Compositions and methods for producing insulin-producing beta cells

Abstract

The present invention provides compositions and methods for producing insulin beta cells from pluripotent stem cells. The compositions and methods of the invention relate to stepwise differentiation when differentiated cells are cultured on lung tissue-derived decellularized scaffolds.

Description

[0001] This application is a divisional application of the invention patent application with a priority date of July 1, 2019, an application date of July 1, 2020, an application number of 202080047874.6, and an invention name of “Compositions and methods for producing insulin beta cells”. Technical Field

[0002] The present invention relates to compositions and methods for generating insulin-producing beta cells from pluripotent stem cells. The compositions and methods of the present invention involve the stepwise differentiation and optional proliferation of cells when cultured on decellularized scaffolds derived from lung tissue. Background Art

[0003] Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) have the unique property of self-renewal and the ability to differentiate into many cell types. They have been proposed for use in generating insulin-producing beta cells for transplantation into patients with diabetes (type 1 diabetes).

[0004] Based on the preliminary work of Rezania et al., 2014, Nat Biotechnol, 32(11): 1121-33, various in vitro differentiation protocols have been developed that can successfully differentiate human ESC / iPSC into single hormone insulin-producing cells that are phenotypically and functionally similar to mature β cells (e.g., Pagliuca et al., 2014, Cell., 159: 428-39; and Russ et al., 2015, EMBO J., 34: 1759-72). An important feature of these protocols is the generation of pancreatic progenitor cells that co-express PDX1 and NKX6.1, thereby increasing the yield of insulin-producing cells. Although the generated insulin-producing cells reverse diabetes after implantation into rodent models and exhibit glucose responsiveness in an in vitro glucose-stimulated insulin secretion (GSIS) assay, perfusion assays indicate that the insulin secretion kinetics and mitochondrial respiration of these cells are functionally immature. This immaturity raises questions regarding the safety of therapeutic use of the cells due to the risk of tumorigenesis of any undifferentiated cells remaining after the differentiation process and possible auto / allogeneic immune responses against the cells after transplantation.

[0005] In the natural environment, β cells are located in aggregates called islets of Langerhans, which are composed of endocrine cells and extracellular matrix (ECM) molecules and are found embedded in pancreatic tissue. In this three-dimensional environment, β cells undergo cell-matrix and cell-cell interactions. Previous studies have shown evidence that the extracellular matrix (ECM) plays a key role in pancreatic cell proliferation and development. It has been shown that cell-matrix interactions improve β cell proliferation, insulin secretion and islet development (Wang et al., 2017, Stem Cells Dev., 26 (6): 394-404; Weber et al., 2008, Tissue Eng Part A, 14: 1959-1968; Hammar et al., 2004, Diabetes, 53: 2034-2041).

[0006] 3D cell culture is an artificially created environment in which cells are allowed to grow or interact with their surrounding environment in all three dimensions. Cells are usually embedded in materials in which they can migrate in all three dimensions and experience cell-matrix interaction and cell-cell contact. 3D cell culture platforms represent an improved in vitro cell culture and differentiation method that can better capture the natural tissue environment. Several studies have used various artificial scaffolds such as PES, PLLA / PVA and PCL / PVA scaffolds to obtain human insulin-producing cells from iPSC (Enderami et al., 2018, ArtifCells Nanomed Biotechnol., 2: 1-8; Abazari et al., 2018, Gene, 5: 50-57; Mansour et al., ArtifCells Nanomed Biotechnol., 2: 1-7). However, such scaffolds have limitations such as non-biodegradable, low potential for attracting cells infiltrated into the scaffold structure and / or poor cell attachment due to hydrophobicity. Such scaffolds lack the structure and characteristics of the natural tissue microenvironment.

[0007] Sionov et al., 2015, Tissue Eng Part A, 21(21-22):2691-702, reported the preparation of endocrine micropancreas (EMP) composed of decellularized pancreas-derived or lung-derived microscaffolds seeded with intact or enzymatically dissociated human pancreatic islets.

[0008] US 7,297,540 discloses the use of micro-organs (MOs) as a (continuous) source of adult stem cells, wherein the MOs are tissue explants that retain the basic cell-cell, cell-matrix, and cell-stroma architecture of the original tissue; the use of the natural multi-signal microenvironment of the micro-organs to induce stem cell differentiation; and the use of the natural three-dimensional structure of the MO decellularized matrix as a scaffold for seeding adult or embryonic stem cells.

[0009] US Pat. No. 10,093,896 discloses a composition of matter comprising a devitalized, decellularized tissue-derived scaffold seeded with differentiated cells, particularly pancreatic islets or pancreatic islet cells, wherein the cells can maintain cell-specific function or structure in culture on the scaffold. Also disclosed are methods for producing the composition and uses thereof.

[0010] There is a need to improve the quality, function and maturity of beta cells generated in vitro from pluripotent stem cells. Summary of the Invention

[0011] The present invention provides compositions, methods, and kits for generating high-quality human beta cells differentiated from human pluripotent stem cells. The compositions, methods, and kits of the present invention utilize stepwise differentiation, wherein multiple differentiation factors are sequentially applied while the differentiated cells are cultured on a decellularized lung tissue-derived scaffold, also known as an acellular micro-organ matrix (MOM). All differentiation steps may or may not be accompanied by cell proliferation.

[0012] The present inventors have utilized a stepwise differentiation process typically performed in 2D cell culture, in which multiple differentiation factors are sequentially applied. The inventors have found that by performing this process while culturing differentiated cells on a three-dimensional (3D) scaffold derived from lung tissue, beta cells with improved function and maturity can be obtained.

[0013] The present invention discloses for the first time the production of beta cells by performing cell differentiation on non-homologous tissue scaffolds, i.e., tissue scaffolds derived from tissues other than the pancreas, particularly lung tissue-derived scaffolds. Previous reports have shown that scaffolds derived from natural tissues influence the differentiation of cells cultured thereon, directing them toward the tissue from which the scaffold originated. Surprisingly, the inventors have discovered that beta cells can be obtained by differentiation on lung tissue-derived scaffolds, characterized by enhanced insulin production compared to cells produced in two-dimensional cell culture using the same differentiation procedure. Thus, the lung origin of the scaffold not only does not negatively impact the differentiation process, but on the contrary, it produces beta cells with improved properties.

[0014] Advantageously, the lung tissue-derived 3D scaffold provides a vast surface area lined with a basement membrane, resembling the natural microenvironment of beta cells in the pancreas. Lung tissue-derived scaffolds were found to be even more advantageous than pancreatic-derived scaffolds. Pancreatic-derived scaffolds primarily contain a matrix derived from the exocrine portion of the pancreas, rather than the endocrine portion containing the islets of Langerhans, as the endocrine portion only accounts for approximately 1-2% of the pancreas. The endocrine portion of the pancreas is characterized by a dense vasculature surrounded by a basement membrane. Therefore, the lung tissue-derived scaffold better mimics the natural microenvironment of pancreatic islet tissue.

[0015] As disclosed herein, the beta cells produced by differentiation on lung tissue-derived scaffolds are fully mature (e.g., they express MAFA transcription factors that are expressed in adult beta cells and not present in developing beta cells and other pancreatic cells) and respond to glucose stimulation to secrete insulin at higher levels than cells produced by differentiation in 2D cell culture. Importantly, the beta cells obtained as disclosed herein secrete insulin in a regulated biphasic manner in response to glucose stimulation, as demonstrated by a dynamic glucose-responsive insulin secretion assay that studies changes in insulin secretion over time in response to glucose. This is in contrast to cells produced by differentiation in 2D cell culture, as illustrated below. The regulated insulin secretion in response to glucose indicates that the obtained cells are functionally mature. In addition, differentiation on lung tissue-derived scaffolds as disclosed herein produces more cells that express insulin than differentiation in 2D culture. Therefore, the differentiation method disclosed herein provides a higher yield of fully differentiated beta cells and increased and improved insulin secretion.

[0016] According to certain embodiments, the lung tissue-derived scaffold is from a non-human source (e.g., porcine) and the differentiated cells are human. Surprisingly, as exemplified below, the non-human lung tissue-derived scaffold has no negative effect on the differentiation of human cells of the pancreatic lineage into beta cells.

[0017] According to one aspect, the present invention provides a method for producing an insulin-producing β-cell population, the method comprising:

[0018] (a) seeding pancreatic lineage progenitor cells onto a devitalized, decellularized, lung tissue-derived three-dimensional scaffold; and

[0019] (b) differentiating the pancreatic lineage progenitor cells into β cells by stepwise differentiation comprising sequentially applying a plurality of differentiation factors, wherein the stepwise differentiation is performed on the lung tissue-derived three-dimensional scaffold such that the cells remain on the scaffold throughout the differentiation process, thereby generating a population of insulin-producing β cells.

[0020] In certain embodiments, the method further comprises differentiating the pluripotent stem cells into progenitor cells of the pancreatic lineage in 2D cell culture prior to step (a).

[0021] In certain embodiments, the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic endoderm cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2) and endocrine precursor cells (also referred to as pancreatic endocrine progenitor cells). Each possibility represents an independent embodiment of the present invention.

[0022] In certain embodiments, the progenitor cells of the pancreatic lineage are selected from the group consisting of definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic endoderm cells, and endocrine precursor cells. Each possibility represents a separate embodiment of the present invention.

[0023] In other embodiments, the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2) and endocrine precursor cells (also known as pancreatic endocrine progenitor cells). Each possibility represents an independent embodiment of the present invention.

[0024] In certain specific embodiments, the progenitor cells of the pancreatic lineage are pancreatic endoderm cells. In certain embodiments, the progenitor cells of the pancreatic lineage are pancreatic endoderm cells, and the sequential application of multiple differentiation factors comprises:

[0025] (i) culturing the scaffold seeded with pancreatic endoderm cells in a culture medium containing one or more endocrine precursor cell differentiation factors to obtain endocrine precursor cells on the scaffold; and

[0026] (ii) culturing the scaffold with the endocrine precursor cells in a culture medium containing one or more β cell differentiation factors to obtain β cells on the scaffold.

[0027] In certain embodiments, the progenitor cells of the pancreatic lineage are pancreatic endoderm cells, and the method further comprises differentiating the pluripotent stem cells into pancreatic endoderm cells in 2D cell culture prior to step (a).

[0028] In certain embodiments, the method further comprises seeding at least one type of supporting cells selected from endothelial cells and mesenchymal stem cells (MSCs) on the scaffold, and performing the differentiation process while co-culturing the supporting cells with the differentiated cells on the scaffold.

[0029] In certain specific embodiments, the method further comprises seeding both endothelial cells and mesenchymal stem cells (MSCs) on the scaffold, and performing the differentiation process while co-culturing the endothelial cells and MSCs with the differentiated cells on the scaffold.

[0030] According to another aspect, the present invention provides a composition for producing insulin-producing β cells, the composition comprising:

[0031] (i) a devitalized, decellularized, lung tissue-derived three-dimensional scaffold; and

[0032] (ii) pancreatic lineage progenitor cells seeded on the scaffold.

[0033] In certain embodiments, the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic endoderm cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2) and endocrine precursor cells (also referred to as pancreatic endocrine progenitor cells). Each possibility represents an independent embodiment of the present invention.

[0034] In certain embodiments, the progenitor cells of the pancreatic lineage are selected from the group consisting of definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic endoderm cells, and endocrine precursor cells. Each possibility represents a separate embodiment of the present invention.

[0035] In other embodiments, the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2) and endocrine precursor cells (also known as pancreatic endocrine progenitor cells). Each possibility represents an independent embodiment of the present invention.

[0036] In certain specific embodiments, the pancreatic lineage progenitor cell is a pancreatic endoderm cell.

[0037] In certain embodiments, the composition further comprises at least one type of supporting cells selected from endothelial cells and mesenchymal stem cells (MSCs) seeded on the scaffold.

[0038] In certain specific embodiments, the composition further comprises both endothelial cells and MSCs seeded on the scaffold.

[0039] According to another aspect, the present invention provides a method for producing insulin-producing beta cells, the method comprising:

[0040] (a) providing a devitalized, decellularized, lung tissue-derived three-dimensional scaffold seeded with pancreatic lineage progenitor cells according to the present invention; and

[0041] (b) differentiating the pancreatic lineage progenitor cells into β cells by stepwise differentiation, wherein the stepwise differentiation is performed on the lung tissue-derived three-dimensional scaffold such that the differentiated cells remain on the scaffold throughout the differentiation process.

[0042] In certain embodiments, in step (a), the scaffold is further seeded with at least one type of supporting cells selected from endothelial cells and mesenchymal stem cells (MSCs), and wherein the stepwise differentiation is performed on the scaffold in the presence of the supporting cells.

[0043] In certain specific embodiments, in step (a), the scaffold is further seeded with both endothelial cells and mesenchymal stem cells (MSCs), and wherein the stepwise differentiation is performed on the scaffold in the presence of the endothelial cells and MSCs.

[0044] According to another aspect, the present invention provides a kit for producing insulin-producing β cells, the kit comprising:

[0045] (i) Devitalized, decellularized, lung tissue-derived three-dimensional scaffolds;

[0046] (ii) a plurality of differentiation factors for effecting stepwise differentiation of progenitor cells of the pancreatic lineage into β cells; and

[0047] (iii) an instruction manual that specifies the technical instructions for performing step-by-step differentiation on the scaffold such that the cells remain on the scaffold throughout the differentiation process.

[0048] In certain embodiments, the scaffold is pre-seeded with progenitor cells of the pancreatic lineage selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic endoderm cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2) and endocrine precursor cells (also referred to as pancreatic endocrine progenitor cells). Each possibility represents an independent embodiment of the present invention.

[0049] In certain embodiments, the scaffold is pre-seeded with progenitor cells of the pancreatic lineage selected from definitive endoderm cells, gastrula cells, posterior foregut cells, pancreatic endoderm cells, and endocrine precursor cells. Each possibility represents a separate embodiment of the present invention.

[0050] In certain embodiments, the scaffold is pre-seeded with progenitor cells of the pancreatic lineage selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2) and endocrine precursor cells (also referred to as pancreatic endocrine progenitor cells). Each possibility represents an independent embodiment of the present invention.

[0051] In some embodiments, the instruction manual further specifies technical instructions for seeding the progenitor cells of the pancreatic lineage on the scaffold prior to the stepwise differentiation. In some embodiments, the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic endoderm cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2), and endocrine precursor cells. Each possibility represents an independent embodiment of the present invention.

[0052] In certain embodiments, the instruction manual further specifies technical instructions for seeding progenitor cells of the pancreatic lineage, wherein the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic endoderm cells, and endocrine precursor cells. Each possibility represents an independent embodiment of the present invention.

[0053] In other embodiments, the instruction manual further specifies technical guidance for seeding progenitor cells of the pancreatic lineage on the scaffold prior to the stepwise differentiation, wherein the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2) and endocrine precursor cells (also referred to as pancreatic endocrine progenitor cells). Each possibility represents an independent embodiment of the present invention.

[0054] In certain embodiments, the kit further comprises a plurality of differentiation factors for performing stepwise differentiation of pluripotent stem cells into progenitor cells of the pancreatic lineage in 2D cell culture prior to seeding on the scaffold. In certain embodiments, the instruction manual further specifies technical instructions for performing stepwise differentiation of pluripotent stem cells into progenitor cells of the pancreatic lineage in 2D cell culture prior to seeding on the scaffold.

[0055] In certain embodiments, the kit further comprises at least one type of supporting cells selected from endothelial cells and mesenchymal stem cells (MSCs) seeded on the scaffold.

[0056] In certain specific embodiments, the kit further comprises both endothelial cells and MSCs seeded on the scaffold.

[0057] In certain embodiments, the kit further comprises one or more cell culture media.

[0058] According to another aspect, the present invention provides a method for producing an artificial micro-organ, the method comprising:

[0059] (a) seeding pancreatic lineage progenitor cells onto a devitalized, decellularized, lung tissue-derived three-dimensional scaffold; and

[0060] (b) differentiating the pancreatic lineage progenitor cells into insulin-producing β cells by stepwise differentiation, wherein a plurality of differentiation factors are sequentially applied, wherein the stepwise differentiation is performed on the lung tissue-derived three-dimensional scaffold such that the cells remain on the scaffold throughout the differentiation process,

[0061] Thereby, an artificial micro-organ is obtained, comprising insulin-producing beta cells cultured on said lung tissue-derived three-dimensional scaffold and maintaining glucose-responsive insulin secretion when cultured on said scaffold.

[0062] In certain embodiments, the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic endoderm cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2) and endocrine precursor cells (also referred to as pancreatic endocrine progenitor cells). Each possibility represents an independent embodiment of the present invention.

[0063] In certain embodiments, the progenitor cells of the pancreatic lineage are selected from the group consisting of definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic endoderm cells, and endocrine precursor cells. Each possibility represents a separate embodiment of the present invention.

[0064] In other embodiments, the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2) and endocrine precursor cells (also known as pancreatic endocrine progenitor cells). Each possibility represents an independent embodiment of the present invention.

[0065] In certain embodiments, the method further comprises seeding at least one type of supporting cells selected from endothelial cells and mesenchymal stem cells (MSCs) on the scaffold, and performing the differentiation process while co-culturing the supporting cells with the differentiated cells on the scaffold.

[0066] In certain specific embodiments, the method further comprises seeding both endothelial cells and mesenchymal stem cells (MSCs) on the scaffold, and performing the differentiation process while co-culturing the endothelial cells and MSCs with the differentiated cells on the scaffold.

[0067] According to another aspect, the present invention provides an artificial micro-organ produced by the method of the present invention, comprising a lung tissue-derived three-dimensional scaffold and insulin-producing β cells cultured thereon.

[0068] According to another aspect, the present invention provides a method for treating diabetes in a subject in need thereof, the method comprising implanting in the subject a therapeutically effective amount of an artificial micro-organ produced by the method of the present invention.

[0069] In certain embodiments, the diabetes is type I diabetes. In other embodiments, the diabetes is type II diabetes. In other embodiments, the diabetes is caused by pancreatitis or other types of pancreatic damage.

[0070] In certain embodiments, the source of pancreatic lineage progenitor cells is autologous to the subject being treated.

[0071] Other objects, features and advantages of the present invention will become apparent from the following description, examples and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1. Differentiation protocol. (A) Standard differentiation protocol in 2D culture; (B) Differentiation protocol according to Example 1, below.

[0073] Figure 2 . A differentiation program from pluripotent stem cells (iPSCs) via definitive endoderm, gastrulation, posterior foregut, pancreatic endoderm and endocrine precursor cells (endo. cells) to insulin-producing β cells (IBs) according to Example 2 below.

[0074] Figure 3 FACS analysis of the expression of the definitive endoderm markers CXCR4 and c-kit (on day 4 of the differentiation protocol according to Example 2 below).

[0075] Figure 4. Insulin expression in β cells differentiated as described in Example 2, below. (A) qPCR analysis of INS mRNA expression in cells differentiated in 2D culture; (B) Insulin / DAPI staining of cells differentiated in 2D culture, 20x magnification; (C) Insulin / DAPI staining of cells differentiated on MOM, 20x magnification.

[0076] FIG5. Glucose-stimulated insulin secretion (GSIS) assay of β cells differentiated as described in Example 2, below. (A) Cells seeded on MOM at day 15 and allowed to complete differentiation on MOM; (B) Cells grown in 2D culture and clustered between days 21 and 24; (C) Cells grown as a monolayer in a 2D culture plate. DETAILED DESCRIPTION

[0077] The present invention relates to the use of a natural matrix derived from decellularized lung tissue to differentiate pluripotent stem cells into functional insulin-producing beta cells. The lung tissue-derived matrix retains its complex organizational structure, which is similar in complexity to the structural microenvironment of beta cells in the pancreas, with a large surface area lined by a basement membrane.

[0078] As disclosed herein, the lung tissue-derived scaffold is seeded with progenitor cells of the pancreatic lineage, and the progenitor cells are induced to differentiate into beta cells through a stepwise differentiation process in which multiple differentiation factors are sequentially applied. The inventors have shown that by performing the differentiation process on the scaffold according to the present invention, improved beta cells can be obtained compared to beta cells obtained by differentiation in 2D culture.

[0079] Surprisingly, the lung tissue origin of the scaffolds had no negative impact on beta cell differentiation and even resulted in improved beta cell differentiation.

[0080] bracket

[0081] As used herein, the term "scaffold" refers to a three-dimensional matrix on which cells can be cultured. Scaffolds according to the present invention are prepared from tissue explants with microscopic thickness, also referred to as "micro-organs" (MOs). Micro-organs retain the basic cell-cell, cell-matrix, and cell-stroma architecture of the original tissue. In order to obtain devitalized, acellular tissue-derived scaffolds according to the present invention, the micro-organ explants are treated to remove cells, producing a "micro-organ-derived matrix," abbreviated as "MOM."

[0082] As used herein, the terms "devitalization" and "decellularization" refer to tissues or structures that have been treated to remove living cellular material (including genetic material). Devitalized, decellularized micro-organs are micro-organ explants that essentially no longer contain any cells or other living material, do not reproduce, do not require a supply of nutrients or gases, and are essentially inert. In certain embodiments, cells are killed and then removed from the tissue, but cells can also be removed without prior killing. Dead cells can fall off in a liquid, or can be removed chemically or mechanically.

[0083] Tissue devitalization methods suitable for the present invention include heat inactivation, radiation, chemical cell stripping by alkali or acid treatment, hypertonic or hypotonic inactivation, mechanical inactivation, detergents, organic solvents, combinations thereof, and the like. It should be understood that since the purpose of devitalization is to provide a decellularized scaffold for cell culture, the devitalization methods suitable for the present invention are selected so as not to destroy the structural and biochemical integrity of the decellularized components of the micro-organ. US 7,297,540 and US 10,093,896 describe exemplary but non-limiting methods for devitalizing cells and removing cells from micro-organs. In an exemplary method, the micro-organ is treated with ammonium hydroxide and a detergent (SDS) and washed thoroughly in saline to remove cellular material. Alternatively, the micro-organ can be treated with 1-2M NaCl and a detergent (e.g., Triton, SDS, etc.). In another embodiment, the micro-organ from the cryopreserved tissue is washed repeatedly and extensively in cold water or 1M NaCl, then with a detergent solution, and finally washed and stored in water with or without a preservative (e.g., antibiotic) before use. In yet another embodiment, the devitalized, decellularized micro-organ matrix is ​​stored frozen until use. Alternatively, the devitalized, decellularized micro-organ matrix is ​​dried (eg, freeze-dried) and rehydrated in water or culture medium before use.

[0084] The dimensions of the MOM are selected to provide sufficient diffusion of nutrients and gases such as oxygen to each cell seeded in the three-dimensional structure and diffusion of cellular waste products out of the MOM to minimize cell toxicity and concomitant death caused by waste products located in the MOM.

[0085] Typically, the dimensions of the devitalized, decellularized, lung tissue-derived three-dimensional scaffold (MOM) according to the present invention are selected such that the deepest point within the scaffold is at least about 100 microns and no more than about 225 microns from the nearest surface of the scaffold. Thus, when populated with cells, the cells at the deepest point within the scaffold are at least about 100 microns and no more than about 250 microns from the cells at the nearest surface formed on the scaffold.

[0086] Thus, in certain embodiments, the dimensions of the decellularized three-dimensional scaffold are selected such that the deepest point within the scaffold is at least about 100 microns and no more than about 250 microns from the nearest surface of the scaffold.

[0087] In one embodiment, the support is a devitalized, decellularized tissue slice with a thickness of 100-500 microns. In another embodiment, the support is a devitalized, decellularized tissue slice with a thickness of approximately 300 microns. In another embodiment, the support is a devitalized, decellularized tissue slice with a thickness of approximately 300 microns. In another embodiment, the support is a devitalized, decellularized tissue slice with a thickness of approximately 300 microns, 5-12mm width, 5-12mm length (comprising each value in the described range). In another embodiment, the support is a devitalized, decellularized tissue slice with a thickness of approximately 300 microns, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50mm length, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50mm width. Every possibility represents an independent embodiment of the present invention.

[0088] Micro-organs suitable for preparing the micro-organ matrices of the present invention can be prepared from lung tissue derived from any animal, preferably a mammal. In certain specific embodiments, the lung tissue is porcine lung tissue. In certain embodiments, the micro-organs are prepared from lungs excised from a recently killed animal under sterile conditions, kept on ice, rinsed with culture medium (e.g., Ringer or DMEM), and cut into 300 micron slices using a tissue slicer. In another embodiment, the micro-organs are prepared from freshly frozen frozen tissue or frozen tissue sections, which are thawed to -2 to -20°C in preparation for sectioning and sectioned using, for example, a pre-cooled tissue slicer or microtome.

[0089] An exemplary procedure for preparing devitalized, decellularized three-dimensional scaffolds is described in detail in US 10,093,896. Briefly, the procedure comprises the following steps:

[0090] First, lung tissue-derived micro-organs are prepared from, for example, porcine lungs. Adult animals are sacrificed and the lungs removed under sterile conditions. The lungs are kept on ice, rinsed, and sliced ​​into 300 μm sections using a tissue slicer to form micro-organs.

[0091] The MO is decellularized and then sterilized for 30 min with, for example, 0.1% PAA and washed 3 times with DDW before storage for 30 min each. In order to decellularize, the MO can be treated with 0.67% ammonium hydroxide in 0.5% SDS. After all cell matter is removed, the remaining extracellular (inactivation, decellularization) material is fully cleaned, for example, in PBS replaced 5 times, after which the matrix is ​​ready to be used as a three-dimensional scaffold. Cells can then be seeded on the MOM. Optionally, the MOM can be frozen at -20 ° C before needing to be used, and melted and cleaned before being used for cultivation, for example, washed 3 times in PBS and washed 2 times in culture medium.

[0092] Alternatively, MO decellularization can be performed by soaking the MO in one of the following solutions for 45 min: (a) 10-50 mM NH4OH + 0.2-3% TritonX-100; (b) 1-2 M NaCl; (c) 1-2 M NaCl + 0.2-3% TritonX-100; or (d) 1-2 M NaCl + 0.01-0.1% SDS. The resulting inactivated, decellularized MOM can then be washed, for example, in sterile distilled H2O for 5 x 15 min. At this stage, the resulting MOM can be frozen and stored at -80°C before use, or rinsed, for example, in PBS for 5 x 15 min.

[0093] Alternatively, the organ (lung) is freshly excised, washed in water, clean, and optionally stored on ice for 1.5 hours at the most. Before sectioning, the organ is cut into 12mm × 12 × 20-40mm strips and frozen at -20°C to -80°C, and kept freezing until needed for MOM preparation. Before sectioning, the organ strips are balanced to -2 to -10°C and then precooled tissue slicer is used to be cut into 200 to 500 μm × 12 × 12mm sections.

[0094] MOMs can then be prepared from the slices as follows:

[0095] a. Wash the sections in cold sterile DDW for 1 hour, changing the water every 15 minutes (approximately 50 ml DDW per wash);

[0096] b. The sections were washed in sterile DDW at room temperature (rt) for 4 hours, with water changed every 20 minutes (approximately 50 ml DDW per wash); and

[0097] c. Store sections in a minimal volume of DDW at -80°C until needed for plating.

[0098] or:

[0099] a. Continue washing the sections in sterile DDW at room temperature for 4 hours, changing the water every 20 minutes (approximately 50 ml DDW per wash); and

[0100] b. Keep the slices in DDW at 4°C overnight;

[0101] c. Sections were then stored in a minimal volume of DDW at -80°C until needed for plating.

[0102] or:

[0103] a. Continue washing the sections in sterile DDW at room temperature for 4 hours, changing the water every 20 minutes (approximately 50 ml DDW per wash); and

[0104] b. Keep the slices in DDW at 4°C overnight;

[0105] c. The sections were stored in PBS containing 10× antibiotics and cells were plated on the resulting MOMs 1 to 10 days later.

[0106] or:

[0107] a. Place the sections in 1M NaCl for 1 hour, changing the solution three times for 20 minutes each.

[0108] b. Transfer the sections to a 0.5% Triton solution in DDW for 3 hours, changing the solution every 30 minutes.

[0109] c. Wash the sections with DDW for 3 x 15 min; and

[0110] d. Sections were stored in PBS containing 10× antibiotics, and cells were plated on the resulting MOMs 1 to 10 days later.

[0111] or:

[0112] a. Place the sections in 1M NaCl for 1 hour, changing the solution three times for 20 minutes each.

[0113] b. Transfer the sections to a 0.5% Triton solution in H2O for 3 hours, changing the solution every 30 minutes.

[0114] c. Wash the sections with H2O for 5 × 15 min; and

[0115] d. Store sections in a minimal volume of DDW at -80°C until needed for plating.

[0116] The method of cell seeding on support is known in the art.Can be by static loading, by being seeded in stirred flask bioreactor, in rotating wall container or use the direct perfusion in the culture medium of cell in bioreactor, by cell seeding on described support.Described cell can be directly seeded on micro-organ matrix support.The cell in culture medium can be adsorbed on the inner surface and outer surface of described support.

[0117] Cells can be seeded at various densities. In some embodiments, cells are seeded at a density of approximately 1×10 4 to about 1×10 6 In another embodiment, cells are seeded at a density of 2×10 cells per 2-4 micro-organ matrices. 5 to about 1×10 6 In another embodiment, cells are seeded at a density of 2×10 cells per 5-7 micro-organ matrices.5 to about 1×10 6 The cells were seeded at a density of 100 cells.

[0118] In certain embodiments, multiple MOMs are cultured in a single cell culture vessel.

[0119] The MOM-cell culture can be maintained in any suitable culture container, such as a 12-well microplate, and can be maintained at 37° C. in 5% CO 2 .

[0120] Cell populations and differentiation programs

[0121] Cell differentiation is the process by which unspecialized (uncommitted) or inadequately specialized cells acquire the characteristics of specialized cells.

[0122] As used herein, the lineage of a cell defines which cells it can give rise to.

[0123] The in vitro differentiation of pluripotent stem cells into insulin-secreting β cells follows a series of developmental stages that mimic pancreatic organogenesis, which begins with differentiation into definitive endoderm (DE) and then sequentially differentiates through several stages, referred to as "stepwise differentiation," in which a variety of differentiation factors are applied sequentially until β cells are obtained. At each stage, a culture medium containing suitable differentiation factors is added to induce the cells to differentiate into the next stage, and the culture medium is then replaced with a culture medium containing the factors required for the cells to differentiate into the next stage, and so on. Each stage is characterized by one or more markers expressed by the cells.

[0124] As used herein, the term "differentiation factor" refers to a molecule, such as a small molecule, protein, or peptide, that induces a cell to differentiate into a desired cell type. For example, a "definitive endoderm differentiation factor" refers to a differentiation factor that induces differentiation into definitive endoderm cells. A "gastrin differentiation factor" refers to a differentiation factor that induces differentiation into gastrocytes, and so on.

[0125] As used herein, a "marker" is a nucleic acid or polypeptide molecule that is differentially expressed in a cell of interest. In this context, differential expression means that the level of a positive marker is increased and the level of a negative marker is decreased compared to cells at a different developmental stage. The detectable level of the nucleic acid or polypeptide marker in the cell of interest is increased or decreased sufficiently compared to cells at a different developmental stage so that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

[0126] As used herein, a cell is "positive" for a particular marker when the marker is detected in the cell. A cell is "negative" for the particular marker when the marker is not detected in the cell. Expression of a marker in a cell population can be determined qualitatively, for example, using immunostaining techniques, or can be determined quantitatively, for example, using FACS, where the percentage of cells in the population expressing the marker can be determined.

[0127] The cells according to the invention are typically human cells.

[0128] "Pluripotent stem cells" are stem cells that have the potential to differentiate into cells of all three germ layers, namely, endoderm, mesoderm, and ectoderm. Markers characteristic of pluripotent stem cells include one or more of the following: Oct4, Nanog, Sox2, Klf4, c-myc, CDH1. Other markers unique to pluripotent stem cells include ABCG2, Cripto, FOXD3, Connexin 43, Connexin 45, hTERT, UTF1, ZFP42 (Rex1), SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81. Markers characteristic of pluripotent stem cells are listed, for example, at the following website: www.rndsystems.com / research-area / embryonic-and-induced-pluripotent-stem-cell-markers.

[0129] Pluripotent stem cells can be readily expanded in culture using a variety of feeder layers or using containers coated with matrix proteins. The containers can be coated with extracellular matrix components, such as those derived from the basement membrane or that can form part of the adhesion molecule receptor-ligand coupling. For example, the container can be coated with the Matrigel® under the trademark TM Matrigel is a reconstructed basement membrane sold under TM is a soluble preparation from Engelbreth-Holm Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane. Other extracellular matrix components and component mixtures known in the art are suitable alternatives.

[0130] The pluripotent stem cells can be plated on the substrate in a suitable distribution and in the presence of a culture medium that promotes cell survival, proliferation, and retention of desired characteristics. Suitable culture media include, for example, feeder-free, serum-free, and complete cell culture media, such as mTeSR TMPluripotent cells can be easily removed from culture plates using enzymes, mechanical means, or using various calcium chelators such as EDTA (ethylenediaminetetraacetic acid). Alternatively, pluripotent cells can be expanded in suspension in the absence of any matrix proteins or feeder layers.

[0131] In certain embodiments, the pluripotent stem cells are embryonic stem cells. In other embodiments, the pluripotent stem cells are not embryonic stem cells. In yet other embodiments, the pluripotent stem cells are induced pluripotent stem cells.

[0132] The type of pluripotent stem cell that can be used includes established pluripotent cell lines, which are derived from tissues formed after pregnancy, including pre-embryonic tissue (such as blastocyst), embryonic tissue or any time during pregnancy, usually but not necessarily fetal tissue obtained before about 10 to 12 weeks of pregnancy. Non-limiting examples are established lines of human embryonic stem cells (hESC) or human embryonic germ cells, such as human embryonic stem cell lines HES-2, H1, H7 and H9. Cells obtained from pluripotent stem cell colonies cultivated in the absence of feeder cells are also suitable. Human embryonic cells are preferably prepared without destroying human embryos, as described in, for example, Chung et al., 2008, Cell Stem Cell., 2 (2): 113-7.

[0133] Induced pluripotent stem cells (iPSCs) or reprogrammed pluripotent cells are also suitable, which can be derived from adult somatic cells using a large number of pluripotency-related transcription factors such as OCT4, NANOG, Sox2, KLF4, and forced expression of ZFP42 (Loh et al., Annu Rev Genomics Hum Genet, 2011, 12: 165-185). The cells can be derived from autologous or allogeneic sources.

[0134] When pluripotent stem cells are to functional beta cell differentiation, they are differentiated by various stages, and each stage can be characterized by the presence or absence of specific markers. The differentiation of cells in these stages is achieved by specific culture conditions, including the presence or absence of certain factors added to culture medium. Suitable growth medium includes chemically defined culture medium containing sufficient amounts of vitamins, minerals, salts, glucose and amino acids. Examples are provided hereinafter.

[0135] In certain embodiments, differentiation from pluripotent stem cells to β cells comprises: differentiating the pluripotent stem cells into definitive endoderm cells; differentiating the definitive endoderm cells into gastrula cells; differentiating the gastrula cells into posterior foregut cells; differentiating the posterior foregut cells into pancreatic endoderm cells; differentiating the pancreatic endoderm cells into endocrine precursor cells (also known as pancreatic endocrine progenitor cells); and differentiating the endocrine precursor cells into β cells.

[0136] In certain embodiments, differentiation from pluripotent stem cells to β cells comprises: differentiating pluripotent stem cells into definitive endoderm cells; differentiating definitive endoderm cells into enterocytes; differentiating enterocytes into posterior foregut cells; differentiating posterior foregut cells into pancreatic progenitor cells 1; differentiating pancreatic progenitor cells 1 into pancreatic progenitor cells 2; differentiating pancreatic progenitor cells 2 into endocrine precursor cells (also known as pancreatic endocrine progenitor cells); and differentiating endocrine precursor cells into β cells.

[0137] When used in this article, " progenitor cell " refers to undifferentiated cell, and it has the cell phenotype that is more primitive (for example, compared with the cell of complete differentiation, be in the earlier step along developmental pathway or progress) relative to the cell that it can produce by differentiation.Some progenitor cell can produce the offspring that can be divided into more than a kind of cell type. It is believed that progenitor cell is devoted to specific differentiation pathway, and finally breaks up along this approach under suitable conditions. Progenitor cell according to the present invention is the progenitor cell of pancreatic pedigree. In some embodiments, the progenitor cell of described pancreatic pedigree is selected from definitive endoderm cell, primitive enterocyte, posterior foregut cell, pancreatic endoderm cell and endocrine precursor cell (also referred to as pancreatic endocrine progenitor cell). In other embodiments, the progenitor cell of pancreatic pedigree according to the present invention is selected from definitive endoderm cell, primitive enterocyte, posterior foregut cell, pancreatic progenitor cell 1, pancreatic progenitor cell 2 and endocrine precursor cell (also referred to as pancreatic endocrine progenitor cell).

[0138] "Definitive endoderm cells" (DE cells) are cells that form the gastrointestinal tract and its derivatives, such as the pancreas or liver. Characteristic markers of definitive endoderm cells include one or more of the following markers: CXCR4, c-kit, PDX1, FoxA2, ​​GP2, Sox17, and GSC. The definitive endoderm cells according to the present invention express one or more, preferably two or more, more preferably three or more, and even more preferably all of the above markers. In certain embodiments, the definitive endoderm cells according to the present invention express markers including CXCR4 and c-kit. Other characteristic markers of definitive endoderm cells include HNF3β, GATA4, Cerberus, OTX2, goosecoid, CD99, and MIXL1. Characteristic markers of definitive endoderm cells are listed, for example, at the following website: www.rndsystems.com / research-area / early-endodermal-lineage-markers.

[0139] Differentiation of pluripotent stem cells into definitive endoderm cells can be performed by plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix and culturing the pluripotent stem cells in a chemically defined serum-free and animal component-free complete medium containing activin A to obtain definitive endoderm cells. Exemplary procedures are described in the Examples section below.

[0140] In certain embodiments, differentiation from pluripotent stem cells into definitive endoderm cells can be performed by a process comprising the following steps:

[0141] (i) plating the pluripotent stem cells on a low-attachment tissue culture substrate, and culturing the pluripotent stem cells in a chemically defined serum-free complete medium containing BMP4 to obtain embryoid bodies;

[0142] (ii) collecting the embryoid bodies and culturing them in a chemically defined serum-free complete medium containing BMP4, bFGF and activin A; and

[0143] (iii) collecting the embryoid bodies from step (ii) and culturing them in a chemically defined serum-free complete medium containing VEGF, activin A and bFGF to obtain definitive endoderm cells.

[0144] In certain embodiments, differentiation from pluripotent stem cells into definitive endoderm cells is performed by a process comprising the following steps:

[0145] (i) plating the pluripotent stem cells on a low-attachment tissue culture substrate, and culturing the pluripotent stem cells in a chemically defined serum-free complete medium comprising glutamine, ascorbic acid, monothioglycerol (MTG) and BMP4 for 24 hours to obtain embryoid bodies;

[0146] (ii) collecting the embryoid bodies and culturing them in a chemically defined serum-free complete medium containing glutamine, ascorbic acid, monothioglycerol (MTG), BMP4, bFGF, and activin A for 48-72 hours; and

[0147] (iii) collecting the embryoid bodies from step (ii) and culturing them in a chemically defined serum-free complete medium containing glutamine, ascorbic acid, monothioglycerol (MTG), VEGF, activin A and bFGF for at least 24 hours to obtain definitive endoderm cells.

[0148] "Primitive gut cells" (PG cells), also known as "primitive gut tube cells" or "gut tube cells," are cells derived from the definitive endoderm that express characteristic markers comprising one or more of the following markers: FoxA1, HNF1-β (HNF1B), HNF4-α (HNF4A). The primitive gut cells according to the present invention express one or more, preferably two or more, and more preferably all of the above markers. Another characteristic marker of primitive gut cells is HNF3-β (FOXA2). Primitive gut cells can give rise to endoderm organs such as the liver, pancreas, stomach, and intestine.

[0149] Differentiation of definitive endoderm into gastrulation cells can be achieved by supplementing the cells with ITS-X, GlutaMAX, TM The cells are cultured in a chemically defined medium supplemented with B27 and / or B27 and containing one or more gastrulation differentiation factors. Examples of gastrulation differentiation factors include KGF, FGF7, and vitamin C. Each possibility represents a separate embodiment of the present invention. In certain exemplary embodiments, the gastrulation differentiation factor is KGF.

[0150] "Posterior foregut cells" (PFG cells), also known as "hindgut tube cells," are cells that express characteristic markers including one or more of the following: PDX1, HNF6, SOX9, PROX1. Posterior foregut cells can give rise to the posterior stomach, pancreas, liver, and part of the duodenum.

[0151] Differentiation of primitive enterocytes into posterior foregut cells can be achieved by supplementing the cells with substances such as ITS-X, GlutaMAX TM The cells are cultured in a chemically defined medium supplemented with B27 and / or B27 and containing one or more posterior foregut differentiation factors. Examples of combinations of posterior foregut differentiation factors include KGF+SANT-1+retinoic acid (RA)+LDN-193189+PdBU; KAAD-cyclopamine+retinoic acid (RA)+LDN-193189; and FGF7+vitamin C+TPB+SANT. Each possibility represents an independent embodiment of the present invention. In certain exemplary embodiments, the posterior foregut differentiation factor is KGF+SANT-1+retinoic acid (RA)+LDN-193189+PdBU.

[0152] In certain embodiments, the enterocytes are differentiated into pancreatic endoderm cells, which are then differentiated into endocrine precursor cells, and finally into beta cells. This procedure is exemplified in Example 2 below. One skilled in the art will recognize that an alternative approach involving two sub-stages as described below can be used: first, the enterocytes are differentiated into pancreatic progenitor cells 1 (PP1 cells), followed by differentiation into pancreatic progenitor cells 2 (PP2 cells), which are then differentiated into endocrine precursor cells, and finally into beta cells. An exemplary procedure is provided in Example 1 below.

[0153] "Pancreatic endoderm cells" are an intermediate cell population in the development of the pancreatic lineage. Characteristic markers of pancreatic endoderm cells include one or more of the following markers: NKx6.1 and PDX1. The pancreatic endoderm cells according to the present invention express one or both of these markers.

[0154] Differentiation from posterior foregut cells into pancreatic endoderm cells can be performed by culturing in chemically defined medium, possibly supplemented with supplements such as ITS-X, and comprising one or more pancreatic endoderm differentiation factors. An exemplary combination of pancreatic endoderm differentiation factors is KGF+SANT-1+RA+iBET151.

[0155] "Pancreatic progenitor cells 1 (PP1 cells)" are another intermediate cell population in the development of the pancreatic lineage. Characteristic markers of PP1 cells include one or more of the following markers: PDX1, NKX6.1, HNF6, Prox1, Sox9, NEUROD1. The PP1 cells according to the present invention express one or more, preferably two or more, more preferably three or more, and even more preferably all of the above markers. Other characteristic markers of PP1 cells include FOXA2, CDX2, SOX9 and then HNF4α. Pancreatic progenitor cells 1 are characterized by the co-expression of PDX1, FOXA2, HNF6 and NKX6-1, wherein the expression of PDX1 is increased compared to intestinal tube cells.

[0156] Differentiation of posterior foregut cells into PP1 cells can be achieved by supplementing the posterior foregut with a TM The cells are cultured in a chemically defined medium supplemented with B27 and containing one or more PP1 differentiation factors. Examples of combinations of PP1 differentiation factors include EGF + FGF7, KGF + retinoic acid (RA) + SANT1 + Y-27632 + LDN-193189 + PdbU, and RA + cyclopamine + Noggin, iBET, and ITS. Each possibility represents a separate embodiment of the present invention.

[0157] Endocrine pancreatic progenitor cells, also known as pancreatic progenitor 2 cells (PP2 cells), are another intermediate cell population in the development of the pancreatic lineage. Characteristic markers of PP2 cells include one or more of the following: NKX6.1, PTF1A, NGN3, and NKX2.2. PP2 cells according to the present invention express one or more, preferably two or more, more preferably three or more, and even more preferably all of the aforementioned markers. Other characteristic markers of PP2 cells include PDX1.

[0158] Differentiation of PP1 cells into PP2 cells can be achieved by supplementing the cells with GlutaMAX TM The cells can be cultured in a chemically defined medium supplemented with B27 and containing one or more PP2 differentiation factors. Examples of combinations of PP2 differentiation factors include an ALK-5 inhibitor + heparin + FGF7 + Y-27632, KGF + retinoic acid + SANT1 + Y-27632 + activin A, iBET, and ITS. Each possibility represents a separate embodiment of the present invention.

[0159] "Endocrine precursor cells" (abbreviated as EN cells), also known as "pancreatic endocrine progenitor cells" and "endocrine progenitor cells", refer to pancreatic endoderm cells that are capable of becoming pancreatic hormone-expressing cells. Characteristic markers of endocrine precursor cells include one or more of the following markers: PDX1, GP2, Nkx6.1, INS, CHGA, GCG and SST. The endocrine precursor cells according to the present invention express one or more, preferably two or more, more preferably three or more, and even more preferably all of the above markers. Other characteristic markers of endocrine precursor cells include NGN3, NKX2.2, NeuroD1, ISL1, PAX4, PAX6, ARX.

[0160] Differentiation of pancreatic endoderm cells into endocrine precursor cells can be performed by culturing in a chemically defined medium that may be supplemented with a supplement such as ITS-X and contains one or more endocrine precursor cell differentiation factors. An exemplary combination of endocrine precursor cell differentiation factors is SANT-1+RA+PI 3-K inhibitor XXI+Alk5 inhibitor II (Alk5iII)+triiodothyronine (T3)+betacellulin.

[0161] Differentiation of PP2 cells into endocrine precursor cells can be achieved by supplementing the cells with GlutaMAX TMThe cells are cultured in a chemically defined medium supplemented with B27 and containing one or more endocrine precursor cell differentiation factors. Examples of combinations of endocrine precursor cell differentiation factors include an ALK5 inhibitor + zinc sulfate + heparin + a gamma secretase inhibitor + Y-27632, or retinoic acid + SANT1 + T3 + XX1 + an ALK5 inhibitor + heparin + betacellulin. Each possibility represents a separate embodiment of the present invention.

[0162] "Beta cells" ("β cells") are pancreatic endocrine cells that express insulin but do not express glucagon, somatostatin, ghrelin, and pancreatic polypeptide. Cells expressing markers characteristic of β cells can be characterized by expression of insulin (INS) and at least one of the following markers: PDX1, GP2, NKX6.1, C-peptide, and MAFA. β cells according to the present invention express one or more, preferably two or more, more preferably three or more, and even more preferably all of the aforementioned markers. β cells can be further characterized by being negative for GCG, PC1 / 3, SST, and CHGA.

[0163] In certain embodiments, the beta cells obtained by the methods of the present invention comprise at least 20% MAFA+ monohormonal cells at the end of the differentiation process, such as at least 30%, at least 40%, at least 50%, at least 60% MAFA+ monohormonal cells at the end of the differentiation process. Each possibility represents a separate embodiment of the present invention.

[0164] In certain embodiments, the β cells obtained by the methods of the present invention comprise at least 10% NKX6-1+ / C-peptide+ monohormone cells at the end of the differentiation process. In further embodiments, the β cells obtained by the methods of the present invention comprise at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% NKX6-1+ / C-peptide+ monohormone cells at the end of the differentiation process. Each possibility represents a separate embodiment of the present invention.

[0165] In certain embodiments, at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%) of the cells in the beta cell population obtained by the methods of the present invention are positive for INS, PDX1, and NKX6.1, and negative for GCG, PC1 / 3, SST, and CHGA. Other characteristic markers include NKX2.2, NeuroD1, ISL1, GLUT2, and PAX6.

[0166] β cells are also characterized by glucose-responsive insulin secretion. Specifically, β cells are characterized by biphasic insulin secretion in response to glucose stimulation. In certain embodiments, at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%) of the cells in the β cell population obtained by the methods of the present invention are insulin-positive, preferably secreting insulin in a biphasic glucose-responsive manner, as tested and exemplified below.

[0167] Differentiation of endocrine precursor cells (pancreatic endocrine progenitor cells) into β cells can be achieved by supplementing the pancreas with GlutaMAX. TM The cells are cultured in a chemically defined medium supplemented with B27 and containing one or more β-cell differentiation factors. Examples of β-cell differentiation medium include CMRL or RPMI plus GlutaMAX. TM , which is supplemented with 10% FBS, 1% B27, and 1% penicillin-streptomycin, and supplemented with differentiation factors such as Y-27632, T3 + ALK5 inhibitor (e.g., 10 μM Alk5i II + 1 μM T3), ALK5 inhibitor + T3 + N-Cys + AXL inhibitor. Each possibility represents a separate embodiment of the present invention. Another example of a β cell differentiation medium is CMRL supplemented with 10% FBS and supplemented with differentiation factors Alk5i II, L-3,30,5-triiodothyronine (T3), and nicotinamide.

[0168] An "insulin-producing beta cell" or "insulin-producing cell" according to the present invention is a functional beta-cell that exhibits glucose-stimulated insulin secretion ("GSIS").

[0169] Differentiation efficiency can be determined by exposing the cell colony to reagents such as antibodies that specifically recognize protein markers expressed by the cells of the differentiation of interest. The method for evaluating the expression of protein and nucleic acid markers in the cells cultivated or separated is a standard method in the art. These methods include RT-PCR, qRT-PCR, microarrays, Northern blotting, in situ hybridization and immunoassays such as immunocytochemical analysis, Western blotting and flow cytometry analysis (FACS) for accessible markers in intact cells.

[0170] In certain embodiments, the method of the present invention comprises seeding the progenitor cells of the pancreatic lineage selected from definitive endoderm cells, primitive intestinal cells, posterior foregut cells, pancreatic endoderm cells and endocrine precursor cells on an inactivated, decellularized, lung tissue-derived three-dimensional scaffold. Each possibility of the progenitor cells to be seeded on the scaffold is an independent embodiment of the present invention.

[0171] In certain embodiments, the methods of the present invention comprise seeding progenitor cells of the pancreatic lineage selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2), and pancreatic endocrine progenitor cells on a devitalized, decellularized, lung tissue-derived three-dimensional scaffold. Each possibility of progenitor cells to be seeded on the scaffold is a separate embodiment of the present invention.

[0172] In certain embodiments, the methods of the present invention comprise inoculating progenitor cells of the pancreatic lineage that are at least at the pancreatic endoderm stage. According to these embodiments, the progenitor cells of the pancreatic lineage can be selected from pancreatic endoderm cells and endocrine precursor cells and any stage therebetween. In certain specific embodiments, the progenitor cells of the pancreatic lineage are pancreatic endoderm cells.

[0173] As described herein, differentiation of pluripotent stem cells into β cells according to the present invention is initiated in 2D culture and continued on a 3D scaffold.

[0174] In certain embodiments, the differentiation method according to the present invention includes: differentiating pluripotent stem cells into pancreatic endoderm cells by stepwise differentiation in 2D cell culture; replacing the culture medium in the 2D cell culture with a culture medium containing one or more endocrine precursor cell differentiation factors, and incubating the cells for 1 day; after incubating in the culture medium containing one or more endocrine precursor cell differentiation factors for 1 day, seeding the cells on the scaffold described in this article in a culture medium containing one or more endocrine precursor cell differentiation factors to obtain endocrine precursor cells on the scaffold; and culturing the scaffold with the obtained endocrine precursor cells in a culture medium containing one or more β cell differentiation factors to obtain insulin-producing β cells on the scaffold.

[0175] In certain embodiments, differentiation of pluripotent stem cells into insulin-producing β cells is performed as described in detail in Example 2 below and in Figure 2 In certain embodiments, the progenitor cells of the pancreatic lineage seeded on the scaffold according to the present invention are any day 4-day 18 cells. In certain specific embodiments, the progenitor cells of the pancreatic lineage seeded on the scaffold according to the present invention are day 14-day 18 cells, meaning that the cells are grown in 2D cell culture until day 14, day 15, day 16, day 17 or day 18, and then seeded on the scaffold and the differentiation process continues on the scaffold. Each possibility represents a separate embodiment of the present invention. In certain exemplary embodiments, according to the detailed description and Figure 2 In the differentiation process described, the pancreatic lineage progenitor cells seeded on the scaffold were day 15 cells.

[0176] In certain embodiments, a method of producing a population of insulin-producing β cells is provided, the method comprising:

[0177] (i) culturing a devitalized, decellularized, lung tissue-derived three-dimensional scaffold seeded with pancreatic endoderm cells in a culture medium comprising one or more endocrine precursor cell differentiation factors to obtain endocrine precursor cells on the scaffold; and

[0178] (ii) replacing the culture medium of step (i) with a culture medium comprising one or more β cell differentiation factors to obtain β cells on the scaffold.

[0179] In certain embodiments, a method of producing a population of insulin-producing β cells is provided, the method comprising:

[0180] (i) culturing a devitalized, decellularized, lung tissue-derived three-dimensional scaffold seeded with definitive endoderm cells in a culture medium comprising one or more primitive gut tube differentiation factors to obtain primitive gut cells on the scaffold;

[0181] (ii) replacing the culture medium of step (i) with a culture medium comprising one or more posterior foregut differentiation factors to obtain posterior foregut cells on the scaffold;

[0182] (iii) replacing the culture medium of step (ii) with a culture medium comprising one or more PP1 differentiation factors to obtain PP1 cells on the scaffold;

[0183] (iv) replacing the culture medium of step (iii) with a culture medium comprising one or more PP2 differentiation factors to obtain PP2 cells on the scaffold;

[0184] (v) replacing the culture medium of step (iv) with a culture medium comprising one or more pancreatic endocrine progenitor cell differentiation factors to obtain pancreatic endocrine progenitor cells on the scaffold; and

[0185] (vi) replacing the culture medium in step (v) with a culture medium comprising one or more β cell differentiation factors to obtain β cells on the scaffold.

[0186] In certain embodiments, the method of the present invention further comprises differentiating pluripotent stem cells into the progenitor cells before seeding the scaffold.

[0187] In certain embodiments, the progenitor cells are definitive endoderm cells, and the sequential application of the plurality of differentiation factors comprises:

[0188] (i) culturing the scaffold seeded with definitive endoderm cells in a culture medium containing FGF7 to obtain primitive gut tube cells;

[0189] (ii) culturing the scaffold with the primitive gut tube cells in a culture medium containing KAAD-cyclopamine, retinoic acid and LDN 193189 to obtain posterior foregut cells;

[0190] (iii) culturing the scaffold with the posterior foregut cells in a culture medium containing EGF and FGF7 to obtain PP1 cells;

[0191] (iv) culturing the scaffold with the PP1 cells in a culture medium containing an ALK5 inhibitor, heparin, FGF7, and Y-27632 to obtain PP2 cells;

[0192] (v) culturing the scaffold with the PP2 cells in a culture medium comprising T3, an ALK5 inhibitor, zinc sulfate, heparin, a γ-secretase inhibitor, and Y-27632 to obtain pancreatic endocrine progenitor cells; and

[0193] (vi) culturing the scaffold with the pancreatic endocrine progenitor cells in a culture medium containing FBS and Y-27632 to obtain β cells.

[0194] An exemplary procedure for the stepwise differentiation and generation of beta cells according to the present invention is described in detail in the Examples section below. Alternative procedures utilizing different differentiation factors at each stage can be used. For example, the following differentiation process can be performed:

[0195] - Differentiating pluripotent stem cells into definitive endoderm (DE) cells by culturing in a medium containing activin A and CHIR-99021;

[0196] - Differentiating DE cells into primitive gastrulation (PG) cells by culturing in a medium containing KGF;

[0197] - Differentiating PG cells into posterior foregut (PFG) cells by culturing in medium containing FGF7;

[0198] - Differentiating PFG cells into pancreatic progenitor cells (PP1) by culturing in medium containing KGF, retinoic acid, SANT1, Y-27632, LDN-193189, and PdbU;

[0199] - Differentiating PP1 cells into endocrine pancreatic progenitor cells (PP2) by culturing in a medium containing KGF, retinoic acid, SANT1, Y-27632, and activin A;

[0200] - differentiating PP2 cells into pancreatic endocrine progenitor cells (EN) by culturing in a medium containing retinoic acid, SANT1, T3, XX1, Alk-5 inhibitor, heparin, and betacellulin; and

[0201] - EN cells were differentiated into β cells by culturing in a medium containing T3, Alk-5 inhibitor and CMRL.

[0202] In certain embodiments, in addition to the above-mentioned culture medium and differentiation factors, at least one type of supporting cell is added to the MOM to support the differentiation and survival of the differentiated cells and subsequently the resulting beta cells. The supporting cells disclosed herein include at least one of endothelial cells and mesenchymal stem cells. In certain embodiments, both endothelial cells and mesenchymal stem cells are seeded on the MOM as supporting cells.

[0203] Endothelial cells such as HUVECs, endothelial cells from pancreas or liver, etc. can be used at a density of, for example, 5000 cells / MOM.

[0204] Mesenchymal stem cells (MSCs), such as those derived from bone marrow, adipose tissue, placenta, and Wharton's jelly (umbilical cord), can be used, for example, at a density of 10,000 cells / MOM.

[0205] Artificial micro-organs

[0206] As used herein, the term "artificial micro-organ" or "engineered micro-organ" refers to a micro-organ scaffold with differentiated β cells according to the present invention cultured thereon, which has β cell-specific functions when cultured and is optionally organized into a micro-organ-like three-dimensional tissue structure.

[0207] In certain embodiments, the artificial micro-organ comprises cells that express at least one cell-specific protein after at least 7 days in culture. In other embodiments, the cells express the at least one cell-specific protein after at least 10, at least 15, at least 20, at least 30, at least 50, and optionally at least 70 days in culture.

[0208] In certain embodiments, characteristic β cell functions include but are not limited to expression of Pdx1 and insulin and glucose-responsive insulin secretion. Methods for monitoring β cell-specific protein expression include but are not limited to RT-PCR, immunohistochemistry, and quantitative immunoassay techniques such as ELISA for transcription of relevant genes.

[0209] Glucose responsive insulin secretion can be determined by changes in insulin secretion from β cell-MOM cultures when the concentration of glucose in the culture medium is raised from "low glucose" to "high glucose" levels, as described, for example, by Marchetti et al. (Diabetes, 1994; 43:827-30). This protocol, using 3 mM glucose as a low level and 16.7 mM glucose as a high level, is currently a standard procedure for testing β cell function before transplantation.

[0210] In certain embodiments, the beta cells of the artificial micro-organs of the present invention are characterized by glucose-responsive insulin secretion after at least 7 days of culture, or at least 10 days of culture, or at least 14 days of culture, or at least 20 days of culture, or at least 24 days of culture, or at least 28 days of culture, or at least 35 days of culture, or at least 40 days of culture, or at least 50 days of culture, or at least 60 days of culture, or at least 70 days of culture, or at least 75 days of culture. Each possibility represents a separate embodiment of the present invention. In certain other embodiments, the beta cells express Pdx1 after at least 7 days of culture, or at least 14 days of culture, or at least 20 days of culture, or at least 28 days of culture, or at least 35 days of culture. Each possibility represents a separate embodiment of the present invention.

[0211] In certain embodiments, the β cell population obtained by the differentiation procedure of the present invention comprises an increased percentage of cells that produce insulin in a glucose-responsive manner compared to a population differentiated in 2D culture.

[0212] In certain embodiments, the β-cell populations obtained by the differentiation procedures of the present invention secrete insulin at higher levels in response to glucose and / or have a better glucose stimulation index, as calculated by the ratio of insulin secreted under high glucose to low glucose, compared to populations differentiated in 2D culture.

[0213] In certain embodiments, the β-cell populations obtained by the differentiation procedures of the present invention exhibit improved insulin secretion kinetics compared to populations differentiated in 2D culture, as determined, for example, by glucose perfusion or stimulation assays using other insulin secretagogues.

[0214] As used herein, "2D culture" (2-dimensional culture) refers to differentiation in a container, such as a plate, comprising wells coated with an extracellular matrix.

[0215] Therapeutic uses

[0216] As used herein, "diabetes mellitus" refers to a disease in an organism, typically a human, that results from an absolute insulin deficiency (type 1 diabetes) or a relative insulin deficiency in the presence of insulin resistance (type 2 diabetes), i.e., an impaired action of insulin, caused by defects in insulin biosynthesis or production. Thus, a diabetic patient has absolute or relative insulin deficiency and exhibits symptoms and signs such as elevated blood glucose levels, the presence of glucose in the urine, and excessive urination.

[0217] In certain specific embodiments, the subject has type 1 diabetes.

[0218] In other specific embodiments, the diabetes is type 2 diabetes.

[0219] In certain embodiments, the diabetes is caused by pancreatitis. Pancreatitis is a condition in which the pancreas becomes inflamed. Damage to the insulin-producing cells in the pancreas caused by chronic pancreatitis can lead to diabetes.

[0220] In certain embodiments, the diabetes is caused by pancreatic inflammation or other causes of pancreatic dysfunction.

[0221] The subject is typically a human subject.

[0222] The term "treating" refers to inhibiting or halting the progression of a disease, disorder, or condition and / or causing alleviation, remission, or regression of the disease, disorder, or condition in an individual suffering from or diagnosed as having the disease, disorder, or condition. Those skilled in the art will recognize various different methods and assays that can be used to assess the progression of a disease, disorder, or condition, and will likewise recognize various different methods and assays that can be used to assess alleviation, remission, or regression of a disease, disorder, or condition.

[0223] As used herein, "transplantation" refers to providing an artificial micro-organ of the present invention to a location in the body of a recipient. For example, the artificial micro-organ can be transplanted subcutaneously (SC) or by intraperitoneal (IP) injection.

[0224] It will be appreciated that more than one micro-organ may be transplanted simultaneously into the same individual. The dosage and characteristics of the micro-organs for transplantation are generally determined based on the patient's weight and disease state, for example, the severity of insulin deficiency.

[0225] In one embodiment, the micro-organ is transplanted into the subject shortly after differentiation into β cells is complete. Alternatively, the differentiated cells can be cultured for hours, days, or weeks prior to transplantation.

[0226] According to one aspect of the present invention, a method for treating diabetes in a subject is provided, the method comprising transplanting a therapeutically effective amount of an artificial micro-organ comprising β cells differentiated on a devitalized, decellularized lung tissue-derived matrix according to the present invention into the subject, thereby treating diabetes.

[0227] The artificial micro-organ can be transplanted into a human subject by itself or in a pharmaceutical composition in which it is mixed with a suitable carrier or excipient.

[0228] As used herein, a "pharmaceutical composition" refers to a formulation comprising the artificial micro-organs described herein and other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the artificial micro-organs to a subject in need thereof.

[0229] The phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier," which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activities and properties of the artificial micro-organ.

[0230] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the artificial micro-organ.

[0231] The pharmaceutical compositions can be manufactured by processes known in the art, such as by utilizing conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0232] Thus, the pharmaceutical compositions for use according to the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and adjuvants. Suitable dosage forms depend on the chosen route of administration.

[0233] For injection, the artificial micro-organs can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.

[0234] Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredient is contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient (engineered micro-organ) that is effective to prevent, alleviate or ameliorate the symptoms of a disorder (e.g., diabetes).

[0235] Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

[0236] Reagent test kit

[0237] In certain embodiments, provided herein is a kit for producing insulin-producing β cells, the kit comprising:

[0238] (i) Devitalized, decellularized, lung tissue-derived three-dimensional scaffolds;

[0239] (ii) a variety of differentiation factors and, optionally, supporting cells (endothelial cells and MSCs) for the stepwise differentiation of pancreatic lineage progenitor cells into β cells; and

[0240] (iii) an instruction manual comprising technical instructions for seeding progenitor cells of the pancreatic lineage on the scaffold and performing step-wise differentiation on the scaffold such that the cells remain on the scaffold throughout the differentiation process.

[0241] In another embodiment, provided herein is a kit for producing insulin-producing beta cells, the kit comprising:

[0242] (i) devitalized, decellularized, lung tissue-derived three-dimensional scaffolds seeded with pancreatic lineage progenitor cells;

[0243] (ii) optionally endothelial cells and MSCs seeded on the lung tissue-derived three-dimensional scaffold, which support the differentiation, generation and survival of the differentiated cells and subsequently the resulting β cells;

[0244] (iii) a plurality of differentiation factors for performing stepwise differentiation of the progenitor cells of the pancreatic lineage into β cells; and

[0245] (iv) an instruction manual that specifies technical instructions for performing step-by-step differentiation on the scaffold such that the cells remain on the scaffold throughout the differentiation process.

[0246] In certain embodiments, the progenitor cell of the pancreatic lineage is selected from the group consisting of a definitive endoderm cell, a primitive enterocyte, a posterior foregut cell, a pancreatic endoderm cell, and an endocrine precursor cell.

[0247] In other embodiments, the progenitor cells of the pancreatic lineage are selected from definitive endoderm cells, primitive enterocytes, posterior foregut cells, pancreatic progenitor cells 1 (PP1), endocrine pancreatic progenitor cells (PP2), and endocrine precursor cells (also known as pancreatic endocrine progenitor cells).

[0248] In certain embodiments, the kit further comprises one or more cell culture media.

[0249] In certain embodiments, the kit further comprises a plurality of differentiation factors for performing stepwise differentiation of pluripotent stem cells into progenitor cells of the pancreatic lineage in 2D cell culture prior to seeding onto the scaffold. In certain embodiments, the instruction manual further specifies technical instructions for performing stepwise differentiation of pluripotent stem cells into progenitor cells of the pancreatic lineage in 2D cell culture prior to seeding onto the scaffold.

[0250] In certain embodiments, the kit further comprises factors and reagents for testing the β cells after differentiation, for example, factors and reagents for testing insulin secretion, factors and reagents for testing the expression of specific markers.

[0251] In certain embodiments, when the kit comprises a scaffold that has not been pre-seeded with cells, the scaffold can be lyophilized.

[0252] The following examples are provided to more fully illustrate certain embodiments of the present invention. However, they should never be construed as limiting the broad scope of the present invention. Those skilled in the art can easily design many variations and modifications of the principles disclosed herein without departing from the scope of the present invention.

[0253] Example

[0254] Example 1

[0255] Differentiation of pluripotent stem cells into insulin-producing cells on lung tissue-derived scaffolds and in 2D cell culture Comparison of differentiation

[0256] General solution

[0257] The procedure was performed using porcine lung-derived decellularized, devitalized micro-organ matrices (MOMs) prepared as previously described (US 10,093,896) and kept frozen until use.

[0258] Pluripotent stem cells, including human induced pluripotent stem cells (iPSCs) selected from iPSCs derived from adult cells such as skin fibroblasts, lymphocytes, and pancreatic cells including beta cells, are suitable.

[0259] A schematic diagram of the standard differentiation protocol in 2D culture is shown in Figure 1A The procedure of this embodiment is shown in Figure 1B middle.

[0260] In this example, the first step of the differentiation program, i.e., differentiation from pluripotent stem cells into definitive endoderm (DE) cells, was performed in 2D culture, and a sample of the DE cells was then seeded on MOM. Further steps were performed in parallel in 2D culture and on MOM. At each differentiation stage, cells were induced to the next differentiation stage in both 2D culture and on MOM.

[0261] In addition, at each differentiation stage, a cell sample was obtained from the 2D culture and seeded on a fresh MOM to continue differentiation on the MOM. The result was a series of MOM cultures, each containing cells seeded on the MOM at a different differentiation stage and completing differentiation on the MOM.

[0262] Step 1. Pluripotent stage (Day 0)

[0263] 2D culture :

[0264] a. Dilute the plate with 1 / 30 Matrigel TM Coat at 37°C for 1 hour

[0265] b. Human pluripotent stem cells were plated in mTeSR at 20-30% confluency. TM Matrigel in culture medium

[0266] c. When the cells reached 80-90% confluence, use TrypLE TM Express enzyme to detach cells

[0267] d. Count the number of cells

[0268] e. The cells were seeded at a density of 1,200,000 (1.2 million) cells per well in Matrigel diluted 1 / 30. TM Coat mTeSR in 6-well plates at 37 °C for 1 h TM Culture medium

[0269] f. The cells were incubated at 37°C, 5% CO2 for 24 h.

[0270] Step 2. Definitive Endoderm (DE) Stage (Days 1-4)

[0271] 2D culture :

[0272] Using STEMdiff TM Definitive endoderm kit (containing Activin A + Chir99021, or GDF8 + MCX-928, or Activin A + Wnt3a).

[0273] a. Day 0-1: Prepare Definitive Endoderm Medium 1 (mix Supplements A and B) according to the manufacturer's instructions and add 2 ml to each well. Incubate cells at 37°C, 5% CO2.

[0274] b. Days 1-2.5: Prepare Definitive Endoderm Medium 2 (mix Supplement B with Definitive Endoderm Basal Medium (1:100)) according to the manufacturer's instructions and add 2 ml to each well (Medium 2 instead of Medium 1). Incubate the cells at 37°C, 5% CO2.

[0275] c. Day 2.5-4: Prepare fresh definitive endoderm medium 2 (mix Supplement B with definitive endoderm basal medium (1:100)) and add 2 ml to each well. Incubate cells at 37°C, 5% CO2 for 36 h.

[0276] Samples of the resulting definitive endoderm (DE) cells were obtained from the 2D culture and the cells were seeded on MOMs in 12-well plates to obtain DE-MOMs as follows:

[0277] 10 5 DE cells / MOM at a density of 3 MOM / well in Definitive Endoderm Medium 2.

[0278] Cells from 2D and MOM cultures are analyzed for expression of one or more DE-specific markers, preferably at least 2-3 markers, selected from the group consisting of CXCR4, PDX1, FoxA2, ​​c-kit, GP2, Sox17, and GSC.

[0279] Step 3. Gastrula (PG) stage (Days 4-6)

[0280] Differentiation from DE to PG was performed in parallel in 2D culture and on MOM seeded with DE cells (DE-MOM) as follows:

[0281] a. Cells were supplemented with GlutaMAX TM The cells were then washed with RPMI 1640 medium containing 1% penicillin-streptomycin.

[0282] b. Add enterocyte culture medium containing:

[0283] i.RPMI 1640 medium + GlutaMAX TM

[0284] ii.1% PS (penicillin-streptomycin)

[0285] iii.1% B27 supplement

[0286] iv. 50ng / ml FGF7

[0287] c. Incubate the cells at 37°C, 5% CO2 for 48 h.

[0288] Samples of the resulting primitive gastrulation (PG) cells were obtained from the 2D culture and the cells were seeded on MOMs in 12-well plates as follows to obtain PG-MOMs:

[0289] 10 5 PG cells / MOM at a density of 3 MOM / well in PG medium as described above.

[0290] Cells from 2D and MOM cultures are analyzed for expression of one or more PG-specific markers, preferably at least two markers, selected from the group consisting of: FoxA1, HNF1B, HNF4A.

[0291] Step 4. Post-foregut (PFG) stage (Days 6-8)

[0292] Differentiation from PG to PFG was performed in 2D culture in parallel on DE-MOM (which now contained PG cells) and PG-MOM as follows:

[0293] a. Replace the PG medium with post-foregut medium and add 2 ml to each well, which contains

[0294] i.DMEM+GlutaMAX TM

[0295] ii.1% PS

[0296] iii.1% B27

[0297] iv. 0.25 μM KAAD-cyclopamine

[0298] v.2uM retinoic acid

[0299] vi.0.26uM LDN-193189

[0300] b. Incubate the cells at 37°C, 5% CO2 for 48 h.

[0301] Samples of the resulting posterior foregut (PFG) cells were obtained from the 2D culture and the cells were seeded on MOMs in 12-well plates as follows to obtain PFG-MOMs:

[0302] 10 5 PFG cells / MOM at a density of 3 MOM / well in PFG medium as described above.

[0303] Cells from 2D and MOM cultures were analyzed for the expression of the PFG-specific marker PDX1.

[0304] Step 5. Pancreatic Progenitor Cell (PP1) Stage (Days 8-12)

[0305] Differentiation from PFG to PP1 was performed in 2D culture on DE-MOM and PG-MOM (which now contained PFG cells) and in parallel on PFG-MOM as follows:

[0306] a. Replace PFG medium with pancreatic progenitor cell 1 medium and add 2 ml to each well, which contains:

[0307] i.DMEM+GlutaMAX TM

[0308] ii.1% PS

[0309] iii.1% B27

[0310] iv. 50ng / ml EGF

[0311] v.25ng / ml FGF7

[0312] b. Incubate the cells at 37°C, 5% CO2 for 48 h

[0313] c. On day 10, the culture medium was renewed and the cells were incubated at 37°C, 5% CO2 for a further 48 h.

[0314] Samples of the resulting pancreatic progenitor (PP1) cells were obtained from the 2D culture and the cells were seeded on MOMs in 12-well plates as follows to obtain PP1-MOMs:

[0315] 10 5 PP1 cells / MOM at a density of 3 MOM / well in Pancreatic Progenitor 1 (PP1) medium as described above.

[0316] Cells from 2D and MOM cultures are analyzed for expression of one or more PPl-specific markers, preferably at least two markers, selected from the group consisting of: PDX1, HNF6, Prox1, Sox9.

[0317] Step 6. Endocrine Pancreatic Progenitor (PP2) Stage (Days 12-13)

[0318] For 2D culture, prepare cell pellets for the PP2 stage as follows:

[0319] a. By adding 2ml AggreWell TM AggreWell cleaning solution TM 4006-well plates were pretreated.

[0320] b. Centrifuge the plate at 1300g for 5 minutes

[0321] c. Prepare PP2 cell pellet culture medium containing:

[0322] i.RPMI+GlutaMAX TM

[0323] ii.1% PS

[0324] iii.1% B27

[0325] iv.1uM ALK5 inhibitor Alk5 receptor inhibitor II (Alk5i II)

[0326] v.10ug / ml heparin

[0327] 25ng FGF7

[0328] vii.10uM Y-27632

[0329] d. Use TrypLE TM Express the PP1 cells obtained in the previous step and detach them from the plate.

[0330] e. Wash the cells with cell pellet culture medium and transfer the cell suspension to AggreWell TM 2 ml of cell pellet culture medium in the wells of a 4006-well plate.

[0331] g. Incubate the cells at 37°C, 5% CO2 for 24 h.

[0332] For MOM culture, the medium from the previous step (PP1 medium) was removed and replaced with PP2 cell pellet medium. The MOM were cultured at 37° C., 5% CO 2 for 24 hours.

[0333] From AggreWell TM Samples of the resulting pancreatic progenitor cells (PP2) were obtained and the cells were seeded on MOMs in 12-well plates as follows to obtain PP2-MOMs:

[0334] 10 5 PP2 cells / MOM at a density of 3 MOM / well in the cell pellet culture medium as described above.

[0335] Analysis from AggreWell TM The cells cultured on plates and MOMs express one or more PP2-specific markers, preferably at least two markers, selected from the group consisting of NKX6.1, PTF1A, NGN3, and NKX2.2.

[0336] Step 7. Pancreatic endocrine progenitor (EN) stage (Days 14-18)

[0337] For 2D culture, perform the following steps:

[0338] a. Prepare pancreatic endocrine progenitor cell stage culture medium containing:

[0339] i.RPMI+GlutaMAX TM

[0340] ii.1% PS

[0341] iii.1% B27

[0342] iv.1uM T3

[0343] v.10uM ALK5 inhibitor

[0344] vi.10uM zinc sulfate

[0345] vii.10ug / ml heparin

[0346] viii. 100 nm γ-secretase inhibitor XXI

[0347] ix.10uM Y-27632

[0348] b. Collect the cell pellet from the previous step in a 50 ml tube, wash with 1 ml of endocrine progenitor stage (EN) medium, and allow to settle by gravity.

[0349] c. Resuspend the pellet (cell pellet) with endocrine progenitor cell stage medium and transfer the cell pellet into a low-attachment 6-well plate

[0350] d. Shake the plate and place in a 37°C, 5% CO2 incubator

[0351] e. Refresh the medium every other day (collect and sediment the cell pellet by gravity).

[0352] For MOM culture, the medium from the previous step (PP2 medium) was removed and replaced with EN medium. The MOM were cultured at 37° C., 5% CO 2 . The medium was refreshed every other day.

[0353] Samples of the resulting pancreatic endocrine progenitor (EN) cell pellets were taken from the plates and seeded onto MOMs in 12-well plates to obtain EN-MOMs as follows:

[0354] 50-100 EN cell clusters / MOM at a density of 3 MOM / well in EN medium as described above.

[0355] Cells from 2D and MOM cultures are analyzed for expression of one or more EN cell-specific markers, preferably at least 2-3 markers, selected from the group consisting of PDX1, GP2, Nkx6.1, CHGA, INS, GCG, and SST.

[0356] Step 8. β-cell stage (Days 18-27)

[0357] Differentiation of ENs into β cells was performed in parallel in 2D and MOM cultures as follows:

[0358] a. Prepare pancreatic β cell stage culture medium containing:

[0359] i.RPMI+GlutaMAX TM

[0360] ii.1% PS

[0361] iii.1% B27

[0362] iv. 10% FBS

[0363] v.10uM Y-27632

[0364] b. The cells were incubated at 37°C, 5% CO2, and the culture medium was refreshed every other day.

[0365] Cells from 2D and MOM cultures are analyzed for expression of one or more β-cell-specific markers, preferably at least 2-3 markers, more preferably at least 3-4 markers selected from the following: INS+, GCG-, PC1 / 3-, SST-, CHGA-, Pdx1+, GP2+, Nkx6.1+C-peptide and MAFA+.

[0366] Example 2

[0367] Differentiation of pluripotent stem cells into insulin-producing cells on lung tissue-derived scaffolds and 2D cell culture Comparison of differentiation in culture

[0368] Specific solutions

[0369] Porcine lung-derived decellularized, devitalized micro-organ matrices (MOMs) were prepared as previously described ( US 10,093,896 ) and kept frozen until use.

[0370] HES-2 cells were used.

[0371] Schematic diagram of the steps in differentiating pluripotent stem cells into insulin-producing cells Figure 2 middle.

[0372] In this example, a complete differentiation process was performed in 2D culture as a control. A differentiation process that was initiated in 2D culture and completed on MOM was performed in parallel. More specifically, differentiation was performed up to the 15th day in 2D culture, and then the differentiated cells were seeded on MOM and continued to differentiate on MOM until the 25th day.

[0373] Differentiation plan

[0374] M1 medium: MCDB131 (Gibco) + 8 mM D-(+)-glucose (Sigma) + 1.23 g / L NaHCO3 (Sigma) + 2% BSA (Sigma) + 0.25 mM vitamin C (Sigma Aldrich) + 1% Pen / Strep (Lonza) + 1% L-glutamine (Lonza)

[0375] M2 medium: MCDB131+20 mM D-glucose+1.754 g / L NaHCO3+2% BSA+0.25 mM vitamin C+heparin 10 mg / ml (Sigma)+1% Pen / Strep+1% L-glutamine.

[0376] Prior to the differentiation process, HES-2 cells were passaged once in animal component-free cell culture medium as follows:

[0377] a. Dilute the plate with 1 / 30 Matrigel TM Coat at 37°C for 1 hour

[0378] b. HES-2 cells were seeded in mTeSR at 20-30% confluence. TM Matrigel in culture medium

[0379] c. When cells reached 80-90% confluence, use TrypLE TM Express enzyme to detach cells

[0380] d. Count the number of cells

[0381] e. The cells were seeded at a density of 1,200,000 (1.2 million) cells per well in Matrigel diluted 1 / 30. TM Coat mTeSR in 6-well plates at 37 °C for 1 h TM Culture medium

[0382] f. The cells were incubated at 37°C, 5% CO2 for 24 h.

[0383] Days 0-3: Definitive endoderm stage

[0384] STEMdiff containing activin A was used according to the manufacturer's instructions. TM Definitive Endoderm Kit (STEMCELL).

[0385] On day 4, the cells were analyzed by FACS for expression of definitive endoderm markers CXCR4 and c-kit. The results are shown in Figure 3As can be seen in the figure, the definitive endoderm stage was reached on day 4 of differentiation, in which approximately 70% of the cells were positive for CXCR4 and c-Kit.

[0386] Days 4-6: Gastrulation stage

[0387] a. Wash the cells with M1 medium

[0388] b. Add enterocyte culture medium (2 ml per well) containing:

[0389] i.M1 medium

[0390] ii. 50ng / ml KGF (Peprotech)

[0391] iii. ITS-X supplement (Invitrogen) 1:50,000

[0392] c. The cells were incubated at 37° C., 5% CO 2 for 24 h, the culture medium was then replaced with fresh culture medium, and the cells were further incubated at 37° C., 5% CO 2 for 24 h.

[0393] The cells were analyzed for the expression of FoxA2 and PDX1 by immunostaining and qPCR on day 4. The expression of FoxA2 and PDX1 was also tested on day 9 to examine the reduction in the expression of these two markers.

[0394] Days 7-8: Post-foregut stage

[0395] a. Replace the enterostasal medium with post-foregut medium and add 2 ml to each well containing:

[0396] i.M1 medium

[0397] ii.50ng / ml KGF

[0398] iii. 0.25 μM SANT-1 (Sigma)

[0399] iv. 2 μM retinoic acid (RA) (Sigma)

[0400] v. 200 nM LDN-193189 (only on day 7) (Sigma)

[0401] vi.500nM PdBU (Millipore)

[0402] vii.ITS-X Supplement 1:200

[0403] b. The cells were incubated at 37°C, 5% CO2 for 24 h, and then the medium was replaced with posterior foregut medium without LDN-193189, and the cells were incubated at 37°C, 5% CO2 for a further 24 h.

[0404] Days 9-13: Pancreatic endoderm stage

[0405] a. Replace the posterior foregut medium with pancreatic endoderm medium and add 2 ml to each well containing:

[0406] i.M1 medium

[0407] ii.50ng / ml KGF

[0408] iii. 0.25 μM SANT-1

[0409] iv.100nM RA

[0410] v.2μM iBET151 (Selleckchem)

[0411] vi.ITS-X Supplement 1:200

[0412] b. The cells were incubated at 37°C, 5% CO2 until day 13 with daily medium changes.

[0413] Days 14-18: Pre-endocrine stage

[0414] a. Replace the pancreatic endoderm culture medium with an endocrine precursor culture medium containing:

[0415] i.M2 medium

[0416] ii. 0.25 μM SANT-1

[0417] iii. 100 nM RA

[0418] iv. 1 μM PI 3-K inhibitor XXI (Millipore)

[0419] v.10 μM Alk5 inhibitor II (Alk5i II) (Selleckchem)

[0420] vi. 1 μM L-3,30,5-triiodothyronine (T3) (Sigma)

[0421] vii. 20ng / ml beta-cell cytokine (R&D)

[0422] viii.ITS-X Supplement 1:200

[0423] b. The cells were incubated at 37°C, 5% CO2 until day 18, with daily media changes. On day 15, a sample of cells differentiated in 2D culture was obtained and plated on MOMs in pancreatic endoderm medium as detailed above. The cells were plated at a density of 50,000-100,000 cells per MOM, with 3 MOMs per well. The next stage of the differentiation process was performed in parallel in 2D culture and on MOMs.

[0424] Days 18-25: β-cell stage

[0425] a. Replace the endocrine precursor culture medium (in 2D culture and in MOM culture) with β-cell culture medium containing:

[0426] i.CMRL medium

[0427] ii. 10% FBS

[0428] iii.10μM Alk5i II (Selleckchem)

[0429] iv. 1 μM L-3,30,5-triiodothyronine (T3) (Sigma)

[0430] v.10mM nicotinamide (Sigma)

[0431] b. The cells were incubated at 37°C, 5% CO2 until day 25 with daily medium changes.

[0432] Cell clusters were generated between day 21 and day 24. More specifically, the 2D culture was split so that a portion of the cells continued to grow as a monolayer and another portion of the cells grew as cell clusters. The cell clusters were generated as follows:

[0433] - Remove the culture medium

[0434] - Add 1ml TrypLE TM Express enzyme and wait 2-3 minutes at room temperature

[0435] -Remove TrypLE

[0436] - Add 2 ml of β-cell culture medium (culture medium described in (a)) to each well and pipette up and down several times to suspend the cells

[0437] - Place the cells in ultra-low attachment wells (transfer each well collected from the 2D culture plate to the corresponding ultra-low attachment well).

[0438] In vitro assay

[0439] Insulin (INS) expression

[0440] The expression of INS mRNA was analyzed by real-time PCR throughout the differentiation process. RNA was extracted using a Qiagen kit according to the kit instructions. Reverse transcription was performed using a Quantabio kit according to the kit instructions. qPCR was performed using Green was used. In addition, insulin expression was analyzed by immunofluorescence staining. To study insulin-positive cells, cells were stained with anti-insulin antibody in PBS-Triton 0.5% at 4°C overnight. The next day, cells were washed several times in PBS and stained with secondary antibody (488) for 45 minutes at RT. Cells were washed several times with PBS and counterstained with DAPI.

[0441] The results are summarized in Figures 4A-4C middle. Figure 4A Figure 2 shows INS mRNA expression in cells differentiated in 2D culture. Unless indicated as "cell clusters," the results are for cells grown as a monolayer. As can be seen in the figure, along the differentiation process, insulin expression increases starting from day 18. Figure 4B Shown is insulin / DAPI staining of the cell mass at day 24. On day 24 of differentiation, some insulin-positive cells were found within the cell mass. Figure 4C Shown are insulin / DAPI staining of cells that completed differentiation on MOM. Described figure shows staining at the 25th day.Compared with 2D culture, MOM improves beta cell differentiation and produces significantly more insulin-positive cells, i.e., compared with only differentiating in 2D culture, significantly more cells express insulin when differentiation is completed on MOM.

[0442] Glucose-stimulated insulin secretion (GSIS) assay

[0443] Cells were treated with low glucose (LG, 2.5mM glucose) for 20 minutes. Cells were then treated with high glucose (HG, 11mM glucose) for 20 minutes, and then treated with high glucose and KCL (being 11mM) for 20 minutes (HG+KCL). Samples were collected at the starting point and in every 10 minutes subsequently, until 60 minutes. The insulin secretion of the samples was then analyzed by ELISA.

[0444] The results are shown in Figures 5A-5C As can be seen in the figure, MOM improves insulin secretion and regulation of differentiated β cells compared to 2D culture. Differentiated β cells seeded on MOM and completed the differentiation process on MOM on day 15 ( Figure 5A ) secrete insulin in a regulated manner, whereas differentiated β cells grown as cell clusters or monolayers in 2D culture plates ( Figures 5B-5C) do not show regulated insulin secretion.

[0445] As mentioned in this article, regulation of insulin secretion means that the amount of insulin secreted in the LG is lower than that in the HG, and the amount secreted in the HG is lower than that in the HG+KCL. This further describes the biphasic secretion of insulin in response to glucose. Increased insulin secretion and the expression of first and second phase insulin release in response to high glucose stimulation are key features of beta cell behavior.

[0446] Example 3

[0447] Differentiation of pluripotent stem cells into insulin-producing cells on lung tissue-derived scaffolds and in 2D cell culture Comparison of differentiation

[0448] Improvement plan

[0449] The protocol for days 0-3 was modified and performed according to the following protocol.

[0450] Day 0 Gathering

[0451] Cell density : One well should have approximately 1 million cells, and there should be spaces between pluripotent stem cell Matrigel colonies (50 to 60% confluence).

[0452] Required culture medium:

[0453] -Accutase TM (Gibco A11105 is stored at -20°C, and cell cultures are stored at 4°C)

[0454] -Stop medium (IMDM:FCS with L-glutamine and P / S; 50:50)

[0455] -IMDM with L-glutamine and P / S

[0456] -D0 Endoderm Induction Medium:

[0457]

[0458] Differentiation plan

[0459] Day 0

[0460] - Remove culture medium from the wells

[0461] -Add 1ml / well Accutase TM (Gibco A11105 stock at -20°C, store at 4°C for cell culture) at room temperature for 1 minute

[0462] - Aspirate Accutase TM

[0463] -Add 1ml / well of stop medium + DNase (200uL / 12ml)

[0464] - Count cells

[0465] - Scrape the wells to generate cell clumps and add 1 ml / well of IMDM supplemented medium

[0466] -Use a 10ml pipette and pipette 2-3 times

[0467] - Add the solution from 6 wells to the 14 ml tube containing IMDM

[0468] - Centrifuge at 1200 rpm for 5 minutes

[0469] - Resuspend in D0 endoderm induction medium and dispense into low cluster plates at 2 ml / well. Use 6-well low cluster plates and set up the same number of plates as Matrigel plates or 6 wells per 10 cm dish.

[0470] - Incubate in a 5% O2 / 5% CO2 / 37°C incubator for 24 hours.

[0471] Day 1: Endoderm induction – complete medium change

[0472] - Remove embryoid bodies (EBs) from the wells, place in 14 ml tubes and allow the EBs to settle completely

[0473] - Centrifuge at 1200 rpm for 5 minutes

[0474] - Prepare Day 1 Endoderm Induction Medium and add 1 ml of Day 1 Medium per well upon EB deposition:

[0475]

[0476] - Incubate in a 5% O2 / 5% CO2 / 37°C incubator for 48-72 hours.

[0477] Day 4: Endoderm induction – complete medium change

[0478] - Remove embryoid bodies (EBs) from the wells, place in 14 ml tubes and allow the EBs to settle completely

[0479] - Prepare Day 4 Endoderm Induction Medium and add 1 ml of Day 4 Medium per well (2 ml / well depending on cell density) upon EB deposition:

[0480]

[0481]

[0482] - Incubate in a 5% O2 / 5% CO2 / 37°C incubator for 72 hours.

[0483] Example 4

[0484] In vitro activity assay

[0485] Beta cells obtained by differentiation on MOMs were analyzed as follows and compared with beta cells differentiated in 2D culture:

[0486] - mRNA gene expression analysis: (a) insulin (INS), glucagon (GCG), and pancreatic polypeptide (PPY); (b) β-cell maturation markers PDX1, NKX6.1, and MAFA; (c) cellular glucose sensing genes (SLC2A1 and GCK); and (d) gap junction genes (CDH1 and CX36). In addition, downregulation of pluripotency and progenitor cell genes was confirmed.

[0487] - Immunochemical analysis to confirm beta cell maturation: insulin (INS), glucagon (GCG) and pancreatic polypeptide (PPY), PDX1, NKX6.1 and MAFA.

[0488] - In vitro assessment of pancreatic function by static and dynamic (perfusion assay) glucose-stimulated insulin / C-peptide secretion assay (GSIS) followed by ELISA.

[0489] - Quantification of insulin content after cell lysis and ELISA.

[0490] - Ultrastructural analysis of differentiated beta cells by transmission / scanning electron microscopy (TEM / SEM) in order to study the secretory vesicles contained within said cells.

[0491] Example 5

[0492] In vivo functional assays

[0493] β cell-MOM compositions (5-10) were transplanted subcutaneously into immunocompromised mice to test their in vivo function. Specifically, it was verified whether human insulin and optionally C-peptide were detectable in the serum of animals transplanted with β cell-MOM compositions.

[0494] After a brief surgical recovery period (2 weeks), mice transplanted with β cell-MOM compositions were injected with glucose and serum was collected 30 min later. ELISA measurements of human insulin and optionally C-peptide were performed to quantify human insulin / C-peptide secreted into the host bloodstream.

[0495] In order to test whether beta cell-MOM compositions is made response to glucose secretion insulin (GSIS in vivo), before acute glucose stimulation (0min) and afterwards (30min) all in the blood stream of a part of mice, measure human insulin / C-peptide.After transplanting 2 weeks, calculate the percentage of transplanted mice that shows human insulin / C-peptide increase in blood stream after glucose stimulation.As another kind of metric of GSIS in vivo, calculate after glucose stimulation than the average ratio of secreted insulin before stimulation, and it should preferably ≥1.For islet transplantation, this stimulation index in vivo is in the scope of 0.4 to 4.3.

[0496] Approximately 1 month after transplantation, the animals were sacrificed and the implanted β cell-MOM composites were removed for histological analysis. IHC assays were performed to examine C-peptide+ / insulin+ cells.

[0497] Harvested implants were also evaluated histologically for the following markers:

[0498] Endocrine (insulin, glucagon, and somatostatin),

[0499] Stem cell properties (Sox2),

[0500] • Pancreatic endoderm (Pdx1, Nkx2.2 and Nkx6.1).

[0501] Beta cells were evaluated for signs of maturity and morphological integration with surrounding tissues and functional integration between the graft and the site vascular network (CD31 staining). The following histopathological assessments were performed:

[0502] a. Degree of implantation and cell survival

[0503] b. Extent of angiogenesis

[0504] c. Cell infiltration in EMP (H&E staining)

[0505] d. EMP fibrosis

[0506] e. Degradation of EMP

[0507] The description of the specific embodiments above sufficiently reveals the general nature of the invention so that others, using existing knowledge, can easily modify and / or adapt such specific embodiments to various applications without undue experimentation and without departing from the general concepts, and therefore, such adaptations and modifications should and are intended to be included within the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that the phrases or terms used herein are for descriptive purposes only and not for limiting purposes. The means, materials, and steps for performing the various functions disclosed may take various alternative forms without departing from the invention.

Claims

1. A method for producing an insulin-producing β-cell population, the method comprising: (a) seeding pancreatic lineage progenitor cells onto a devitalized, decellularized, lung tissue-derived three-dimensional scaffold, wherein the pancreatic lineage progenitor cells are pancreatic endoderm cells; and (b) differentiating the pancreatic lineage progenitor cells into beta cells by stepwise differentiation comprising sequentially applying a plurality of differentiation factors, wherein the stepwise differentiation is performed on the lung tissue-derived three-dimensional scaffold such that the cells remain on the scaffold throughout the differentiation process, This results in a population of insulin-producing beta cells. 2 . The method of claim 1 , further comprising differentiating pluripotent stem cells into progenitor cells of the pancreatic lineage in 2D cell culture before step (a).

3. The method of claim 1, further comprising seeding at least one type of supporting cells selected from endothelial cells and mesenchymal stem cells (MSC) on the scaffold in conjunction with seeding the progenitor cells of the pancreatic lineage, and performing the differentiation process when the supporting cells are co-cultured with differentiated cells on the scaffold; preferably, seeding endothelial cells and mesenchymal stem cells (MSC) on the scaffold in conjunction with seeding the progenitor cells of the pancreatic lineage, and performing the differentiation process when the endothelial cells and MSC are co-cultured with differentiated cells on the scaffold.

4. A composition for producing insulin-producing β cells, comprising: (i) a devitalized, decellularized, lung tissue-derived three-dimensional scaffold; and (ii) pancreatic lineage progenitor cells seeded on the scaffold, wherein the pancreatic lineage progenitor cells are pancreatic endoderm cells.

5. The composition of claim 4, further comprising at least one type of supporting cells selected from endothelial cells and mesenchymal stem cells (MSCs) seeded on the scaffold, preferably further comprising endothelial cells and MSCs seeded on the scaffold.

6. A method for producing insulin-producing β cells, the method comprising: (a) providing a devitalized, decellularized, lung tissue-derived three-dimensional scaffold seeded with pancreatic lineage progenitor cells according to claim 4; and (b) differentiating the pancreatic lineage progenitor cells into β cells by stepwise differentiation, wherein the stepwise differentiation is performed on the lung tissue-derived three-dimensional scaffold such that differentiated cells are retained on the scaffold throughout the differentiation process.

7. The method of claim 6, wherein in step (a) the scaffold is further seeded with at least one type of supporting cells selected from endothelial cells and mesenchymal stem cells (MSC), and wherein the stepwise differentiation is performed on the scaffold in the presence of the supporting cells; preferably wherein in step (a) the scaffold is further seeded with endothelial cells and mesenchymal stem cells (MSC), and wherein the stepwise differentiation is performed on the scaffold in the presence of the endothelial cells and MSC.

8. A kit for producing insulin-producing β cells, the kit comprising: (i) Devitalized, decellularized, lung tissue-derived three-dimensional scaffolds; (ii) a plurality of differentiation factors for performing stepwise differentiation of a progenitor cell of the pancreatic lineage into a β cell, wherein the progenitor cell of the pancreatic lineage is a pancreatic endoderm cell; and (iii) an instruction manual that specifies the technical instructions for performing step-by-step differentiation on the scaffold such that the cells remain on the scaffold throughout the differentiation process.

9. A method for producing an artificial micro-organ, the method comprising: (a) seeding pancreatic lineage progenitor cells onto a devitalized, decellularized, lung tissue-derived three-dimensional scaffold, wherein the pancreatic lineage progenitor cells are pancreatic endoderm cells; and (b) differentiating the pancreatic lineage progenitor cells into insulin-producing β cells by stepwise differentiation, wherein a plurality of differentiation factors are sequentially applied, wherein the stepwise differentiation is performed on the lung tissue-derived three-dimensional scaffold such that the cells remain on the scaffold throughout the differentiation process, Thereby, an artificial micro-organ is obtained, comprising insulin-producing beta cells cultured on said lung tissue-derived three-dimensional scaffold and maintaining glucose-responsive insulin secretion when cultured on said scaffold.

10. The method of claim 9, further comprising seeding at least one type of supporting cells selected from endothelial cells and mesenchymal stem cells (MSC) on the scaffold, and performing the differentiation process when the supporting cells are co-cultured with differentiated cells on the scaffold; preferably, it further comprises seeding endothelial cells and mesenchymal stem cells (MSC) on the scaffold, and performing the differentiation process when the endothelial cells and MSC are co-cultured with differentiated cells on the scaffold.

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