken therefrom, producing compounds or separated cellular material; (b) From digested or dissociated cellular material: (i) Tra-1-60, Tra-1-81; OCT4, SOX2, NANOG, EpCAM, SOX9, CK7, CK7, Lgr5, These cells or cells express one or more of NIS, CD44 and CK19. At least a portion, a substantial portion, or a majority of the cells are Tra-1-60, Tra-1- 81, OCT4, SOX2, NANOG, EpCAM, SOX9, CK7, CK7, Lg a population of cells expressing r5, NIS, CD44, and CK19; and / or (ii ) these cells, or at least a portion of the cells, that express both SOX17 and PDX1 A substantial or majority of the population of cells expressing both SOX17 and PDX1 obtaining a

[0140] In some embodiments of the method for isolating BGSCs or a population of cells comprising BGSCs, The intestine, any part thereof, any specimen taken therefrom, any remains and / or any digesta or separated material The cells or tissue material, or a combination thereof, may be treated with a disinfectant or sterilizing medium, solution or is contacted with the agent.

[0141] As used herein, the term "disinfectant" refers to an agent that can be used to disinfect bacteria, viruses, fungi, etc. Any medium that destroys pathogens and / or pathogenic and / or beneficial microorganisms. A medium, solution or agent intended to contain and kill bacteria or fungal spores. In some embodiments, the disinfectant is a hypochlorous acid solution. In some embodiments, the disinfectant is about 0.01% to about 0.1% sodium hypochlorite. It is a solution containing about 0.1% to about 0.2% sodium hypochlorite. In embodiments, the disinfectant is a 0.01% sodium hypochlorite solution, a 0.02% hypochlorous acid solution, Sodium solution, 0.05% sodium hypochlorite solution, 0.1% sodium hypochlorite solution, 0.15% sodium hypochlorite solution, or 0.2% sodium hypochlorite solution In a preferred embodiment, the disinfectant is a 0.05% sodium hypochlorite solution.

[0142] Alternatively, in some embodiments, the disinfectant may be alcohol, sodium hydroxide, aldehyde, , oxidizing agents, peroxyacids and peroxoacids, phenols, quaternary ammonium compounds , inorganic compounds (chlorine, iodine, acids and bases, metals, etc.), or terpenes, etc. As used herein, a disinfectant may be a medium, solution or agent selected from the group consisting of: Also, penicillins, polymyxins, rifamycins, lipoamycins, quinolones, or sulfonamides.

[0143] The duodenum, a portion thereof, or a specimen taken therefrom may be placed in a disinfectant or sterile medium, solution or or a drug, resulting in the resulting BGSC, and The population may be substantially free of pathogens and / or free of pathogenic and / or beneficial microorganisms. It is the knowledge of the present disclosure that the present invention does not include biological and and chemical phenomena rarely, very rarely, complete and / or proceed completely, or with absolute certainty. It will be understood that the objective of the invention is to achieve or avoid the results of the The terminology is used to pick up on the possible lack of completeness inherent in many biological and chemical phenomena. For example, in some embodiments, "substantially free of pathogens" refers to and / or the absence of pathogenic and / or beneficial microorganisms" is intended to The presence of pathogens and / or pathogenic and / or beneficial microorganisms may be insufficient for the desired use. or may not interfere with the desired use of the BGSC or a population of cells containing the BGSC. low levels or undetectable in the sample of interest as determined by commonly known methods Such methods may refer to situations where the bacteria is at a gram-positive, gram-negative, aerobic, Standard sterility tests for virulent and anaerobic bacteria, mycoplasma and endotoxin This includes the use of non-BGSCs derived from tissues, tissue sections or specimens. "Substantially free of pathogens and / or pathogen-associated" cells or populations of cells This term applies to the term "absence of microbial and / or beneficial microorganisms."

[0144] Thus, the compositions of the present disclosure include Tra-1-60, Tra-1-81, OCT4, S OX2, NANOG, EpCAM, SOX9, CK7, Lgr5, NIS, CD44, C Expresses one or more markers of K19 and both SOX17 and PDX1 , substantially free of pathogens and / or free of pathogenic and / or beneficial microorganisms The cell population may include BGSCs that do not contain BGSCs or cell populations that contain BGSCs.

[0145] The compositions of the present disclosure also include isolated BGS in combination with a physiologically acceptable medium. As used herein, the present invention may include, for example, C or other pluripotent cells, or cell populations comprising such cells. When used, the term "physiologically acceptable medium" refers to a medium that is acceptable to human patients as conventional pharmaceutical practice. "Vehicle" refers to any vehicle used to formulate a pharmaceutical composition for administration to a subject, such as a human. The physiologically acceptable medium is glucose, mannose, sucrose or dextran. , carbohydrates such as mannitol, proteins, polypeptides or amino acids such as glycine Acids, antioxidants, vitamins, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and saline containing other adjuvants such as preservatives. The compositions of the present invention may be administered to the circulation or target organ or tissue of a subject. The cells may be formulated for injection of BGSCs or cell populations containing BGSCs directly into the mouse.

[0146] Thus, the present disclosure relates to Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCAM, SOX9, CK7, Lgr5, NIS, CD44, CK19 Pathogens that express one or more markers and both SOX17 and PDX1 and / or are free of pathogenic and / or beneficial microorganisms The method may include the steps of: (a) administering to the patient a BGSC or a cell population comprising a BGSC; and (b) administering to the patient a physiologically acceptable medium. stomach.

[0147] Methods for using isolated Brunner's gland stem / progenitor cells (BGSCs) The BGSCs and / or populations of cells comprising BGSCs disclosed herein may be used in medical treatments. It is intended for use in

[0148] For example, the present disclosure relates to the liver, pancreas, stomach, duodenum, small intestine, large intestine, rectum, and / or other organs. Treating a subject diagnosed with a disease or condition that involves or affects endodermal tissue The method comprises administering to a subject in need thereof an effective amount of a population of BGSCs. , relates to a method.

[0149] In some embodiments, the present disclosure provides a method for the detection of inflammatory bowel disease in the liver, pancreas, stomach, duodenum, small intestine, large intestine, rectum, and and / or other patients diagnosed with a disease or condition that involves or affects endodermal tissue. A method of treating an elephant comprising administering to the animal a therapeutically effective amount of Tra-1-60, Tra-1-81, OCT4, SOX2 , NANOG, EpCAM, SOX9 and CK7, Lgr5, NIS, CD44, CK 19, and SOX17 and PDX1 At least a portion, or a substantial portion, or a majority of BGSCs expressing both The present invention relates to a method comprising administering an effective amount of a population of cells comprising:

[0150] In another aspect, the present disclosure provides Tra-1-60, Tra-1-81, OCT4, SOX2 , NANOG, EpCAM, SOX9 and CK7, Lgr5, NIS, CD44, CK 19, and SOX17 and PDX1 They express both IL-1 and IL-2 and proliferate with limited or minimal differentiation under culture conditions that direct self-renewal. an effective amount of a BGSC, or at least a portion of a BGSC, characterized as the ability to Administration of a population of stem / progenitor cells isolated, in part or in large part, from the duodenum. Some embodiments of autologous cell or gene therapy include In some embodiments, the cells are genetically engineered or modified cells.

[0151] In another aspect, the present disclosure provides Tra-1-60, Tra-1-81, OCT4, SOX2 , NANOG, EpCAM, SOX9 and CK7, Lgr5, NIS, CD44, CK 19, and SOX17 and PDX1 They express both IL-1 and IL-2 and proliferate with limited or minimal differentiation under culture conditions that direct self-renewal. an effective amount of a BGSC, or at least a portion of a BGSC, characterized as the ability to Administration of a population of stem / progenitor cells isolated, in part or in large part, from the duodenum. Some embodiments of allogeneic cell or gene therapy include In some embodiments, the cells are genetically engineered or modified cells.

[0152] In another aspect, the present disclosure provides a method for the detection of a tumor in the liver, pancreas, stomach, duodenum, small intestine, large intestine, rectum, and / or for the treatment of diseases or conditions involving or affecting other endodermal tissues, and and / or optionally, autologous or allogeneic cell lines using cells that have been genetically engineered or modified. Tra-1-60, Tra-1-81, OC for use in cell or gene therapy T4, SOX2, NANOG, EpCAM, SOX9 and CK7, Lgr5, NIS, One or more markers selected from the group consisting of CD44, CK19, and SOX 17 and PDX1, and under culture conditions that direct self-renewal, limited or minimal The present invention relates to the use of BGSCs, which are characterized as their ability to proliferate with differentiation.

[0153] In another aspect, the present disclosure provides a method for the detection of a tumor in the liver, pancreas, stomach, duodenum, small intestine, large intestine, rectum, and / or for the treatment of diseases or conditions involving or affecting other endodermal tissues, and and / or optionally, autologous or allogeneic cell lines using cells that have been genetically engineered or modified. Tra-1-60, Tra-1-81, OC for use in cell or gene therapy T4, SOX2, NANOG, EpCAM, SOX9 and CK7, Lgr5, NIS, One or more markers selected from the group consisting of CD44, CK19, and SOX At least a portion, or a substantial portion, of BGSCs express both 17 and PDX1 The present invention relates to the use of a population of cells that contains a majority, or a proportion, of the

[0154] As used herein, "treating" a disease in a subject or "treatment" of a disease refers to ) A symptom or disease is diagnosed in a subject who has not yet shown symptoms of the disease or who shows limited symptoms. (2) to inhibit or arrest the onset of a disease or (3) referring to the regression of a disease or the amelioration or causing of symptoms of a disease. As understood in the art, "treatment" refers to the attainment of beneficial or desired results, including clinical results. A useful or desired result for the purposes of this technique is a detection Alleviation or relief of one or more symptoms, whether detectable or undetectable, or improvement or Rest, reduction in the severity of the condition (including disease), stabilization of the state of the condition (including disease) (i.e. , not worsening), delaying or slowing down a condition (including a disease), progressing, improving, or alleviating a condition This may include one or more of the following: (including disease), condition, and remission (partial or complete) The following are possible examples, but are not limited to these.

[0155] As used herein, the terms "effective amount" or "effective amount" are used with emphasis. An effective amount refers to an amount sufficient to treat the disease or condition being treated. Such delivery may be by application, application or dose. The duration of administration will depend on many variables, including the bioavailability of the composition, the route of administration, etc. However, the particular amount of the composition for any particular patient will depend on the activity of the particular agent used, The patient's age, weight, health status, comorbidities, sex, and diet, time of administration, metabolism, and and / or rate of excretion, the combination of compositions, the particular disease or condition being treated (e.g. It is understood that the efficacy and safety of the drug will depend on a variety of factors, including the severity of the disease (e.g., liver disease), as well as the mode of administration. will be done.

[0156] How disclosure will be carried out The present disclosure provides: i) human and / or animal duodenum is pluripotent or multipotent; Endoderm positive for markers, and other biomarkers of stem cells or "stemness" The term "Brunner's gland stem / progenitor cells" as used herein refers to cells that have the phenotypic characteristics of Brunner's gland stem / progenitor cells. ii) these cells contain cells, including Brunner's gland cells, termed GSCs; , Tra-1-60, Tra1-81, OCT4 and CK7 expression, but are not limited to these. iii) B has a phenotype that is distinct from that of intestinal stem cells in the mucosal crypts, SSCs express both SOX17 and PDX1, and these cells also differentiate into hepatic stem cells (SOX 17 but not PDX1), and pancreatic stem cells (which express PDX1 but not PDX1) iv) BGSCs (which express SOX17 but not SOX17) have a phenotype different from that of BGSCs. destroys, at least in part, mucosal epithelial cells (villi and crypts) Isolation by chemical, mechanical and / or surgical procedures or methods that preserve the submucosa v) BGSCs can be cultured in the form of spheroids, organoids, cell aggregates or other cell types. The ability to form and grow cell populations, which shows self-renewal properties, pluripotency, and differentiation potential. vi) BGSCs can be engrafted in tissues and selected in vitro; and vi) BGSCs can be engrafted in tissues in vivo and selected in vitro. For example, they can engraft in the liver of SCID mice and differentiate into lineages related to the tissue. , and differentiate towards mature hepatocytes.

[0157] Brunner's glands are unique mucous glands located in the submucosa of the duodenum. These glands It was not found in the stomach, but was found in other parts of the small intestine (i.e., the jejunum and ileum) and in the large intestine. Its main known function is to protect the duodenal mucosa from the acidity of the material leaving the stomach. The number of Brunner's glands increases from the pyloric opening to the duodenal jejunal curvature. It gradually decreases and almost disappears in the lower and ascending duodenal parts. The intestinal glands are separated by the intestinal crypts (or glands) and the muscularis mucosae, but there is no direct contact between them. The intestinal crypts are in perfect anatomical continuity with the intestinal epithelium, which is a continuous remodeling of the intestinal epithelium along the crypt-to-villus axis. It contains a specific population of stem cells that are involved in life.

[0158] The data provided herein suggest that, alongside mucous cells, Brunner's gland expresses SOX9 , Lgr5, EpCAM, CD44, and / or both SOX17 and PDX1. It is established that the subject has a population of cells that express a particular set of stem cell markers. These cells are small, have little cytoplasm, and a high cytoplasm-to-nucleus ratio; their phenotype resembles that of the ventral endoderm of the embryo. Moreover, a limited subpopulation of these cells (approximately 5%) was found to be 4A, SOX2, Tra-1-60, and Tra-1-81. Furthermore, BGSSCs show expression of the proliferation marker PCNA, which indicates that It exhibits a replicative activity, which is probably involved in the replication of mucin-producing cells. Expression of stem cell markers is precisely distributed, and pluripotency markers are located in deeper acini Lgr5 and PCNA are expressed in cells adjacent to the muscularis mucosae and in cells associated with intestinal crypts. This distribution suggests the existence of two distinct but overlapping BGSC populations. One population has a primitive phenotype, is quiescent, and is located deep within the submucosa, while the other population is They show a transient amplification characteristic, cross the muscularis mucosae, and are spatially associated with the intestinal crypts. Both the “stopping” and transiently amplifying BGSC populations were expressed in intestinal crypt cells (Lgr5 + / CK7 - / CK 19 + / Tra-1-60-), a phenotype that is clearly distinct from + / - / CK7 + / CK19 + / Tra-1-60 + ) was shown.

[0159] The embodiments disclosed herein include a method for removing a mucosal layer to at least partially remove the surface epithelium. Processes involving chemical, mechanical or surgical destruction, which may expose the submucosal layer; digestion of the submucosa or isolation; collection of cells, including cells, expressing the markers described in this disclosure. and isolating a portion of the BGSC or at least one portion of the BGSC, while leaving the remainder intact. The method was developed to isolate a portion, or a substantial portion, or a majority of the human duodenum. This method is directed to an approach to the development of spheroids maintained in serum-free Kubota medium. The culture selection conditions may include culture conditions for cytoplasmic cells, such as cytoplasmic cells, organoids, cell aggregates, or cell populations. Once isolated, the cells retain the phenotypes expressed in vitro in the organelles (e.g., Lgr5 + / - / CK7 + / CK19 + / Tra-1-60 + / Pluripotency genes + ) and cell preparation In vitro, BGSCs are depleted from spheroids, osteoclasts, and They grow as luganoids, cell aggregates or cell clusters, and maintain their undifferentiated phenotype. There was no expression of mature cell markers and no evidence of mucin production. When transferred to specific differentiation conditions, BGSCs differentiate into at least hepatocytes, cholangiocytes and endogenous cells. They could rapidly mature toward various fates, including the secretory pancreatic lineage.

[0160] Interestingly, previous reports have shown that human gastric epithelial cells and duodenal cells can reprogram. showed that the cells did not exhibit stem cell behavior and pluripotency without ramification (see table). The results disclosed herein, in contrast, show that stem / progenitor cells (BGSCs) are It has been shown that it is found in the duodenum of humans and animals, including the duodenal gland. Considering their location, these BGSCs or cell populations containing these BGSCs are They can be easily isolated using the methods described in the disclosure and do not require reprogramming. However, they essentially exhibit the properties, characteristics and capabilities of endodermal stem / progenitor cells and have pluripotent properties. do.

[0161] In this study, the differentiation potential of BGSCs to a mature endothelial fate was tested. The drug has been injected into the mouse liver via the nascent route.

[0162] In the field of liver disease, orthotopic liver transplantation is currently the treatment of choice for acute liver failure and end-stage chronic liver disease. Liver transplantation is limited by a severe shortage of organ donors. Therefore, cell therapy strategies may be a viable alternative to support liver function while awaiting organ allocation. However, regenerative medicine approaches to liver disease are sustainable, easy to treat, and may represent an alternative option. This requires the identification of an available cell source.

[0163] The present disclosure provides a novel stem cell microenvironment with pluripotent potential and applicable cells for postnatal development. The present invention provides a method for isolating human BGSCs from the duodenum of a human donor. Represents a readily available source of cells that do not require genetic reprogramming or extensive manipulation. This makes it easier to implement in clinical programs compared to reprogrammed cells. should be possible (and potentially a safer approach). They can be harvested using endoscopy and then used for autologous or allogeneic cell and gene therapy. These cells have unique potential as a source of cells that can be used to

[0164] Abbreviation AFP, alpha-fetoprotein; ALB, albumin; BTSC, biliary stem cell CD, common determinant; CD44, hyaluronan receptor; CD133, prominin; CFTR, cystic fibrosis transmembrane conductance regulator, cGMP, Good Manufacturing Practice; C K, cytokeratin protein; CXCR4, CXC-chemokine receptor 4 (fusin or also called CD184; also called platelet factor 4; DAPI, 6-diamidino- 2-phenylindole; DPBS, Dulbecco's phosphate buffered saline; EGF, epithelial growth factor EpCAM, epithelial cell adhesion molecule; FBS, fetal bovine serum (or FCS, fetal calf serum); bovine serum; FGF, fibroblast growth factor (FGF10 is one of many types of FGF );HB, hepatoblasts;HDM, hormonally defined medium;HDM-C, cells differentiated into cholangiocytes HDM, engineered to be lineage-restricted to hepatocytes; HDM-H, engineered to be lineage-restricted to hepatocytes HDM-P, lineage-engineered HDM that restricts cells to a pancreatic fate; HGF; Hepatocyte growth factor; HpSC, hepatic stem cells; IF, immunofluorescence staining; IHC, immunohistochemistry; K M, Kubota medium, a serum-free medium designed for endodermal stem cells; KRT, cytokeratin Gene; Lgr5, leucine-rich repeat-containing G protein that binds R-spondin Coupling receptor 5; MKM, calcium, copper, and bFGF-supplemented Kubota medium Modified Kubota's medium; NANOG, a transcription factor critically involved in self-renewal; NCA M, neural cell adhesion molecule; NIS, sodium / iodide symporter; OCT4, (octamer binding transcription factor 4), also known as POU5F1 (POU domain, class 5, transcription factor 1) K, genes expressed by stem cells; PBS, phosphate-buffered saline; PDX1, pancreatic and duodenal homeobox 1, a transcription factor important for pancreatic development; PBG, peribiliary gland, bile duct Stem cell microenvironment of stem cells; RMPI, Roswell Park Memorial Institute - Researchers at the institute have established Acronym for various basal media established; RT-PCR, reverse transcription polymerase chain reaction; SALL 4. Sal-like protein 4 (SOX, Sry) found to be important for stem cell self-renewal HMG box-related; SOX2, maintenance of self-renewal, or regulation of embryonic and committed stem cells A transcription factor essential for pluripotency in endoderm tissues (liver, biliary system and pancreas). SOX17, a transcription factor essential for hepatic differentiation; VEGF, a vascular Skin cell growth factor.

[0165] material and method Human Tissue Sourcing The human duodenum was cultured at the Department of General Surgery and Cardiology, Sapienza University of Rome, Rome, Italy. The tissue was collected from organ donors at the Department of Organ Transplantation "Paride Stefanini". Informed consent to use the tissue for the purposes of our transplant program was provided. The protocol was approved by the Institutional Review Board and procedures were performed in accordance with current good manufacturing practice. The study protocol was conducted at the University of Rome. The study was reviewed and approved by the Ethics Committee of the Umberto I Polyclinic.

[0166] Media and solutions All media were sterile filtered (0.22 μm filter) and kept at 4°C in the dark before use. RPMI-1640, the basal medium for all cell cultures, and fetal bovine serum (FBS) were used. ) from GIBCO / Invitrogen (Carlsbad, CA). All reagents were obtained from Sigma (St. Louis, MO) unless otherwise specified. Growth factors were obtained from R&D Systems, Inc., except as noted. (Minneapolis, MN).

[0167] Kubota's medium (KM) is copper-free, low calcium (0.3 mM), 10 -9 M Select 0.1% bovine serum albumin (BSA), 4.5 mM nicotinamide, 0.1 nM sulfur Zinc acetate heptahydrate, 10 -8 M Hydrocortisone (or dexamethasone), 5 μg / ml transferrin / Fe, 5μg / ml insulin, 10μg / ml high density lipoprotein The soluble fatty acid complex consists of a mixture of added fatty acids bound to purified human serum albumin and a lipid permeabilization agent. The medium is made of any basal medium (RPMI 1640 in this example). Coll was first reported by Kubota and Reid as a defined medium for hepatoblasts. 2 Kubota's medium was subsequently used to generate mouse, rodent, and human hepatic stem cells, biliary stem cells, and hepatoblasts. It has been shown to be effective for gallbladder-derived stem cells and pancreatic progenitor cells. 3~9 .

[0168] For differentiation studies, serum-free KM was supplemented with calcium (final concentration: 0.6 mM), copper (10 -12 M), and 20 ng / ml bFGF were added, and this was designated as modified Kubota's medium (MKM). Based on MKM, and with specific supplements, it is possible to target islet fate (HDM-P) Hormones used to induce selective differentiation of BG cells towards a hepatic fate (HDM-H) Mon defined medium (HDM) was prepared: HDM-H for hepatocyte differentiation contains MKM with 7μg / L glucagon, 2g / L galactose, 1nM triiodothyroxine 3 (T3), 10ng / ml oncostatin M (OSM), 10ng / ml epidermal growth factor (EGF), 20ng / ml hepatocyte growth factor (HGF), and 1 μM dexamethasone. HDM-P for islet cell differentiation: MKM without hydrocortisone, 2% B27, 0.1 0.5 mM ascorbic acid, 0.25 μM cyclopamine, and 1 μM retinoic acid were added to the first bFGF was added for 4 days, followed by 50 ng / ml exendin-4 and 20 ng / ml It was replaced with HGF.

[0169] Magnetic sorting method Cells were identified by the manufacturer (Miltenyi Biotec Inc., Germany). By using magnetic bead immunoselection according to the protocol described above, EpCAM or TR Briefly, positive cells were screened for EpCAM microbeads. (Miltenyi Biotec Inc., Catalog No. 130-061-101 ) or TRA-1-60 microbeads (Miltenyi Biotec Inc. The cell suspension was then magnetically labeled with MAC S LS column (Miltenyi Biotec Inc., Cat. No. 130-0 42-401) and placed in the magnetic field of the MACS separator. The labeled cells were retained within the column, and the unlabeled cells passed through the column. The column was then removed from the magnetic field. After removal of the positively selected cells, the magnetically retained cells were eluted as a positively selected cell fraction. The cells were assessed by cell count and cell viability as previously described. Positive cells were counted in 1 ml The cells were suspended in basal medium at a concentration of 300,000 cells per 1000 ml and used as the final cell suspension. An N.4 aliquot containing 200,000 cells was collected for flow cytometry.

[0170] Cell isolation and sterility testing under GMP conditions To produce BG stem / progenitor cells under cGMP conditions for future clinical applications, The European Commission has established the European Union's Medicinal Products Regulation and the European Commission's Manufacturing Control and Regulation of Pharmaceuticals for Human Use. European guidelines on standards for manufacturing and quality control (EudraLex-Vol. 4, Manufacturing Control and Sterility testing was performed under cGMP conditions in a "direct The "Medicinal Products for Human and Veterinary Use" and the "Guidelines for Manufacturing Control of Pharmaceuticals" This was done according to guidelines.

[0171] Cell culture and clonal expansion Unsorted and sorted cells (approximately 3 × 10 5 100 cells / ml) were seeded onto a 3 cm diameter plastic culture dish and incubated overnight in KM containing 10% FBS ( The cell cultures were then maintained in serum-free KM for at least 2 months. To test for clonal expansion, single cell suspensions were obtained and cultured in self-renewal medium. 500 cells / cm in serum-free KM 2 Cells were seeded at a clonal seeding density of 1000×.

[0172] Organoid preparation and culture After centrifugation, the cell pellet was suspended in KM and diluted to 3 x 10 5 The cells are 2.2 in diameter. The cultures were then placed in 12-well plastic culture dishes of 200 mm diameter and incubated overnight (approximately 10 min) in KM containing 10% FBS. The cells were then maintained in KM for 3 h at 4 °C for 12 h, after which the cultures were fed serum-free KM. The cells were cultured for one week in an incubator at 7°C with atmospheric oxygen and 5% CO2. After 7 days, the cells were removed from the 12-well plate and The cell pellet was then immersed in cold Matrigel (Corning Matrigel Basement t Membrane Matrix Growth Factor Reduced, The applicant embedded the cells in 400 μl of phenol red-free water. Winning 2 x 10 5 The gel was seeded with 400 μl of cells. After incubation at 7°C, the gel was covered with 500 μl of organoid medium. 7, N2 (Life Technologies), and 1.25 mM N-acetylglucosamine Lucysteine ​​(Sigma-Aldrich), 10 nM gastrin (Sigma-Aldrich), ldrich), and growth factor: 50ng / ml EGF (Peprotech) ), 1 μg / ml recombinant human R-spondin-1 (Perotech), 100 ng / ml FGF10 (Peprotech), 25ng / ml HGF (Peprotech) ech), 10 mM nicotinamide (Sigma-Aldrich), 5 μM A8 3-01 (Tocris), and 10 μM forskolin (FSK) Based on d-DMEM / F12 (Life Technologies). The medium was changed every 2-3 days while the size and number of organoids were controlled microscopically.

[0173] After 10-14 days, the cells were cultured using cell recovery solution (Corning) and ice-cold PBS. The organoids were removed from the Matrigel. The organoids in the culture gel were then collected in cell recovery solution ( Corning) to gently disrupt the organoids while preserving them as whole spheres. The regel was broken down into small fragments. The organoids were then gently centrifuged to the bottom of the tube. The majority of the supernatant was removed with a pipette, and the organoids were then collected in a centrifuge. Ganoid pellets were fixed in 4% formalin for further analysis.

[0174] Positive control NTERA-2 clone D1 pluripotent human embryonic cell line (Sigma Aldrich, St. Louis, Missouri, USA (Code: 01071221) is a company that specializes in flow cytometry. Pluripotency markers (SOX2, OCT4A and NANOG) was used as a positive control. 10 In addition, human seminoma testis fragments It has been used as a positive control in immunohistochemistry experiments for pluripotency markers.

[0175] Human colon adenocarcinoma cell line (LGC Standards SrL, Milan, Italy; HT-29 (Code: ATCC-HTB-38) was used for flow cytometry and R It was used as a positive control for the Lgr5 antibody for T-PCR experiments. It was used as a control in the differentiation experiments and was It was purchased from rodoLab (HIR-001).

[0176] Primary human hepatocytes (Clonetics™ Human Hepatocyte C Cell Systems NHEPS™ cells (Code: CC-2591S) are Onza (Basel, Switzerland) was purchased as a positive control for hepatocyte differentiation experiments. It is used as such.

[0177] Normal islet cells have been used as a control in experiments on islet differentiation and are The antibody was purchased from ProdoLab, Inc., Irvine, California (HIR-001).

[0178] Light microscopy (LM), immunohistochemical staining (IHC), immunofluorescence staining (IF) Specimens were fixed in 10% buffered formalin for 2–4 h and then transferred to low-temperature liquid paraffin (55–5 7°C) and sections at 3–4 μm were prepared by hematoxylin- The IHC was stained with eosin and Sirius red / fast green. Intrinsic peroxidase activity was determined by incubation with methanolic hydrogen peroxide (2.5%) for 30 min. Blocked by incubation with proteinase K as indicated by the vendor. (Dako, code S3020) was applied for 10 minutes at room temperature to retrieve the antigen. Sections were then incubated with primary antibodies overnight at 4°C (Supplementary Table 1). The samples were rinsed twice for 5 min with PBS and incubated with the secondary biotinylated antibody (LSAB+System- HRP, Dako, code K0690; Glostrup, Denmark) Streptavidin-HRP (LSAB+System-HRP, Dako, code K0690) at room temperature for 20 minutes. Cell culture was performed using 100% ethanol (Osaka, Japan) as a substrate and sections were counterstained with hematoxylin. For immunofluorescence staining in vitro, the chamber slides were fixed in acetone for 10 min at room temperature. The plates were then rinsed with PBS-Tween 20. Nonspecific protein binding was determined by 5% The cells were blocked with normal goat serum. The fixed cells were incubated with the primary antibody. The cells were then washed and incubated with a labeled isotype-specific secondary antibody (anti-mouse anti-AlexaFluor-546, anti-mouse AlexaFluor-488, anti-rabbit A lexaFluor-488, Antibody Goat AlexaFluor-546, Invitro gen, in collaboration with Life Technologies Ltd, Paisley, UK The cells were incubated for 1 hour with 4,6-diamidino-2-phenylindole (4,6-diamidino-2-phenylindole) for visualization of the cell nuclei. The cells were counterstained with DAPI. For all immunoreactions, negative controls were also included. This consisted of replacing the primary antibody with preimmune serum. Sections were then cut using the Jenoptik Pro Equipped with a g Res C10 Plus Videocam (Jena, Germany), eica Microsystems DM 4500 B light and fluorescence microscope (We The IF staining was also performed according to the method coded by the International Society of Clinical Oncology (IHC) of The results were analyzed by a confocal microscope (Leica TCS-SP2). LM, IHC and I F is the image analysis system (IAS - Delta Sistemi, Rome, Italy). ) and were conducted independently by two investigators in a blinded manner.

[0179] The area occupied by the BG was analyzed by an image analysis system (IAS-Delta Sistemi, Using this, the applicants determined that the volume occupied by BG was It was calculated as the total area occupied by the tufts and expressed as a percentage of the total duodenal submucosa. All counts were performed on six non-overlapping fields (magnification × 100) for each slide. 20) and at least three different slides were taken from each specimen.

[0180] For IHC / IF staining, positive cell counts were calculated for each slide / culture in six replicates. Cells were counted randomly and blindly in untreated fields (magnification ×20), and data were reported as % positive cells. IF staining was performed using a digital scanner (Aperio Scanscope FL System, Aperio Technologies, Inc., Oxford The images were scanned by the National Astronomical Observatory (North York, UK) and processed by ImageScope. Using a quantification algorithm, the percentage of positive pixel area for a single fluorophore or for two fluorophores is calculated. The area of ​​colocalization of the molecules was quantified. To test the glycogen storage capacity, the manufacturer's procedure was used. The staining was performed using a periodic acid Schiff (PAS) staining system (Sigma Aldrich, I NC, Cat. No. 395) and α-amylase (Sigma Aldrich, I NC, Cat. No. A 3176) digestion method (followed by PAS staining) was used.

[0181] Flow cytometry (FC) analysis Cells in culture are trypsinized and detached by gentle pipetting. Approximately 2×10 in PBS 5 The isolated cells were incubated with fluorescent primary antibodies or For intracellular antigens, cells were stained with 4% paraformaldehyde. The sections were fixed in 0.5% PBS-saponin before incubation with primary antibodies. The cells were permeabilized with 10% FCS. The primary antibody was EpCAM (EpCAM-FITC, MiltenyiBiotec Inc., Catalog No. 130-080-301) Lgr5 (Lgr5-PE, Origene Technologies Inc., Rockville, MD, USA, Catalog No. TA400001), TRA-1-60 (TRA-1-60-PE, Miltenyi Biotec Inc., Catalog No. The cells were analyzed using a BD FACScanto™ filter. Flow cytometer (Becton, Dickinson and Company, The analysis was conducted by BD FAC (New Jersey, USA). 10,000 events were acquired and SDiva(TM) software (Becton, Dickinson and Com The analysis was performed by Pany, NJ, USA.

[0182] Reverse transcription polymerase chain reaction (RT-PCR) analysis RNA extraction was performed on mice maintained in serum-free KM for 6 days and then transferred to either KM or HDM. The study was performed on tissues or cultures that had been incubated for an additional 7 days in either Total RNA was extracted according to the method of Chomczynski and Sacchi. was 11 The quality and quantity of RNA was determined using the previously described RNA StSens analysis chip (B io-Rad Laboratories, Hercules, CA, USA) RNA extraction was performed using the Experion automated electrophoresis system equipped with a The cells were maintained in serum-free KM for 6 days and then incubated in either KM or HDM for an additional 7 days. The study was performed on cultures incubated for a total of 13 days. Albumin (ALB), cysteine cytochrome P450 (CYP3A4), insulin (INS), glucagon (GLUC), The expression of PDX-1, SOX17, OCT4A, SOX2, and NANOG genes was significantly increased in cytoplasmic For total RNA samples extracted from cells and tissues, p RT-PCR by Qiagen, Hamburg, Germany) and reverse transcription These genes were amplified by PCR. Gene expression was measured using the Experion System ( On-chip capillary microelectrophoresis was performed using a 100-milliamp microelectrophoresis system (Bio-Rad, UK). The expression of the gene of interest was measured by quantification of the amplicon used. The ratio of the concentration of β-aminobutyric acid to the reference gene GAPDH (reported by the instrument in nmol / L) was used to measure the was calculated (Supplementary Table 2).

[0183] In vivo transplantation of BGSCs into the liver of normal mice Five male SCID (severe combined immunodeficiency) mice were incubated at 22°C with a 12-h light / dark cycle. They were housed in a room with an average constant temperature of 10°C and had free access to standard pelleted chow and water. The study protocol was performed in accordance with the guidelines of our institution. - EU Directive 2010 of the Sapienza University of Rome and the Umberto I University Hospital of Rome Approved by the Ethics Committee on Animal Experiments of the EU Convention on Animal Health (Prot. No.: 541). A suspension of 2 × 10 human BGSCs in 100 μl of saline was delivered to the liver via the splenic artery. 6 of Sham-control mice were injected with 100 μl of saline alone. All animals were They were kept under close observation until their recovery and had free access to food and water. No mortality was observed. Ta.

[0184] One month after transplantation, the animals were sacrificed and the livers were harvested. Liver fragments were analyzed for histology and immunohistochemistry. Placed in 10% buffered formalin for histochemistry or in Trizol reagent for gene expression analysis. Necrosis and fibrosis were visualized by hematoxylin and eosin (H&E) and Sirius staining, respectively. Engraftment of human BGSCs in mouse liver and their differentiation were evaluated by thread staining. were anti-human antibodies (anti-human mitochondrial, anti-human Immunohistochemistry was performed using the anti-HepPar-1 and anti-human albumin (anti-Human albumin) immunohistochemistry. Slides stained with histochemical staining (anti-human mitochondrion) were placed on ImageScope. Digital scanner processed by Aperio Scanscope CS System tem, Aperio Technologies, Inc., Oxford, UK) The image analysis algorithm was used to identify cells positive for anti-human mitochondria. The objective was to quantify the percentage of area occupied.

[0185] RT-PCR for human albumin in mice was performed as previously described. Briefly, specific primers for human albumin (Supplementary Table 2) were programmed Universal Probe Library Assay Using the ay Design Center (Roche), The human albumin gene was specifically identified from the offspring.

[0186] statistical analysis Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS statistical software. The analysis was performed using SPSS Inc., Chicago, IL, USA. Between-group differences in non-significant parameters were tested by the Mann-Whitney U test. Scientific significance was set at a p-value <0.05.

[0187] Example 1 - Isolation of cells from mucosa Human duodenum, including the hepato-pancreatic ampulla and pancreas, was cultured at the University of Rome, Rome, Italy. University of Pienza, Department of General Surgery and Organ Transplantation "Paride Stefanini" The tissue was collected from donors who provided informed consent for the use of the tissue for research purposes. All samples were obtained from adults aged 19–73 years. The protocol was approved by the Institutional Review Board of the present inventors, and the treatment was performed according to the current The study protocol was in compliance with current Good Manufacturing Practice (cGMP). The study was reviewed and approved by the Ethics Committee of the Umberto I University Hospital in Rome. The duodenum was carefully separated from the pancreas, and the intestine including the hepatopancreatic ampulla was surgically removed. The intestine was cut into slices with a scalpel. The tissue samples were then processed as previously described. The tissue was then incubated with 0.1% bovine serum albumin, 1 nM selenium, antibiotics, and type I collagenase. (300 collagen digestive units / ml) (Sigma-Aldrich, Italy), 0 .3mg / ml deoxyribonuclease (Sigma-Aldrich, Italy) Digest the cells in RPMI 1640 supplemented with 100% CO at 37°C for 30-45 min with frequent mixing. The suspension was filtered through an 800 micron metal mesh filter (IDEALE ACLRI9 Filtration through inox stainless steel, centrifugation at 270 g for 10 min, then The cell suspension was then filtered through 100 and 30 micron mesh filters. Serial passages were performed and then cell counts were performed using a Fast-Read 102 (Biosigma Srl, Venice, Italy) and cell viability was assessed by trypan blue assay. The viability (expressed as the percentage of viable cells relative to the total cells) was measured. Instead, the same methods already developed and utilized for the liver and bile ducts are used to treat These macroaggregates were always (N=10) isolated from non-viable cells. The cells are coated and disrupted, leading to cell death and making it impossible to obtain a cell culture. This is because the digestive tissue is highly saturated with mucus and decomposition products released by the mucosal epithelium. The physical and chemical properties of the tissue create molecular wafers that encapsulate the cells that are disrupted during the procedure. In fact, the isolation of cells from the intestine requires the use of animal or human The methods established in avoid destruction of the mucosal epithelium.

[0188] Another feature of the cultures obtained by this method was the frequent contamination (6 / 10). Based on these results, in order to preserve the Brunner gland and avoid the above-mentioned problems of microbial contamination, Strategies to separate the epithelium from the submucosa were adapted. Four different strategies were used: 1) external 2) surgical incision (N=3), 2) mucosal resection with prior injection of saline under the mucosa (N= 3) 3) Mucosal scraping (N=3), 4) selective solubilization of the mucosa (N=10) were tried. The method involves selective solubilization of the duodenal mucosa by a specific surfactant solution injected into the intestinal lumen. Except for the above, the results were illogical and were identical to those of the initial method.

[0189] Failed and suboptimal isolation methods The human duodenum was carefully separated from the pancreas and the entire intestine, including the hepatopancreatic ampulla, was surgically excised. The duodenum was cut into slices with a scalpel. The tissue specimens were then diluted with 0.1% bovine serum albumin (BSA). Serum albumin, 1 nM selenium, antibiotics, type I collagenase (300 collagen digestion unit) 0.3 mg / ml (Sigma-Aldrich, Italy), RPMI 164 supplemented with ribonuclease (Sigma-Aldrich, Italy) The digestion was carried out in 0 at 37°C for 30-45 min with frequent mixing. Ron's metal mesh filter (IDEALE ACLRI9 inox stainless steel) The cells were then filtered through a filtration membrane, centrifuged at 270 g for 10 min, and then resuspended. The suspension is passed successively through 100 and 30 micron mesh filters and then Cell counts were performed using a Fast-Read 102 (Biosigma Srl, Venice, Italy). The results were analyzed by trypan blue assay to determine cell viability (viable cells / total cells). The concentration of α-tocopherol (expressed as a percentage of α-tocopherol) was measured.

[0190] When used in the human duodenum (N=5), the results were consistent with previous studies developed for the liver and bile duct, Using the same method successfully used, 40,816,000 cells (standard deviation) were isolated. Of these, only half were viable by trypan blue assay (viability 43±1.0). Regardless of the viable cell count, isolated cells were coated with macroaggregates and disrupted. It was revealed that the cells contained oxidized cells, leading to cell death and making it impossible to obtain cell cultures. In the cultures obtained by this method, the cells were unable to adhere and survive. was always contaminated with bacteria (5 / 5).

[0191] This unsuccessful outcome was due to the fact that the mice were highly saturated with mucus and digestive products released by the mucosal epithelial cells. This may be due to the physical and chemical properties of the digested tissue that is reconstituted, which may result in the formation of a complex of cells within the mucosal debris. This leads to containment and is shattered during the procedure.

[0192] Another important point was the presence of an intestinal stem cell microenvironment (intestinal crypts) within the mucosal layer. Based on these findings, we decided to separate the mucosal epithelium from the submucosa. Strategies: 1) surgical incision (N=3), 2) mucosa after submucosal saline injection resection (N=3), 3) mucosal scraping (N=3), and 4) selective solubilization of the mucosal layer (N= 10) These were carried out. Strategies 1 to 3 were used to study the mucosal layer with the presence of intestinal crypts. This resulted in partial removal of the mucosa, so the best strategy in terms of mucosal removal was strategy In addition, strategy number 4 was superior in terms of cell isolation and viability. It provided the best possible approach.

[0193] Selective solubilization of duodenal mucosa by specific detergent solutions infused into the intestinal lumen. After isolating the duodenum near the head of the pancreas, the entire ampulla of the pancreas is removed and the intestine is clamped with a surgical clamp. The duodenal limbs were opened and the tissue was closed by pushing it from the upper part to the lower part. The intestinal mucus was removed by squeezing it out from the lower calcified area. This part is very important because it should contain as little mucus as possible. Using a pharmacy pipette, add approximately 200 ml of distilled water (Gibco, Italy). The water was poured into the duodenum through the incision in the upper limb, and the water was collected by clamping the incision in the lower limb (Figure 8 A) After that, both limbs are clamped for about 20 minutes to selectively induce osmotic damage in mucosal epithelial cells. The intestine was completely filled with distilled water by this method. (Figure 8B). After opening the lower limbs and removing the water, the rats were then swabbed with a 25 ml serological pipette. The duodenum was washed twice with 100 ml of DPBS (Gibco). Using a pharmacy pipette, approximately 200 ml of DPBS (Gibco, Italy) was The same procedure was adopted to drain the intraduodenoid spleen. The duodenum was filled with phosphatidylcholine (Sigma-Aldrich, Italy) 0. 5ml, Deoxycholic acid (Sigma-Aldrich, Italy) 20mg, DP BS (Gibco, Italy) 99.5 ml of surfactant solution (100 ml) The filling was maintained for 1 min. The solution was again removed by opening the crura and the internal duodenum. The duodenum was then washed with 100 ml of DPBS. Finally, the duodenum was placed in a 10 cm sterile Petri dish. The organ was then transferred to a cerebrospinal fluid (ECF) tube and opened by a longitudinal incision (Figure 8C).

[0194] Remove the mucus from the small folds in the tissue, taking great care to remove it from the upper Using a sterile scalpel, carefully cut the small folds of tissue ( Further dissection of the mucosa was performed by removing the mucus from the folds (G The tissue was washed in a sterile container containing 100 ml of 0.01% PBS (Ibco, Italy). Immerse in 200 ml of 5% sodium hypochlorite for a few seconds, then rinse with DPBS solution. The tissue was then cleaned and aseptically passed using sterile scissors and a scalpel. After mechanical and chemical techniques to remove the mucosa, the tissue was cut into small pieces. Specimens were collected and processed for histomorphometric analysis. Tissue samples were then processed as previously described. Briefly, tissue fragments were placed in two M tubes ( The tissue was collected in a flask (Milteny Biotec, Germany) and shaken until the tissue was in a dissociated state. To achieve this, we programmed the MACS separator (Milteniy Biotec, Germany). One or two cycles were performed (the opacity of the solution was noted along with the production of very small tissue fragments). The digestion buffer was pre-warmed to 34°C for 10 minutes (the time when the enzyme is most effective). After mechanical separation, the solution containing the tissue fragments was diluted with DTT (Sigma-Aldrich Dilute with a solution containing 100% EDTA (Italy) (see composition details below) and dilute with two 50 ml bottles The Falcon tube was rotated at 1,300 rpm (300 The pellet was collected and centrifuged at 75 cm in the presence of 150 ml of digestion buffer. The flask was wrapped with parafilm (Parafilm, USA). Seal and incubate at 37°C and 5% CO2 for approximately 30 min with occasional shaking to control digestion. The flask was then placed horizontally on a water heater for approximately 10 min, after which the cells were allowed to settle by gravity. The floating supernatant on the surface of the solution contained impurities and was precipitated using a 10 ml serological pipette. may be discarded using

[0195] After enzymatic digestion, the buffer containing the tissue fragments was transferred to four 50 ml Falcon tubes. The Falcon tube was centrifuged at 1,300 rpm (300 g) for 5 minutes. The pellets were collected in two 50 ml Falcon tubes and diluted with a solution containing DTT (hereafter (see composition details below) and then rotate the tube at 1,300 rpm (300 g). The supernatant was collected and filtered through an 800 micron metal mesh filter (IDEA The cells were placed onto a LE ACLR I9 inox stainless steel plate and filtered with fresh cell washing fluid. The filtrate was collected in a sterile container. A scraper and syringe (Terumo #SS-20 The plunger of the ES2 was used to filter the flow-through, and the tissue was further disaggregated during filtration. The obtained suspension, 200 ml on average, was diluted with DNase Pulmozyme 2500 U / mL. Two vials of 2.5 ml (Roche, Italy) were treated (Figure 8D).

[0196] Isolation of BGSCs from fetal duodenum and endoscopic duodenal biopsies performed in adult subjects Methods for isolating BGSCs from duodenal biopsies and fetal organs are different from those from intact adult duodenum. This is less complicated than the optimized methods for obtaining cells from cultures.

[0197] Duodenal biopsies were performed during gastroscopy in the Department of Transplantation and Precision Medicine, at the level of the bulb, using forceps. The samples were collected remotely using a computer. This method was used to obtain the desired results. The procedure was performed using a flexible endoscope under endoscopic guidance. The endoscope was introduced orally. Biopsies were obtained by visual inspection, thereby avoiding both arteries and veins. Histological examination of the biopsies obtained revealed that BG was present in the biopsy obtained from the bulbus but not in the distal tent. It was absent in the biopsy obtained from the bidenum (Fig. 15A), and BG was located in the submucosa just below the muscularis mucosa. Next, during this procedure, 1000 pieces of tissue were removed from the duodenal bulb per patient. Two to four biopsies were collected and used for cell isolation.

[0198] The resection is performed proximally at the level of the pylorus and distally at the level of the ligament of Treitz, The duodenum was collected from fetuses (18-22 weeks: therapeutic abortion in the obstetrics and gynecology department). and the intraductal bile duct were removed by removing them at the level of the hepatopancreatic ampulla.

[0199] Subsequently, the whole fetal duodenum or the whole duodenal biopsy is separated using a scalpel and MACS separation. The cells were further dissociated using a 300U / ml I Type 1 collagen (Sigma Aldrich) and 0.3 mg / ml deoxyribonucleic acid in a buffer containing clease (Sigma Aldrich) at 37°C for 20–30 min. Freshly isolated cells were then lysed using magnetic beads (Miltenyi Biotec). were used to immunoselect for TRA1-60 positive cells.

[0200] Sorting yielded an average of 12 million viable cells (N=3) from fetal duodenum and 100% viable cells from duodenum. 100,000 viable cells (N=2) were obtained from intestinal bulb biopsies. The isolation procedure took an average of The incubation time was 5 h. Cells were cultured in sterile 10% glucose solution at 1 million cells per mL. The cells were suspended in 0.5% CO and maintained at a controlled temperature of 4°C for 45 minutes before incubation. Self-replicating Brunner's gland cells are shown in Figure 15B. Regulations Concerning the Regulations Concerning the Control and Quality Control of Pharmaceuticals for Human Use European guidelines for standards (EudraLex-Vol. 4, on manufacturing and quality control) The cell products were classified into gram-positive, gram-negative, aerobic and anaerobic cells. Standard sterility tests for bacteria, fungi, and endotoxins were evaluated and TR Flow cytometry of A1-60 (Miltenyi Biotec, human, dilution ratio 1:50) The cells were immediately characterized by flow cytometry (FC).

[0201] Example 2 - Tissue and Cell Characterization Studies in human duodenal tissue Adult human duodenal mucosa (N=10) was elevated up to the intestinal villi, transected, and fixed. Deep within the stratified intestine are infolded intestinal glands (crypts) (Figure 1A). In the upper and descending parts, the submucosa is divided into glandular elements (duodenal glands or Brunner's glands: BG) This collectively represents 9.95±2.68% of the submucosal area and 4.62± The BG consisted mainly of PAS-positive mucous cells, which accounted for 1.93% of the total wall area (Figure 1 A) The BG is anatomically continuous with the intestinal crypts via the muscularis mucosae. There were few BG acini located in the distal duodenum and they were continuous with the intestinal crypts (Fig. 1A). In the lower and ascending parts, BG gradually disappeared, and glandular components were hardly observed in the lower part. (Approximately one per 20x field), and few glands were observed in the ascending portion.

[0202] In the human duodenum, immunohistochemical analysis revealed that BG cells and intestinal crypts were partially expressed The cytokeratin expression was similar in both the intestinal crypts and BG. However, they were CK19 positive; in contrast, CK7 was specifically expressed by a portion of BG cells. Both intestinal glands and BG were expressed in the IL-11A / IL-11B / IL-11C / IL-11C / IL-11D / IL-11E / IL-11E / IL-11F ... However, they contained cells expressing SOX9 (a marker for endodermal stem cells, Fig. 1C), and BG In this study, SOX9 was co-expressed with CK7 in the same cells.

[0203] Furthermore, intestinal glands and BG express the proliferation markers PCNA, and CD44, EpC Cells expressing AM, and several other stem / progenitor cell markers such as Lgr5 In the BG, Lgr5 colocalized with SOX9, and its expression was significantly higher in the mucosal higher in acini located within the muscularis and deeper within the submucosa The crypts were more continuous with the intestinal crypts than with the nuclei in the nuclei (Fig. 10A).

[0204] A subpopulation of BG cells expressed pluripotency markers (Figure 2). 1-60 and Tra-1-81 were expressed by BG cells but not by cells within the intestinal crypts. Tra-1-60 was not expressed by SO in the same BG cells (Fig. 2A). Finally, BG colocalized with X2 and Oct4A in cells expressing NIS (Fig. 2B). This included the vesicles, which were also expressed by intestinal crypts (FIG. 10B).

[0205] In summary, semiquantitative analysis of stem / progenitor cell marker expression revealed that BG is a + ( 9.12%±3.30) cells and proliferating cells (PCNA + : 4.82% ± 1.33) Furthermore, the BG microenvironment was found to contain Lgr5 (4.76% ± 1.04), EpCAM (8.80% ± 0.65), and other endodermal stem cells Approximately 5% of the cells contained cells expressing Tra-1-60, Tra-1-81 Interestingly, they expressed pluripotency markers such as IL-1, Oct4A, and SOX2. PCNA + / SOX9 + / Lgr5 + The cells are located deeper in the submucosa (SO X9 + / Lgr5 + : 4.80% ± 1.50; PCNA + :0.98%±0.62;p< 0.05) was more prevalent in acini directly connected to the intestinal glands than in acini directly connected to the intestinal glands (SOX 9 + / Lgr5 + : 11.80% ± 4.40; PCNA + :5.95%±2.25;p< In contrast, multipotent cells are predominant in acini located deeper within the submucosa. There were more.

[0206] Studies in rodent duodenal tissue Similar to humans, the rodent duodenum contains mucous glands located submucosally. Rodent SGs were directly associated with intestinal crypts that did not contain an intact muscularis mucosa. Due to their fluid content they were distinguishable from crypts by virtue of their clear cytoplasm (FIG. 13A). SGs were restricted to the proximal duodenum in rodents. Expression of SOX9 and PCNA was studied. In the present study, SOX9+ cells were present in SGs, whereas PCNA+ cells were mainly located in the crypts ( 26.1±5.7%), whereas only a small number of SG cells were PCNA positive (6.7±2.2%). , p<0.01 for crypts). Duodenal SGs and crypts also showed a significant difference in Ck19 expression. The former was almost negative, whereas the latter was positive (Figure 13A). The crypts contained cells that were SOX9+, PCNA+, and Ck19+ (Figure 1 Based on this phenotypic characteristic and the low percentage of PCNA+ cells in SGs, We introduced the Krt19CreTdTomatoLSL mouse lineage tracing model to investigate the role of S We assessed whether G replication proceeded from Ck19+ / PCNA+ cells within duodenal crypts. First, the jejunum was analyzed to estimate the recombination efficiency in the intestinal crypts (Figure 13C). The percentage of Tomato (Td-Tom) positive crypts was 72±6%, and the percentage of negative crypts was Villi above td-Tom+ crypts were always located next to td-Tom+ crypts. This resulted in the td-Tom-crypts being negative for the vertical and horizontal regions immediately above them. When mouse duodenum was examined, almost all Ck19-SG was 130td. -Tom+ crypts, including the cells located immediately beneath them, were td6 Tom- (Fig. 13C). Consistently, PCNA+ and SOX9+ cells in SGs were td-Tom- (Fig. 13D). Collectively, these data suggest that cell proliferation rates in rodent SGs are related to duodenal fibroblast proliferation. Moreover, under physiological conditions, SG replication is significantly lower in the SG subpopulations than in the intestinal crypts. SOX9+ and PCNA+ cells in the SG, but not in the crypt cells, express Ck1 Not derived from 9+ crypt cells.

[0207] Successful BGSC isolation and culture method After chemical and mechanical treatment of the duodenal tissue of Example 1, the surface epithelium and almost All crypts were removed, while the lamina propria and muscularis mucosae connective tissue remained (FIG. 3A). This allows for the preservation of the BG by virtue of its endoscopic location beneath the muscularis mucosae and within the submucosa. Thus, the BG remains intact and their CK7 + (Fig. 3B), Tra- 1-60 + (Figure 3C), and SOX9 + (not shown) Cells were retained.

[0208] The duodenal submucosa was further processed as described in the methods and showed a viability of >80% (85 Approximately 350 ± 100 million cells were isolated with a FC of 40.0 ± 5% of freshly isolated cells. ±18.5% were EpCAM + The cells were immunoselected against EpCAM. When the cells were cultured, the cell population was 70.3 ± 19.3% EpCAM. + Concentrate in cells (before sorting) p<0.05), of which 46.3% ± 7.3 of these cells were also Lgr5+ Cells isolated from the duodenum were also analyzed by flow cytometry for Tra-1-60 expression. The FC was 5.8 ± 1.6 for freshly isolated cells. % is Tra-1-60 + The cells were immunoselected against Tra-1-60. When separated, the cell population was 30.4±19.8% Tra-1-60 + Concentrate in cells (selection p<0.05 compared to before, Tra-1-60 + 7.3% ± 4.2% of cells were also infected with EpCA M + (Representative scatter plots are shown in Fig. 4B). After magnetic immunoselection, the contaminating cells The population was cultured using two different selection approaches: on plastic and as organoids. First, a single cell suspension was obtained and cultured in serum-free Kubota's medium. 500 cells / cm on plastic 2 The cells were seeded at a clonal seeding density of 1000×1000×1000, and the medium Allows survival and self-renewal of endodermal stem / progenitor cells, but not mature cell survival and self-renewal Replication is not possible, and either survival or self-renewal of mesenchymal cells is not possible. Under these conditions, Tra-1-60 + Only the cells were able to proliferate (Figure 4C), and after 1–2 days After a lag phase, they began to proliferate and formed small clusters of 10–15 cells after 6–8 days of culture. After 14 days, large colonies were observed (Figure 4C). Each colony contained high cell counts. Small (diameter = 12.06 ± 5.76 mm), dense, uniform cells with a plasma-to-nucleus ratio The self-renewal culture conditions were established in the culture selection of BTSCs. As mentioned above, this resulted in the loss of almost all mesenchymal cells (not shown). In culture conditions adjusted for loganoid formation, single BGSCs increased in size and number. In general, organoids are characterized by their self-organization into spherical structures that expand over time. They were visible after 5 days, and their average diameter reached 2.3±5 mm within approximately 13–14 days. Organoid formation is mediated by Tra-1-60, which represents the dominant cell phenotype that forms organoids. + Thin The enrichment of vesicles was determined (Figure 4D).

[0209] Phenotypic characterization of 2D colonies of BGSCs and BGSC-derived organoids Phenotypic analysis revealed that on plastic and in KM (self-renewing conditions, Figure 5A), The cultures express CK7, SOX9, EpCAM, Lgr5, and pluripotency markers (SO X2, Tra-1-60, and Tra-1-81) were expressed in the .

[0210] In parallel, organoids (Figure 5B) were cultured using CK7 + and CK19 + It is made up of cells, Organoid cells express endodermal stem cell (EpCAM, Pdx1) and pluripotency markers The organoids expressed markers for PAS (Oct4A and Tra-1-60). Negative (goblet cell characteristic) and negative for villin, CFTR, albumin, Hep-Par1, and Some mature cell markers, such as insulin, were negative (data not shown). .

[0211] The BGSC phenotype was further examined by RT-PCR and comparison with appropriate positive controls. , BGSCs in monolayers on plastic and in organoids expressed endodermal biomarkers ( e.g., EpCAM, SOX17, and PDX1, Figure 5C) and pluripotency (e.g., S OX2, OCT4A, and NANOG (Figure 5D). Under these conditions, markers of hepatocyte lineage (i.e., albumin), cholangiocyte lineage (i.e., cholangiocyte lineage), and cholangiocyte lineage (i.e., cholangiocyte lineage) were expressed. markers for the β-pancreatic cell lineage (i.e., CFTR), and markers for the β-pancreatic cell lineage (i.e., The cells were nearly negative for threonine (data not shown).

[0212] In vitro differentiation potential of BGSCs The differentiation potential of cells isolated from BG was determined to be hepatic (HDM-H) or endocrine pancreatic (HDM-P) By transferring them to separate media specifically tailored to induce lineage differentiation. The results were evaluated as follows.

[0213] After 7–14 days in HDM-H (Fig. 6A), the morphology of most cells was small spindle-shaped cells. The cells changed significantly from cystic to polygonal (cuboidal) cells. These cells aggregated to form multicellular cortex. The cells formed pores and had a larger diameter than those cultured in KM (p<0.01), and the cell size was These large IF cells were found to have a cell size consistent with that of normal diploid adult human hepatocytes. The polygonal cells expressed albumin (Figure 6A). Furthermore, PAS staining showed that the H We showed the presence of PAS-positive cells in DM-H ( Fig. 6A ), but not in KM ( After digestion with α-amylase, no visible PAS staining was detectable. This supports the glycogen storage ability of cells cultured in HDM-H. RT-PCR analysis (Figure 6A) showed that cells in HDM-H were significantly more sensitive to IL-1 than cells in KM. However, albumin (approximately 2-fold), transferrin (intermediate or zone 2; >100-fold), and and hepatic CYP3A4 (drug metabolism, late or zone 3 gene; >100-fold) genes It was shown to increase the expression of cell-specific genes.

[0214] After 14 days on HDM-P, islet-like structures were observed, and these structures expressed Ngn3 and islet The HDM-Ps were composed of confluent cells expressing surin (Figure 6B). After 7 days in HDM-P, RT -PCR analysis revealed that PDX1 expression, but not insulin and glucagon gene expression, , which was increased compared to the findings in KM cells (Figure 12). After 14 days of incubation, the levels of PDX1, insulin and glutamate were significantly higher than those of KM cells. A significant increase in kagon gene expression was detected (Figure 6B).

[0215] Tra-1-60+ duodenal SG cells rapidly commit to a β-pancreatic fate in vitro and SGs can spontaneously generate insulin-expressing cells in vivo. The in vitro differentiation potential of cells isolated from duodenal SGs was examined by culture them in conditioned medium (PM). After 7 days in PM, rare islets were detected in the 14-well plate. The presence of pancreatic islet-like structures was observed (1.4±0.5 per culture) (FIG. 14A). PCR analysis revealed that the PDX1 gene, but not the insulin gene, was more prevalent in PM than in KM. In parallel, islet-like structures were identified by immunofluorescence analysis. showed expression of PDX1, but not insulin, as determined by After 14 days in PM, the number of islet-like structures was significantly increased compared to 7 days (Figure 14B). 4.8±0.8 per culture, p<0.01). PDX1 gene expression was significantly higher in KM than in were higher in the 14-day PM compared with the 7-day PM based on RT-PCR analysis. (Figure 14B). Insulin and glucagon gene expression increased significantly at 14 days. (FIG. 14C), reaching the level of the islets used as controls. and glucagon+ cells appeared in pancreatic islet-like structures (Fig. 14C).

[0216] To study the ability of duodenal SG cells to commit to an endocrine pancreatic fate in vivo, we The inventors demonstrated that experimentally induced diabetes in mice induces the acquisition of specific pancreatic traits. Therefore, we investigated whether two different streptozotocin (STZ) models could be used. The high-dose STZ model was characterized by a rapid increase in blood glucose levels and a high mortality rate. The low-dose STZ model showed a slower and less pronounced increase in glycemia and a longer survival time. The mice were treated with high STZ doses, which extended the observation period. When sacrificed 14 days later, duodenal SG volume was increased compared to controls (FIG. 14D). Furthermore, a higher percentage of SG cells expressed PCNA, PDX1 and NG compared with controls. Finally, 2 / 5 S1-related IL-1 expression was observed in the control group, whereas 2 / 5 S1-related IL-1 expression was observed in the control group (Figure 14E-F). In TZ-treated mice, insulin+ and glucagon+ cells were observed in the duodenal SG. (Figure 14E-F). However, no correlation with blood glucose characteristics was observed. , a rodent duodenum-derived mutant characterized by increased expression of genes associated with pancreatic endocrine fate This was consistent with the RT-PCR analysis of the specimens (Figure 14G). These characteristics did not emerge (data not shown). Finally, type 2 diabetes (T2D) We investigated insulin expression in human duodenum from patients with rare (<5%) insulinomas. Insulin+ cells are found in duodenal SGs from T2D patients but not from normal subjects This was not observed in the control group (Fig. 14H).

[0217] In vivo transplantation of undifferentiated human BGSCs into mouse liver The ability of BGSCs to generate mature hepatocytes in vivo was examined by injecting them via the vascular route (splenic injection). The study was carried out by transplantation into the liver of SCID mice via intravenous injection. , human antigens (i.e., anti-human mitochondrial, anti-human nuclear, anti-human albumin, and Immunostaining with a specific antibody that reacts only with anti-human HepPar-1 (BGS Engraftment of C. human (h) mitochondria was assessed in the mouse liver one month after cell injection. + The cells were observed (Figure 7A), and the positive cells were mainly located around the portal vein, with some cells also located in the lobules. In the injected mice, approximately 5.1±1.3% of the host liver cells were expanded toward the cardiac position. antigen + In addition, h-mitochondria were expressed by + The engrafted cells were The cells were positive for mature hepatocyte markers, such as HepPar-1 (Fig. 7D) and HepPar-1 (Fig. 7E). Rarely, anti-human nuclear positive cells were observed in the interlobular bile ducts. These cells were CK19 positive. The results were consistent with those of the control group (data not shown).

[0218] To confirm the effective engraftment and differentiation of transplanted hBGSCs, Applicant performed a 3D model of mouse liver Human albumin mRNA expression in the liver was further investigated. This was measurable in livers harvested from injected mice (Figure 7C), but not in sham control mice (fed with saline). No detection was observed when salt water was injected. References Kubota,H.& Reid,L.M.Clonogenic hepatobla sts,common precursors for hepatocytic an d biliary lineages,are lacking classical major histocompatibility complex class I antigens.Proc.Natl.Acad.Sci.(USA)97,12 132-12137(2000). Furth,M.E.,et al.Stem Cell Populations G iving Rise to Liver,Biliary Tree and Pan creas.in The Stem Cells Handbook,2nd Edi tion(ed.Sell,S.)75-126(Springer Science Publishers,NY,NY,NYC,NY,2013). Harrill,J.A.,et al.Lineage Dependent Eff ects of Aryl Hydrocarbon Receptor Agonis ts Contribute to Liver Tumorigenesis.Hep atology 61 548-560(2015). 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Claims

1. expressing one or more markers selected from the group consisting of Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCAM, SOX9 and cytokeratin 7 (CK7); capable of differentiating into pancreatic and hepatic lineages, capable of proliferation with limited or minimal differentiation under culture conditions that support self-renewal; Brunner's gland stem / progenitor cells (BGSCs) isolated from the duodenum.

2. 2. The isolated BGSC of claim 1, capable of proliferation with limited or minimal differentiation for at least 1 month, at least 2 months, at least 6 months or at least 12 months.

3. The isolated BGSC of claim 1 , wherein the culture conditions that support self-renewal comprise a serum-free medium.

4. The isolated BGSC of claim 1 , wherein the culture conditions that support self-renewal include a medium containing serum.

5. expressing one or more markers selected from the group consisting of Lgr5, NIS, CD44 and CK19; capable of proliferation with limited or minimal differentiation under culture conditions that support self-renewal; Brunner's gland stem / progenitor cells (BGSCs) isolated from the duodenum.

6. 6. The isolated BGSC of claim 5, capable of proliferation with limited or minimal differentiation for at least 1 month, at least 2 months, at least 6 months or at least 12 months.

7. The isolated BGSC of claim 5 , wherein the culture conditions that support self-renewal comprise a serum-free medium.

8. The isolated BGSC of claim 5 , wherein the culture conditions that support self-renewal include a medium containing serum.

9. express both SOX17 and PDX1, capable of differentiating into pancreatic and hepatic lineages, capable of proliferation with limited or minimal differentiation under culture conditions that support self-renewal; Brunner's gland stem / progenitor cells (BGSCs) isolated from the duodenum.

10. 10. The isolated BGSC of claim 9, capable of proliferation with limited or minimal differentiation for at least 1 month, at least 2 months, at least 6 months or at least 12 months.

11. The isolated BGSC of claim 9 , wherein the culture conditions that support self-renewal comprise a serum-free medium.

12. The isolated BGSC of claim 9 , wherein the culture conditions that support self-renewal include a medium containing serum.

13. expressing one or more markers selected from the group consisting of Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCAM, SOX9, CK7, Lgr5, NIS, CD44 and CK19; capable of differentiating into pancreatic and hepatic lineages, capable of proliferation with limited or minimal differentiation under culture conditions that support self-renewal; Brunner's gland stem / progenitor cells (BGSCs) isolated from the duodenum.

14. 14. The isolated BGSC of claim 13, capable of proliferation with limited or minimal differentiation for at least 1 month, at least 2 months, at least 6 months or at least 12 months.

15. The isolated BGSC of claim 13 , wherein the culture conditions that support self-renewal comprise a serum-free medium.

16. The isolated BGSC of claim 13 , wherein the culture conditions that support self-renewal include a medium containing serum.

17. An isolated BGSC described in any one of claims 1 to 16, which is substantially free of pathogens and / or pathogenic and / or beneficial microorganisms.

18. An isolated BGSC described in any one of claims 1-17, capable of proliferation with limited or minimal differentiation for at least 2 months, at least 6 months or at least 12 months.

19. A population of Brunner's gland stem / progenitor cells (BGSCs) isolated from the duodenum, comprising: at least a portion, or a substantial portion, or a majority of the cells express one or more markers selected from the group consisting of Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCAM, SOX9, and CK7; BGSCs can differentiate into pancreatic and hepatic lineages. Group.

20. 20. The population of claim 19, which is capable of proliferation with limited or minimal differentiation for at least 1 month, at least 2 months, at least 6 months, or at least 12 months.

21. 20. The population of claim 19, wherein the culture conditions that support self-renewal comprise serum-free medium.

22. 20. The population of claim 19, wherein the culture conditions that support self-renewal comprise a medium containing serum.

23. A population of Brunner's gland stem / progenitor cells (BGSCs) isolated from the duodenum, comprising: at least a portion, or a substantial portion, or a majority of the cells express one or more markers selected from the group consisting of Lgr5, NIS, CD44, and CK19; BGSCs can differentiate into pancreatic and hepatic lineages. Group.

24. 24. The population of claim 23, which is capable of proliferation with limited or minimal differentiation for at least 1 month, at least 2 months, at least 6 months, or at least 12 months.

25. 24. The population of claim 23, wherein the culture conditions that support self-renewal comprise serum-free medium.

26. 24. The population of claim 23, wherein the culture conditions that support self-renewal comprise a medium containing serum.

27. A population of Brunner's gland stem / progenitor cells (BGSCs) isolated from the duodenum, comprising: at least a portion, or a substantial portion, or a majority of said cells express both SOX17 and PDX1; BGSCs can differentiate into pancreatic and hepatic lineages. Group.

28. 28. The population of claim 27, which is capable of proliferation with limited or minimal differentiation for at least 1 month, at least 2 months, at least 6 months, or at least 12 months.

29. 28. The population of claim 27, wherein the culture conditions that support self-renewal comprise serum-free medium.

30. 28. The population of claim 27, wherein the culture conditions that support self-renewal comprise a medium containing serum.

31. A population of Brunner's gland stem / progenitor cells (BGSCs) isolated from the duodenum, comprising: at least a portion, or a substantial portion, or a majority of the cells express one or more markers selected from the group consisting of Tra-1-60, Tra-1-81, OCT4, SOX2, NANOG, EpCAM, SOX9, CK7, Lgr5, NIS, CD44, and CK19; BGSCs can differentiate into pancreatic and hepatic lineages. Group.

32. 32. The population of claim 31, which is capable of proliferation with limited or minimal differentiation for at least 1 month, at least 2 months, at least 6 months, or at least 12 months.

33. 32. The population of claim 31 , wherein the culture conditions that support self-renewal comprise serum-free medium.

34. 32. The population of claim 31 , wherein the culture conditions that support self-renewal comprise a medium containing serum.

35. A population described in any one of claims 19-34, which is substantially free of pathogens and / or pathogenic and / or beneficial microorganisms.

36. A population described in any one of claims 19-35, capable of proliferation with limited or minimal differentiation for at least 2 months, at least 6 months, or at least 12 months.

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