In vitro derivation of pancreatic islets from human pluripotent stem cells

Abstract

The present invention relates to a method for generating pancreatic lineage cells, e.g., pancreatic β cells, comprising culturing a population of posterior foregut cells for up to about 84 hours under conditions permissive for differentiation into pancreatic progenitor cells. The present invention also relates to the pancreatic lineage cells obtained by the method and their medical uses.

Description

[Technical Field]

[0001] The present disclosure relates to a method for generating pancreatic lineage cells, e.g., pancreatic β cells, comprising culturing a population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for up to about 84 hours. The present disclosure also relates to the pancreatic lineage cells obtained by the method and their medical uses. [Background technology]

[0002] According to the International Diabetes Federation (IDF), diabetes is a major global health crisis affecting more than 200–300 million people worldwide. Type 1 diabetes is caused by autoimmune destruction of insulin-producing pancreatic beta cells, while type 2 diabetes is characterized by peripheral insulin resistance and the inability to produce enough insulin to overcome this resistance. Other less common forms of diabetes associated with impaired insulin production include gestational diabetes, maturity-onset diabetes of the young, neonatal diabetes, and pancreatic islet loss due to pancreatitis. Patients with type 1 diabetes are treated with exogenous insulin injections, which can provide some control of blood glucose levels and significantly reduce the diabetic condition, but it is not a curative treatment and is associated with short- and long-term complications. Therefore, although insulin treatment has saved countless diabetic patients from premature death, it is a palliative treatment, not a cure.

[0003] Pancreatic islets, also called islets of Langerhans, are regions of the pancreas that contain endocrine cells. They are densely packed throughout the human pancreas and play a key role in glucose metabolism. Hormones produced in the islets are secreted directly into the bloodstream by (at least) five types of cells: The cellular structure of the pancreatic islet is important for regulating intercellular communication and hormone secretion: α cells, which produce glucagon; β cells, which produce insulin and amylin; δ cells, which produce somatostatin; ε cells, which produce ghrelin; and PP cells (γ cells or F cells), which produce pancreatic polypeptide.

[0004] Diabetic patients, particularly those with type 1 diabetes, may be cured by transplantation of insulin-producing pancreatic beta cells (β cells). The unlimited generation of human β cells from pluripotent cells could potentially provide a cure for millions of patients. Therefore, diabetes could be cured by replacing lost β cells in patients in need. This approach was demonstrated in animal models several decades ago. Rats induced with streptozotocin-induced diabetes could be cured by infusion of allogeneic pancreatic islets (reviewed in Murtaugh 2007). Transplantation of human pluripotent stem cell-derived pancreatic progenitor cells and / or islet-like cell aggregates is a promising method for treating diabetes. However, reliable and safe strategies for obtaining the necessary cell quantities are needed, which rely on efficient differentiation protocols that can be scaled up to meet therapeutic needs.

[0005] Pluripotent cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (hereinafter referred to as iPS cells or iPSCs), have the ability to differentiate into any somatic cell type, and the potential for harnessing the therapeutic potential of pluripotent cells has attracted significant scientific and societal interest.

[0006] Over the past decade, various studies have demonstrated that pancreatic cells, including polyhormonal and monohormonal insulin-expressing cells, can be generated from hPSCs (U.S. Patent Application Publication No. 2019 / 0359943, U.S. Patent Application Publication No. 10,253,298, U.S. Patent Application Publication No. 2011 / 0280842, Nostro et al., (2015), D'Amour et al., (2006)).

[0007] However, conventional methods do not yield pancreatic β cells with the efficiency and / or reproducibility required for clinical applications. Therefore, alternative methods are needed to more efficiently derive desired cell types from pluripotent cells. Maximizing stem cell utilization requires (i) developing in vitro differentiation methods that ensure the generation of enriched cell populations of specific desired cell types, (ii) ensuring the identity and functionality of in vitro-generated cells, and (iii) removing contaminating, undesired cell types that may impair the function of the desired cell type. The presence of undesired cell types, such as polyhormonal pancreatic cells, in cultures can pose safety concerns for potential replacement therapies or adversely affect the results of drug screening or disease modeling.

[0008] It would therefore be desirable to provide a differentiation strategy that overcomes the above-mentioned drawbacks as is evident from various parts of this background description. Summary of the Invention

[0009] It is an object of the present disclosure to provide a differentiation strategy for generating cells of the pancreatic endocrine lineage, e.g., pancreatic β cell lineage, which overcomes and / or mitigates the above-mentioned and other drawbacks of current strategies.

[0010] An object of the present disclosure is to provide an efficient and reproducible in vitro differentiation protocol for producing cells of the pancreatic lineage, such as pancreatic β cells, that exhibit the desired ability to produce insulin in response to glucose stimulation.

[0011] It is an object of the present invention to provide an efficient and reproducible in vitro differentiation protocol that allows for the production of large numbers of cells of the pancreatic endocrine lineage, such as pancreatic β cells.

[0012] Furthermore, it is an object of the present disclosure to provide in vitro differentiation protocols that allow for obtaining cultures with a high percentage of cells of the pancreatic endocrine lineage, such as pancreatic monohormonal β cells, and a low percentage of contaminating cell types.

[0013] It is also an object of the present disclosure to provide differentiated cell populations that have a high proportion of cells that exhibit functional characteristics of cells of the pancreatic endocrine lineage, such as pancreatic β cells.

[0014] An object of the present disclosure is to provide an in vitro differentiation protocol that enables the production of high-quality islet-like cell aggregates, e.g., exhibiting a high proportion of pancreatic monohormonal beta cells and a low proportion of contaminating cell types. The islet-like cell aggregates also contain monohormonal alpha cells. Another object of the present disclosure is to provide cultures that contain a high proportion of cells of the pancreatic endocrine lineage, e.g., pancreatic monohormonal beta cells, and a low proportion of contaminating cell types. An object of the present disclosure is to provide high-quality islet-like cell aggregates, e.g., exhibiting a high proportion of pancreatic monohormonal beta cells and a low proportion of contaminating cell types. The islet-like cell aggregates also contain monohormonal alpha cells. It is particularly important that the cells of interest are viable and healthy. Such cultures may be useful for many applications, including therapeutic and scientific / biotechnological applications, such as in vitro drug development and screening. For example, such cultures can be used for cell transplantation into patients in need of cell transplantation.

[0015] These and other objects will become apparent to those skilled in the art from this disclosure and are accomplished by the various aspects of the present invention as set forth in the appended claims and generally disclosed herein.

[0016] The methods disclosed herein for differentiating cells of the pancreatic endocrine lineage, such as the pancreatic β cell lineage, from pluripotent cells involve the use of specific culture conditions, e.g., a combination of soluble factors and environmental conditions and timing, which induce a significantly higher percentage of pluripotent cells to differentiate into cells of a desired cell fate. This disclosure is based on the surprising realization that short-term culture of posterior foregut (PF) cells under conditions permissive for differentiation into pancreatic progenitor (PP) cells yields a higher number of endocrine precursor (EP) cells (also referred to herein as endocrine precursor cells) than corresponding methods involving longer culture times, although fewer PP cells are obtained. The terms "endocrine precursor cells" and "endocrine precursor cells" are used interchangeably herein. Without being bound by theory, it appears that PP cells obtained by the methods of the present invention, including those obtained through short culture times, are capable of developing / differentiating into EP cells. Importantly, EP cells obtained by the methods disclosed herein have the potential to develop into mature, functional pancreatic β cells.

[0017] Thus, in a first aspect of the present invention, there is provided a method for producing cells of the pancreatic endocrine lineage, such as the pancreatic β cell lineage, comprising the steps a) to c) of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for no more than about 78 hours; and c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1.

[0018] Specifically, the cells of the cell population of posterior foregut (PF) cells characterized by the expression of PDX1 provided in step a) do not express NKX6.1, in other words, lack the expression of NKX6.1.

[0019] Thus, in one embodiment of the method disclosed herein, the cell population of posterior foregut (PF) cells provided in step a) is characterized by expression of PDX1 and lack of expression of NKX6.1. In one embodiment, the cell population of PF cells provided in step a) is further characterized by expression of HNF6. Thus, it will be understood that posterior foregut cells may be characterized by expression of HNF6, PDX1, or co-expression of PDX1 and HNF6.

[0020] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized ("less committed") cell acquires the characteristics of a specialized ("more committed") cell, such as a pancreatic cell. A differentiated cell is one that occupies a more specialized ("committed") position within the lineage of a cell.

[0021] As used herein, the term "commitment," when applied to the process of differentiation, refers to cells that have progressed in the differentiation pathway to a point where, under normal circumstances, they would continue to differentiate into a particular cell type or subset of cell types, but are unable under normal circumstances to differentiate into a different cell type or revert to a less differentiated cell type.

[0022] As used herein, the term "lineage" of a cell refers to the genetics of the cell, i.e., what cells it is derived from and what cells it can give rise to. The lineage of a cell places the cell within a genetic scheme of development and / or differentiation in vivo or in vitro.

[0023] As used herein, the term "lineage-specific marker" refers to a feature that is specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of non-committed cells to the lineage of interest.

[0024] Those skilled in the art are familiar with the different developmental stages of the pancreatic endocrine lineage, including the pancreatic β cell lineage.

[0025] As used herein, the term "pancreatic β cell lineage" refers to a genetic scheme of in vivo or in vitro development and / or differentiation that ultimately leads to the provision of cells that exhibit characteristics of pancreatic monohormonal β cells, such as the production of insulin and the expression of at least one of PDX1, NKX6.1, and NEUROD1. One skilled in the art will understand that a cell of the pancreatic β cell lineage can be any cell at an early developmental stage of said cell.

[0026] As used herein, the term "progenitor cells thereof" relates to cells of the pancreatic lineage, such as progenitor cells of pancreatic beta cells, and refers to any cell that can differentiate into a pancreatic beta cell, including, for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut cell, a posterior foregut cell, a pancreatic progenitor cell, or an endocrine progenitor cell, when cultured under conditions suitable for and / or permissive for the progenitor cell to differentiate into a pancreatic lineage, such as a progenitor cell of a pancreatic beta cell.

[0027] Differentiation of cells along the pancreatic β-cell lineage involves differentiation from low-commitment to high-commitment cell types. Briefly, the development of insulin-producing pancreatic β-cells is the culmination of a complex developmental program, involving in vivo stages in which cells of the posterior foregut acquire pancreatic identity, the pancreatic primordium expands and commits to an endocrine fate, and a subset of these progenitor cells becomes capable of generating β-cells. Factors, e.g., transcription factors, that have been shown to be important for the development of the pancreatic β-cell lineage include PDX1 (pancreatic and duodenal homeobox 1), PTF1A (pancreas-specific transcription factor 1a), NGN3 (neurogenin 3), NEUROD1 (neuronal differentiation 1), and NKX6.1 (homeobox protein NKX-6.1). This list of factors is not considered to be exhaustive, and additional factors are discussed below.

[0028] Furthermore, development of cells along the pancreatic β cell lineage requires signaling from exogenous factors, including, but not limited to, transforming growth factor β (TGFβ) and retinoic acid (RA). Recent studies suggest that TGFβ signaling induces definitive endoderm in mouse and human embryonic stem (ES) cells, and that RA treatment promotes PDX1 expression and pancreatic specification in ES cell-derived endoderm. Those skilled in the art will appreciate that in vitro differentiation of cells along the pancreatic β cell lineage requires the addition of exogenous factors to the cell growth medium at appropriate / permissive concentrations at appropriate stages in the differentiation process, which in principle mimics the in vivo development of said cells.

[0029] Thus, one skilled in the art will understand that "conditions permissive for differentiation" into a recited cell type refers to conditions that allow cells to exhibit characteristics of said cell type, and may include combinations of cell culture media, the presence or absence of exogenous factors, as well as the timing thereof.

[0030] The following is a brief summary of the stages of pancreatic development. Those skilled in the art will appreciate that such development can be described in terms of developmental stages. Each developmental stage, 0-6, is characterized by the expression of a set of factors (often referred to as markers).

[0031] Those skilled in the art will appreciate that the process of differentiating pluripotent stem cells in vitro into functional pancreatic endocrine cells, e.g., monohormonal pancreatic β cells, can in some embodiments be viewed as progressing through six sequential stages, each corresponding to a stage of in vivo development, as shown in the schematic diagram of Figure 1. The stages of in vivo development are well understood by those skilled in the art and are considered to be within the common general knowledge in the art.

[0032] In this stepwise progression, stage 0 refers to undifferentiated hES cells. Stage 1 refers to cells expressing markers characteristic of definitive endoderm (DE) cells. Stage 2 refers to cells expressing markers characteristic of primitive gut cells (PGT). Stage 3 refers to cells expressing markers characteristic of posterior foregut (PF) cells. Stage 4 refers to cells expressing markers characteristic of pancreatic progenitor cells (PP). Stage 5 refers to cells expressing markers characteristic of pancreatic endocrine progenitor cells (EP). Stage 6 refers to cells expressing markers characteristic of endocrine islet cells, such as pancreatic beta cells. Stage 6 cells, as defined herein, have the potential to form islet-like cell aggregates in vitro (in vitro cell aggregates), which mimic pancreatic islets found in vivo.

[0033] Those skilled in the art understand that not all cells within a particular population progress through these stages at the same rate, i.e., some cells may be progressing through the differentiation pathway slower or faster than the majority of cells present in the population.

[0034] The following provides a non-exhaustive description of characteristics associated with cells at various stages of in vitro culture, as described above. Those skilled in the art will understand that the various stages of in vitro culture correspond to stages of in vivo development. Those skilled in the art will understand that by selectively choosing which proteins to monitor for expression, it is possible to track developmental progression along a pancreatic endocrine lineage, such as the pancreatic beta cell lineage. If desired, the expression of multiple protein characteristics at developmental stages can be assessed. As cells develop, the expression of certain proteins is up-regulated and down-regulated, making them suitable as markers for various developmental stages.

[0035] As used herein, the term "germ cell" refers to a cell that develops from the epidermis during germ layer formation and exhibits characteristics of cells that form the digestive tract and its derivatives. Definitive endoderm cells express at least one, two, or all three of the following markers: CXCR4, FOXA2, and SOX17. Thus, definitive endoderm cells can be identified by the expression of at least one, two, or all three of the markers CXCR4, FOXA2, and SOX17. In particular, definitive endoderm cells can be identified by the expression of SOX17.

[0036] As used herein, the term "primitive gut cells" refers to cells derived from the definitive endoderm that can give rise to all endodermal organs, such as the lung, liver, pancreas, stomach, and intestine. Primitive gut cells express at least one or two markers, HNF1β and HNF4α. Thus, primitive gut cells can be identified by the expression of HNF1β, HNF4α, or both HNF1β and HNF4α.

[0037] As used herein, the term "posterior foregut cells" refers to endodermal cells that form part of the stomach, liver, pancreas, gallbladder, and duodenum. Posterior foregut cells express PDX1 or both PDX1 and HNF6. Thus, posterior foregut cells may be identified by the expression of PDX1, HNF6, or both PDX1 and HNF6.

[0038] As used herein, the term "pancreatic progenitor cells" refers to cells that express at least one, two, three, four, five, or all six of the following markers: PDX1, PTF1A, NKX6.1, SOX9, CPA, and HNF6. As shown in Figure 1, pancreatic progenitor cells express PDX1, NKX6.1, PTF1A, and SOX9. In particular, pancreatic progenitor cells co-express PDX1 and NKX6.1.

[0039] Therefore, pancreatic progenitor cells can be identified by the expression of PDX1 and NKX6.1.Pancreatic progenitor cells can be identified by the expression of PDX1, NKX6.1, and one or both of PTF1A and SOX9.

[0040] The term "endocrine precursor cells" as used herein refers to pancreatic endoderm cells that can become pancreatic hormone-expressing cells. Pancreatic endocrine precursor cells express at least one, two, three, or all four of the following markers: PDX1, NKX6.1, NGN3, and NEUROD1. Endocrine precursor cells can be identified by the expression of NXK6.1 and NEUROD1. In particular, endocrine precursor cells can be distinguished from pancreatic precursor cells by the expression of NEUROD1 and NGN3, which are not expressed in pancreatic precursor cells. Endocrine precursor cells can also be identified by the expression of PDX1, NKX6.1, and NGN3, or the expression of PDX1, NKX6.1, NGN3, and NEUROD1.

[0041] As used herein, the term "pancreatic islet cells" refers to cells capable of expressing at least one of the hormones insulin, glucagon, somatostatin, ghrelin, and pancreatic polypeptide. In addition to these hormones, markers characteristic of pancreatic endocrine cells include one, two, or all three of PDX1, NKX6.1, and NEUROD1. NEUROD1 is naturally expressed in all endocrine islet cells, while PDX1 and NKX6.1 are specific to stage 6 pancreatic beta cells.

[0042] As used herein, the term "islet-like cell aggregate" or "islet-like aggregate" refers to a cellular aggregate of pancreatic cells that exhibits the characteristics of a pancreatic islet in vivo. In particular, the aggregate exhibits the desirable properties of a high number of monohormonal beta cells, a desired number of monohormonal alpha cells, a low number of polyhormonal cells (including a low number of polyhormonal beta cells and a low number of polyhormonal alpha cells), a low number of non-endocrine cells, and a low number of proliferating cells. In particular, it is highly desirable for the islet-like cell aggregates obtained in vitro to mimic the characteristics of in vivo pancreatic islets in terms of both the distribution and functional properties of the cell types present. Those skilled in the art will understand that the percentages described herein relate to the average proportions exhibited by the islet-like cell aggregates. Thus, for example, 100 aggregates are analyzed, and the average number of the cell types is as set forth herein. In this context, the following listed characteristics refer to the average proportions in a population of islet-like cell aggregates according to the present disclosure.

[0043] In particular, as used herein, the term "pancreatic beta cells" or "monohormonal pancreatic beta cells" refers to cells that express insulin but do not express glucagon or somatostatin. As used herein, the terms "pancreatic beta cells" and "monohormonal pancreatic beta cells" are used interchangeably. Thus, pancreatic monohormonal beta cells can be identified by the lack of insulin expression and glucagon and / or somatostatin expression.

[0044] Pancreatic β cells are monohormonal, i.e., they express only one hormone, insulin. In contrast, pancreatic β cells are polyhormonal, expressing multiple hormones, such as at least two of insulin, glucagon, and somatostatin.

[0045] For example, as shown in Figure 1, progression through the above-mentioned developmental stages can be tracked by the expression of markers. For example, progression from stage 0 to stage 1 is associated with down-regulation of OCT4, NANOG, and SOX2 expression, and up-regulation of SOX17, FOXA, and CXCR4 expression. Progression from stage 1 to stage 2 is associated with up-regulation of HNF1β and HNF4α expression. Progression from stage 2 to stage 3 is associated with up-regulation of PDX1 and HNF6 expression.

[0046] Progression from stage 3 to stage 4 is associated with the maintenance of expression of PDX1 and HNF6, and the upregulation of expression of NKX6.1, PTF1A, and SOX9. Progression from stage 4 to stage 5 is associated with the downregulation of expression of PTF1A and SOX9, the maintenance of expression of PDX1 and NKX6.1, and the upregulation of expression of NGN3 and NEUROD1. Progression of pancreatic β cells from stage 5 to stage 6 is associated with the downregulation of expression of NGN3, the maintenance of expression of PDX1, NKX6.1, and NEUROD1, and the upregulation of expression of insulin and C-peptide. Thus, for example, PDX1 + Upregulation of NKX6.1 in cells indicates progression to stage 4.

[0047] Those skilled in the art will appreciate that the developmental progression of cells of the pancreatic lineage can be further divided into additional stages, for example, based on the expression levels of markers. As an example, a review article by Verhoeff et al. (Stem Cell Reviews and Reports (2022): 18, 2683-2698) outlines seven stages of differentiation into pancreatic islet-like clusters, with stages 1-4 and 7 corresponding to stages 1-4 and 6 used in the present application. According to Verhoeff, stage 5 is characterized by expression of NKX6.1 and low expression of NGN3, while stage 6 is characterized by high expression of NGN3 and expression of endocrine hormones. Stage 5, as defined in the present disclosure, corresponds to Verhoeff's stages 5 and 6.

[0048] The resulting cells are identified or characterized by phenotypic characteristics, morphological characteristics, and / or expression of cellular markers, which are readily understood by one of skill in the art in evaluating such cells. As used herein, the term "marker" refers to a nucleic acid or polypeptide molecule that is differentially expressed in a cell of interest.

[0049] Non-limiting examples of markers of the beta cell lineage discussed in the present disclosure include CXCR4, FOXA2, SOX17, HNF1β, HNF4α, PDX1, HNF6, PDX1, PTF1A, NKX6.1, SOX9, NGN3, NEUROD1, and insulin. As discussed above, combinations of such markers are characteristic of different developmental stages along the beta cell lineage. As discussed above, those skilled in the art will understand that markers can be selected such that expression or lack of expression of a combination of markers allows differentiation of cells at different developmental stages along the pancreatic endocrine lineage (see FIG. 1 ). The inventors have used expression of different markers to differentiate cells at different developmental stages, as illustrated in the accompanying Examples. As used herein, the term "characterized by expression of" when referring to cells of a cell population is to be interpreted as relating to the expression of a given marker or set of markers. Conversely, the terms "characterized by lack of expression of" or "lacking expression of" or "does not express," when referring to cells of a cell population, are to be interpreted as relating to lack of expression of a particular marker or set of markers. Those skilled in the art are well aware of the use of marker expression as a method of distinguishing cells with different characteristics, such as cells at different developmental stages of the pancreatic beta cell lineage.

[0050] Those skilled in the art will understand that when analyzing cell populations using a method that limits the number of markers that can be simultaneously scored, for example, due to limitations of the method itself or the reagents used, it is possible to select a subset of markers that can distinguish a first cell population from a second cell population. As an example, a cell population of pancreatic progenitor cells characterized by the expression of PDX1 and NKX6.1 can be distinguished from a population of endocrine precursor cells characterized by the expression of PDX1, NKX6.1, NEUROD1, and NGN3. For example, pancreatic progenitor cells are positive for PDX1 or NKX6.1 and negative for either NGN3 or NEUROD1. EP cells can be scored as double-positive for NKX6.1 and at least one marker not expressed by pancreatic progenitor cells (e.g., double-positive for NKX6.1 and NEUROD1, or double-positive for NKX6.1 and NGN3). This principle is applied in the Examples section to distinguish cells with different characteristics. Importantly, when the cells are scored by the other markers listed above, it will show the complete marker profile of a particular population.The fact that only a subset of markers is used in an experimental setting should never be interpreted as representing the absence of expression of the remaining characteristic markers.Those skilled in the art will understand that marker expression can be evaluated at the nucleotide level, for example, at the mRNA level, or at the protein level.Well-known methods for evaluating marker expression include, but are not limited to, immunohistochemistry, in situ hybridization, FACS, RNA sequencing, the use of arrays such as microarrays, and quantitative PCR.Those skilled in the art will recognize these and other suitable methods.

[0051] In this context, differential expression means an increased level of a positive marker and a decreased level of a negative marker compared to undifferentiated cells or cells at a different developmental stage, where the detectable level of the marker nucleic acid or polypeptide is sufficiently high or low in the cell of interest compared to other cells 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.

[0052] As used herein, a cell is "positive for" or "positive" a particular marker if the particular marker is sufficiently detected within the cell, in other words, expressed by the cell. Thus, a cell characterized by expression of a particular marker is positive for that marker. Conversely, a cell is "negative for" or "negative" a particular marker if the particular marker is not sufficiently detected within the cell. Thus, a cell characterized by the absence of expression of a particular marker is negative for that marker. When a "+" or "-" symbol is used in connection with a marker, this means positive or negative for that marker (e.g., NKX6.1 + The cells are positive for the marker NKX6.1).

[0053] When using any method of in vitro differentiation, the ability to obtain a population containing cells that exhibit the same or similar characteristics at a particular time point is crucial, as cells at different developmental, maturational, or functional stages may respond differently to external and internal stimuli. Specifically, factor X may lead to the specification of cell types A and B during development, while treatment with the same factor X may lead to the selective cell death of mature cell type A. Thus, not only is it important to know the downstream functional consequences of subjecting cells to a particular factor, but it is equally important to subject the cells to that factor within a time window that allows the cells to respond to that factor in the desired manner. Thus, those skilled in the art will understand the importance of obtaining a synchronized cell population to ensure the desired response throughout the cell population. For example, it may be desirable for the majority of cells in culture at a particular time point to be progenitor cells.

[0054] In one embodiment of the first aspect disclosed herein, the method is provided wherein, in step b), the cell population is cultured for about 75 hours or less, for example, about 72 hours or less, for example, about 66 hours or less, for example, about 60 hours or less, for example, about 48 hours or less. In another embodiment, the method is provided wherein, in step b), the cell population is cultured for a period of about 42 to 78 hours, for example, about 44 to 76 hours, for example, about 46 to 74 hours, for example, about 48 to 72 hours. In one embodiment, the cell population is cultured for a period of about 40 to 78 hours, for example, about 42 to 76 hours, for example, about 44 to 74 hours, for example, about 48 to 72 hours. In one embodiment, the cell population is cultured for a period of about 42 to 54 hours, for example, about 44 to 52 hours, for example, about 46 to 50 hours, for example, about 48 hours.

[0055] In one embodiment of the method disclosed herein, the cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of PDX1 and NKX6.1 in step c) is further characterized by expression of at least one marker selected from the group consisting of PTF1A, SOX9, HNF6, and CPA, such as a marker selected from the group consisting of SOX9 and PTF1A. In one embodiment of the method disclosed herein, the cell population of pancreatic progenitor cells in step c) is further characterized by expression of PTF1A and SOX9.

[0056] As described above, "conditions permissive for differentiation" refers to conditions that allow cells to exhibit characteristics of the cell type, and may include the combination of cell culture medium, the presence or absence of exogenous factors, and the timing thereof. In one embodiment, the culture medium in step b) is a culture medium suitable for culturing posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells. Non-limiting examples of culture media for step 4 (S4) are as defined in the accompanying Examples. The culture medium in step b) may be supplemented with other factors as defined herein. Those skilled in the art will understand that other suitable media may also be used. In particular, culturing under conditions permissive for differentiation into pancreatic progenitor cells in step b) disclosed herein may involve culturing a cell population in a culture medium in the presence of specific exogenous factors, such as epidermal growth factor (EGF) and nicotinamide (NIC) or a derivative or agonist thereof. Thus, in one embodiment of the methods disclosed herein, the conditions permissive for differentiation into pancreatic progenitor cells comprise culturing the cell population in a culture medium in the presence of an effective amount of epidermal growth factor (EGF), such as human EGF, or a derivative or agonist thereof, and an effective amount of nicotinamide (NIC), or a derivative or agonist thereof. In one embodiment of the methods disclosed herein, step b) comprises culturing the cell population in a culture medium in the presence of an effective amount of EGF, such as human EGF, and an effective amount of NIC.

[0057] Epidermal growth factor (EGF) is a protein that stimulates cell proliferation and differentiation by binding to the receptor EGFR. Human EGF is a 6-kDa protein with 53 amino acid residues and three intramolecular disulfide bonds. Receptor binding stimulates ligand-induced dimerization, activates intrinsic protein tyrosine kinase activity, and initiates a signaling cascade that results in various biochemical changes within the cell—increased intracellular calcium concentration, increased glycolysis and protein synthesis, and increased expression of certain genes, including the EGFR gene—ultimately leading to DNA synthesis and cell proliferation. EGF is a member of the EGF family of proteins. Members of this protein family share very similar structural and functional properties. Besides EGF itself, other family members include heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor alpha (TGF-α), amphiregulin (AR), epiregulin (EPR), epigen, betacellulin (BTC), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), and neuregulin 4 (NRG4).

[0058] Those skilled in the art will understand that the term "epidermal growth factor (EGF) or its derivative or agonist" as used herein is meant to include factors that enhance or replace EGF signaling. Such factors may be involved in downstream signaling of EGF or may be small molecule agonists. A non-limiting list of EGF derivatives or agonists includes high-affinity EGFR ligands such as TGF-α, BTC, and HB-EGF, and low-affinity ligands such as AR, EPR, and Epigen. Thus, in one embodiment of this aspect of the invention, the EGF or its derivative or agonist is selected from the group consisting of EGF, TGF-α, BTC, HB-EGF, AR, EPR, and Epigen, e.g., EGF. In one particular embodiment, the EGF is human EGF.

[0059] Nicotinamide (NIC), also known as NAM, is a type of vitamin B. Nicotinamide's structure consists of a pyridine ring with a primary amide group attached at the meta position, making it an amide of nicotinic acid. Nicotinamide is well known as a cell culture supplement used in the differentiation of embryonic stem cells and induced pluripotent stem cells and has been shown to regulate stem cell differentiation in various applications, including pancreatic cell differentiation. Those skilled in the art will understand that the term "nicotinamide (NIC) or its derivative or agonist" as used herein is intended to include factors that enhance or replace NIC signaling. Non-limiting examples of such derivatives or agonists include NIC, niacin (nicotinic acid), nicotinamide riboside, NAD / NADP, and tryptophan, a precursor of NIC. Thus, in one embodiment of this aspect of the present invention, the NIC or its derivative or agonist is selected from the group consisting of NIC, niacin, nicotinamide riboside, NAD / NADP, and tryptophan, e.g., the NIC or its derivative or agonist is NIC.

[0060] In one embodiment of the method, the effective amount of EGF or a derivative or agonist thereof is about 50 to 200 ng / mL, for example, about 50 to 150 ng / mL, for example, about 75 to 125 ng / mL, for example, about 100 ng / mL.

[0061] In one embodiment of the method, the effective amount of the NIC or a derivative or agonist thereof is about 5 nM to 20 nM, for example, about 5 to 15 mM, for example, about 8 to 12 mM, for example, about 10 mM.

[0062] Step b) of the methods disclosed herein may include culturing the cell population in a culture medium containing additional factors such as one or more factors selected from KGF and derivatives and agonists thereof, ActA and derivatives and agonists thereof, retinoic acid and derivatives and agonists thereof, SANT-1 and derivatives and agonists thereof, PDBu and derivatives and agonists thereof, and LDN and derivatives and agonists thereof, e.g., one or more factors selected from KGF, ActA, retinoic acid, SANT-1, PDBu, and LDN. In one embodiment, step b) comprises culturing the cell population in a culture medium further comprising KGF or a derivative or agonist thereof, ActA or a derivative or agonist thereof, retinoic acid or a derivative or agonist thereof, SANT-1 or a derivative or agonist thereof, PDBu or a derivative or agonist thereof, and LDN or a derivative or agonist thereof, e.g., a culture medium further comprising KGF, ActA, retinoic acid, SANT-1, PDBu, and LDN. It will be understood that the culture medium may comprise a mixture of factors and their derivatives and / or agonists.

[0063] Those skilled in the art will appreciate that the cell culture medium and the factors contained therein may be adapted by substituting derivatives and / or agonists thereof, such as those described below.

[0064] Keratinocyte growth factor (KGF), also known as fibroblast growth factor 7 (FGF7), is a member of the FGF family of proteins. It is a bioactive protein commonly used in cell culture applications. KGF binds to fibroblast growth factor receptor 2b (FGFR2b). KGF induces proliferation of many epithelial cells, but not fibroblasts or endothelial cells. KGF is the primary growth factor for skin keratinocytes and is also used in the culture and differentiation of pluripotent cells. Those skilled in the art will understand that KGF can be substituted with its derivatives or agonists in the cell culture methods described herein. Non-limiting examples of such agonists include other factors that bind to FGFR2b and signal through the receptor, such as FGF10.

[0065] In one embodiment, the KGF or derivative or agonist thereof is selected from the group consisting of KGF and FGF10, for example FGF10.

[0066] In one embodiment, the culture medium in step b) contains 25 to 75 ng / mL of KGF or a derivative or agonist thereof, for example, 50 ng / mL of KGF or a derivative or agonist thereof. In one embodiment, the culture medium in step b) contains about 25 to 75 ng / mL of KGF, for example, about 50 ng / mL of KGF.

[0067] ActA, or activin A, is a member of the TGF-β superfamily. Both activin and Nodal ligands can signal through the same receptors and effectors to regulate transcription. Often, the effects of signaling through Nodal and activin are indistinguishable, hence the term activin / Nodal pathway. Activin / Nodal binds to type II activin receptors (ActRII / IIB), triggering the recruitment, phosphorylation, and activation of type I activin receptors (activin receptor-like kinases, or ALKs, including ALK1-7), particularly ALK4. In turn, the serine / threonine kinase receptors ActRII / IIB and ALK4 / 7 induce phosphorylation of the Smad transcription factors Smad2 and Smad3.

[0068] TGFβ signaling is known in the art to be involved in embryogenesis, cell differentiation, apoptosis, and other functions. The Activin / Nodal and TGFβ pathways share the downstream effectors Smad2 and Smad3.

[0069] Activin / Nodal has been reported to be involved in maintaining stem cell pluripotency, but Activin / Nodal signaling is also required for endoderm differentiation.

[0070] Those skilled in the art will understand that ActA can be substituted with its derivatives or agonists in the cell cultures described herein. Examples of such agonists include downstream effector molecules, such as Nodal and Smad 2 and 3, which signal through the receptor, as well as TGFβ, which signals through the same effector molecules. Additional derivatives or agonists include, but are not limited to, TGFβ1-3 (TGFβ1, TGFβ2, TGFβ3), Nodal, activin A, GDF-1, GDF-8, and GDF-11, which activate the Smad 2 / 3 / 4 complex.

[0071] In one embodiment, the ActA or a derivative or agonist thereof is selected from the group consisting of ActA, GDF-8, Nodal, and TGFβ1-3 (TGFβ1, TGFβ2, TGFβ3). In one embodiment, the medium in step b) contains about 1 to 10 ng / mL, for example, 2.5 to 7.5 ng / mL, for example, about 5 ng / mL, of ActA.

[0072] Retinoic acid (RA) is a metabolite of vitamin A and mediates the functions of vitamin A required for growth and development. Retinoic acid is known to be involved in specifying the position along the anterior-posterior axis of the embryo, also known as patterning, and is also known to play a role in promoting the generation of pancreatic endocrine precursor cells and their differentiation into pancreatic islets and β cells during later stages of pancreatic development.

[0073] Retinoic acid acts by binding to retinoic acid receptors (RARs), which bind to DNA as heterodimers with retinoid X receptors (RXRs) at a region called the retinoic acid response element (RARE). Binding of a retinoic acid ligand to RARs alters the structure of the RAR, affecting the binding of other proteins that induce or repress transcription of nearby genes. Those skilled in the art will understand that the term "retinoic acid or its derivatives or agonists," as used herein, is intended to include factors that enhance or substitute for retinoic acid signaling. Such factors may be involved in signal transduction downstream of retinoic acid or may be small molecule agonists. A non-limiting list of retinoic acid derivatives or agonists includes all-trans retinoic acid, synthetic retinoids ec23, Ch55, TTNPB, fenretinide, RAR agonists such as RARA agonists and RARB agonists AC261066, adapalene, AC55649, AM80, AM580, BMS753, tazarotene, and Ro41-5253. Thus, in one embodiment of this aspect of the invention, the retinoic acid or derivative or agonist thereof is selected from the group consisting of retinoic acid, all-trans retinoic acid, synthetic retinoids ec23, Ch55, TTNPB, fenretinide, RAR agonists such as the RARA and RARB agonists AC261066, adapalene, AC55649, AM80, AM580, BMS753, tazarotene, and Ro41-5253.

[0074] In one embodiment, the retinoic acid (RA) or derivative or agonist thereof is selected from the group consisting of retinoic acid, all-trans retinoic acid, and the synthetic retinoid ec23. In one embodiment, the retinoic acid or derivative or agonist thereof is selected from the group consisting of retinoic acid and all-trans retinoic acid. In one embodiment, the retinoic acid or derivative or agonist thereof is all-trans retinoic acid. In one embodiment, the medium in step b) contains about 50 to 150 nM RA or a derivative or agonist thereof, for example, about 100 nM RA or a derivative or agonist thereof.

[0075] In one embodiment, the medium in step b) contains about 50 to 150 nM RA, for example about 100 nM RA.

[0076] Hedgehog (HH or Hh) signaling is known to play an important role in regulating vertebrate organogenesis, such as digit growth and brain organization. The vertebrate Hedgehog protein family consists of Sonic hedgehog (SHH), Indian hedgehog (IHH), and Desert hedgehog (DHH), which signal through similar pathways and share many functional characteristics. Because Sonic hedgehog (SHH) is a key negative regulator of pancreatic development, it is important to suppress SHH during the initiation of pancreatic development, and maintaining Hedgehog suppression is necessary to ensure proper pancreatic development.

[0077] Briefly, Hh signals through interaction with the Hh receptor complex, which consists of two components, Patched (Ptc) and Smoothened (Smo), which transmit the Hh signal to the cell. Ptc is thought to inhibit Hh signaling by binding to Smo in the plasma membrane. In the presence of Hh ligand, this inhibition is relieved, allowing Smo to signal. In vertebrates, the zinc finger proteins Gli1, Gli2, and Gli3 are downstream mediators of Hh signaling and are involved in regulating the transcriptional response of target genes in an Hh-dependent manner.

[0078] SANT-1 is an inhibitor of Hedgehog (Hh) signaling and acts by antagonizing Smoothened activity. Non-limiting examples of Hedgehog signaling inhibitors include cyclopamine, IHR1, IHR-Cy3, itraconazole, Jervine, M25, MRT10, PF04449913 maleate, PF5274857 hydrochloride, SANT-1, and SANT-2.

[0079] In one embodiment, the SANT-1 or derivative or agonist thereof is selected from the group consisting of cyclopamine, IHR1, IHR-Cy3, itraconazole, jervine, M25, MRT10, PF04449913 maleate, PF5274857 hydrochloride, SANT-1, and SANT-2.

[0080] In one embodiment, the medium in step b) contains about 0.10 to 0.50 μM SANT-1, for example, about 0.25 μM SANT-1.

[0081] PDBu (phorbol-12,13-dibutyrate) is a potent promoter of nitric oxide (NO) synthesis and a potent activator of protein kinase C. PDBu has been reported to be a tumor promoter, activating various cellular responses, including proliferation. PBDu can be substituted with other activators of protein kinase C, such as, but not limited to, phorbol 12-myristate 13-acetate (PMA) and TPPB. In one embodiment, the PDBu or its derivative or agonist is selected from the group consisting of PDBu, PMA, and TPPB. In one embodiment, the medium in step b) contains about 0.25 to 0.75 μM PDBu, for example, about 0.5 μM PDBu.

[0082] LDN193189 (hereinafter referred to as LDN) is an inhibitor of the bone formation (BMP) pathway and acts by inhibiting ALK2 and ALK3. LDN functions primarily by inhibiting the phosphorylation of Smad1, Smad5, and Smad8. LDN is an analog of dorsomorphin and noggin, and dorsomorphin can be a substitute for LDN. In one embodiment, the LDN or its derivative or agonist is selected from the group consisting of LDN, noggin, and dorsomorphin. In one embodiment, the medium in step b) contains about 100 to 300 nM LDN, for example, 100 to 250 nM LDN, for example, 150 to 250 nM LDN, for example, about 200 nM LDN.

[0083] In one embodiment, step b) of the method disclosed herein comprises culturing the cell population in a culture medium comprising about 50 ng / mL KGF, about 5 ng / mL ActA, about 100 nM retinoic acid, about 0.25 mM SANT-1, about 500 nM PDBu, about 200 nM LDN, about 100 ng / mL EGF, and about 10 mM NIC.

[0084] In tissue engineering, stem cell, and molecular biology research, cell culture is primarily performed on flat dishes / cell culture plates made of plastic or glass. These cultures are considered adherent cultures. This technique is commonly known as two-dimensional (2D) cell culture. Herein, the term "2D" in the context of cell culture refers to culture on flat cell culture plates, where cells adhere directly or indirectly to the culture plate, e.g., via a cell culture substrate. The plate may be coated with a cell culture substrate. Cell culture substrates fall into two main categories: natural and synthetic. The most commonly used natural substrates are collagen, fibronectin, and laminin, which constitute part of the extracellular matrix. Cells interact with these matrix components through cell surface receptors, such as integrins. Receptors bind to domains in collagen, fibronectin, and laminin, triggering intracellular signaling pathways that promote the formation of adhesion complexes and may induce cell proliferation or differentiation. The most commonly used synthetic substrates for 2D culture include polylysine, a polymer of lysine containing positively charged amino groups. The choice of substrate is an important parameter to consider as it can have a significant impact on cell proliferation and attachment.

[0085] In one embodiment of the method disclosed herein, in step b) the cells are cultured on a 2D substrate. In one embodiment, the cells are adherent to the 2D substrate. The 2D substrate may comprise one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and Matrigel™ (Matrigel). For example, the 2D substrate may comprise one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen, gelatin, and Matrigel™. For example, the 2D substrate may comprise one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and Matrigel™. For example, the 2D substrate may comprise one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and Matrigel™. In particular, the fragment may comprise a functional domain of a protein. Thus, those skilled in the art will understand that the fragments may correspond to or include functional domains, e.g., may include functional domains and further include additional N-terminal and / or C-terminal amino acids. Matrigel™ is derived from the murine Engelbreth-Holm-Swarm tumor and contains many unknown components. Laminin is known to be the main component of Matrigel. For cell culture, particularly for therapeutic purposes, it may be beneficial to avoid animal-derived products in addition to using a defined medium to achieve a defined and reproducible product and avoid potential patient safety issues. As used herein, functionalized silk refers to a recombinant fusion protein comprising a silk protein and a cell-binding motif. In certain embodiments, the functionalized silk is: RGD, IKVAV (SEQ ID NO: 1), YIGSR (SEQ ID NO: 2), EPDIM (SEQ ID NO: 3), NKDIL (SEQ ID NO: 4), GRKRK (SEQ ID NO: 5), KYGAASIKVAVSADR (SEQ ID NO: 6), NGEPRGDTYRAY (SEQ ID NO: 7), PQVTRGDVFTM (SEQ ID NO: 8), AVTGRGDSPASS (SEQ ID NO: 9), A cell-binding motif selected from TGRGDSPA (SEQ ID NO: 10), CTGRGDSPAC (SEQ ID NO: 11), and C1X1X2RGDX3X4X5C2 (SEQ ID NO: 12), preferably selected from C1X1X2RGDX3X4X5C2, GRKRK, IKVAV, RGD, and CTGRGDSPAC, wherein X1, X2, X3, X4, and X5 are each independently selected from naturally occurring amino acid residues other than cysteine, and C1 and C2 are linked via a disulfide bond.

[0086] In certain embodiments, the functionalized silk comprises a spidroin fragment and a cell-binding motif defined as comprising the amino acid sequence C1X1X2RGDX3X4X5C2 (SEQ ID NO: 12); X1, X2, X3, X4, and X5 are each independently selected from natural amino acid residues other than cysteine, and C1 and C2 are linked via a disulfide bond.

[0087] The functionalized silk is described in WO 2016 / 207281 and WO 2017 / 137611, the disclosures of which are incorporated herein in their entirety. In particular, the functionalized silk may be a recombinant polypeptide comprising the amino acid sequence C1X1X2RGDX3X4X5C2 (SEQ ID NO: 12); X1 is S or T; X2 is G, A, or V; X3 is S or T; X4 is G, A, V, or P; X5 is G, A, or V, and C1 and C2 are linked via a disulfide bond. and the spidroin fragment is composed of protein parts REP and CT, REP is a repeat fragment of 70 to 300 amino acid residues selected from the group consisting of L(AG)nL, L(AG)nAL, L(GA)nL, and L(GA)nGL, where n is an integer of 2 to 10; each A segment is an amino acid sequence of 8 to 18 amino acid residues, in which 0 to 3 of the amino acid residues are not alanine and the remaining amino acid residues are alanine; each G segment is an amino acid sequence of 12 to 30 amino acid residues, in which at least 40% of the 25 amino acid residues are glycine; and each L segment is a linker amino acid sequence of 0 to 30 amino acid residues; and CT is a fragment of 70-120 amino acid residues and has at least 70% identity to SRLSSPSAVSRVSSAVSSLVSNGQVNMAALPNIISNISSSVSASAPGASGCEVIVQALLEVITALVQIVSSSSVGYINPSAVNQITNVVANAMAQVMG (SEQ ID NO: 13). In one embodiment, the CT fragment has at least 70%, such as at least 80%, for example at least 85%, preferably at least 90%, for example at least 95% identity to SEQ ID NO: 13. In certain embodiments, the spidroin fragment has at least 70%, such as at least 80%, for example at least 85%, preferably at least 90%, for example at least 95% identity to amino acid residues 18-277 of SEQ ID NO: 16 or SEQ ID NO: 14. In certain embodiments, the cell-binding motif comprises the amino acid sequence CTGRGDSPAC (SEQ ID NO: 11).

[0088] In certain embodiments, the functionalized silk comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15. In certain embodiments, the functionalized silk has at least 70%, such as at least 80%, such as at least 85%, preferably at least 90%, such as at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15.

[0089] Thus, in one embodiment, the 2D substrate does not contain animal-derived components. In one embodiment, the 2D substrate is not Matrigel™. In other words, components of the 2D substrate may be recombinantly produced components.

[0090] Thus, in one embodiment, the 2D substrate may comprise or consist of laminin (LN) and fragments thereof, such as recombinantly produced laminin (LN) and fragments thereof.

[0091] Laminins are macromolecular proteins of the extracellular matrix. They are major components of basement membranes, the underlying protein network of most cells and organs. Laminins are an important biologically active part of basement membranes and influence cell differentiation, migration, and adhesion. Therefore, the selection of laminins for use as cell culture substrates is crucial for providing cells with an appropriate chemo- and mechanosensitive microenvironment and optimal culture conditions.

[0092] Each laminin isoform consists of three intercoiled chains (α, β, and γ chains), with five, four, and three genetically distinct variants, respectively. Laminin isoforms are named according to their chain composition. For example, the combination of α5, β2, and γ1 chains forms laminin 5-2-1 (LN-521). The trimeric protein forms a cruciform structure that can bind to other extracellular matrix molecules and various cell membrane receptors.

[0093] Therefore, the choice of laminin or its fragments for use as a 2D substrate is important and influenced by the cell type being cultured. Furthermore, differences between cell lines, such as different ES or iPS cell lines or primary cells, can affect the selection of the optimal 2D substrate. While full-length laminin is useful as a cell culture substrate, its fragments can also be used for cell culture. Different domains of laminin have been identified that mediate various activities, including cell adhesion and influencing cell proliferation, differentiation, and motility.

[0094] In one embodiment of the method disclosed herein, the laminin (LN) and fragments thereof are selected from the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, LN-121 and fragments thereof, and LN-111 and fragments thereof, such as the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof, such as the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, and LN-332 and fragments thereof, such as the group consisting of LN-521 and fragments thereof, or the group consisting of LN-511 and fragments thereof.

[0095] In one embodiment, the laminin and fragments thereof are selected from the group consisting of LN-521, LN-511, LN-332, LN-421, LN-121, and LN-111, such as the group consisting of LN-521, LN-511, LN-332, LN-421, and LN-121, such as the group consisting of LN-521, LN-511, and LN-332, such as the group consisting of LN-521 and LN-511, e.g., the laminin and fragments thereof are LN-521, or e.g., the laminin and fragments thereof are LN-511.

[0096] Laminin E8 fragments, truncated proteins consisting of the C-terminal regions of the α, β, and γ chains, are known. These laminin fragments contain the laminin globular 1-3 domain of the α chain and an active integrin-binding site containing the C-terminal glutamic acid residue of the γ chain, but lack other activities associated with full-length laminin, such as heparin / heparan sulfate binding activity. The E8 fragment represents a functionally minimal form that retains the full ability to bind to α6β1 integrin. In one embodiment, the fragment(s) are E8 fragment(s).

[0097] Thus, in one embodiment, the laminin and fragments comprise an E8 fragment of laminin, such as an E8 fragment selected from the group consisting of an E8 fragment of LN-511, an E8 fragment of LN-521, an E8 fragment of LN-332, an E8 fragment of LN-421, an E8 fragment of LN-121, and an E8 fragment of LN-111, such as an E8 fragment of LN-511, an E8 fragment of LN-521, an E8 fragment of LN-332, an E8 fragment of LN-421, and an E8 fragment of LN-121, such as an E8 fragment of LN-511, an E8 fragment of LN-521, and an E8 fragment of LN-332, such as an E8 fragment of LN-511 and an E8 fragment of LN-521, such as an E8 fragment of LN-511 or an E8 fragment of LN-521.

[0098] As noted above, the present disclosure is based on the surprising realization that short-term culture of posterior foregut (PF) cells under conditions permissive for differentiation into pancreatic progenitor (PP) cells results in fewer PP cells but higher numbers of endocrine progenitor (EP) cells compared to corresponding methods involving longer culture times.

[0099] Thus, in one embodiment of this aspect of the invention, there is provided a method of differentiating at least about 70%, such as at least about 75%, such as at least about 80% of the posterior foregut cells of a) into pancreatic progenitor cells of c).

[0100] In one embodiment, there is provided a method disclosed herein, wherein in step c), at least about 80%, for example, about 80-85%, for example, about 80-90% of the total cell population expresses PDX1. In one embodiment, there is provided a method disclosed herein, wherein in step c), up to about 10%, for example, up to about 8%, for example, up to about 7%, for example, up to about 5%, for example, up to about 3% of the total cell population expresses NEUROD1. In one embodiment, there is provided a method disclosed herein, wherein in step c), up to about 10%, for example, up to about 8%, for example, up to about 7%, for example, up to about 5%, for example, up to about 3% of the total cell population expresses NEUROD1 and does not express NKX6.1. In one embodiment, there is provided a method disclosed herein, wherein in step c), about 25-50%, for example, about 25-48%, for example, about 25-45% of the total cell population expresses NKX6.1. In one embodiment, there is provided a method as disclosed herein, wherein in step c), about 30-50%, for example about 35-45%, for example about 40-45% of the total cell population expresses NKX6.1. In one embodiment, in step c), at least about 40%, for example at least about 50%, for example at least about 60% of the cells do not express NEUROD1 and NKX6.1.

[0101] In one embodiment, said posterior foregut cells are characterized in a) by expression of PDX1 and lack expression of NKX6.1.

[0102] In another embodiment of the method disclosed herein, the method comprises, prior to steps a) to c), the following steps a-1) to c-1): a-1) providing a cell population of primitive intestinal cells, such as primitive intestinal cells, characterized by the expression of HNF1β and / or HNF4α; b-1) culturing the population of primitive intestinal cells for about 54 hours or less under conditions that allow differentiation into posterior foregut cells; and c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1.

[0103] It will be appreciated that posterior foregut cells may similarly be characterized by HNF6 or co-expression of PDX1 and HNF6.

[0104] As used herein, each method step is designated by a letter code, such as a), b), c), etc. A complete method group for culturing a cell population at a particular developmental stage under appropriate conditions to differentiate into a population at a next developmental stage includes steps a), b), c), etc. Each step may be further designated by a letter code with an associated numeric indicator. The numeric indicator indicates whether each step within the complete method group precedes (negative numeric indicator) or follows (positive numeric indicator) the complete method group for culturing posterior foregut (PF) cells to generate pancreatic progenitor cells (PP). The method steps associated with a complete method group for culturing a cell population at a certain developmental stage under appropriate conditions to differentiate into a population at a next developmental stage are set forth below in Table 1.

[0105] [Table 1]

[0106] In one embodiment of the method disclosed herein, the cell population of primitive intestinal cells provided in step a-1) is characterized by expression of HNF1β, HNF4α, or both HNF1β and HNF4α.

[0107] In one embodiment, the posterior foregut cell population is characterized by expression of PDX1 or HNF6. In one embodiment, the posterior foregut cell population is characterized by expression of PDX1 and HNF6. In one embodiment of the method disclosed herein, the cell population of posterior foregut cells in step c-1) is characterized by expression of PDX1 and lack of expression of NKX6.1.

[0108] In one embodiment, the posterior foregut cell population in c-1) is characterized by expression of PDX1 and HNF6 and lacks expression of NKX6.1. Said population of posterior foregut cells does not express NKX6.1, PTF1A, and SOX9.

[0109] Without being bound by theory, it is believed that in order to obtain a posterior foregut cell population capable of developing into later stages of the β cell lineage, it is beneficial that step b-1) of culturing the cell population of primitive intestinal cells under conditions permissive for differentiation into posterior foregut cells does not exceed 54 hours. It is assumed that primitive intestinal cells cultured for longer than 54 hours under conditions permissive for differentiation into posterior foregut cells have a low ability to develop into later stages of the β cell lineage. In one embodiment of the method disclosed herein, the cell population in step b-1) is cultured for about 52 hours or less, for example, about 50 hours or less, for example, about 48 hours or less, for example, about 44 hours or less, for example, about 40 hours or less, for example, about 36 hours or less, for example, about 32 hours or less, for example, about 28 hours or less, for example, about 26 hours or less, for example, about 24 hours.

[0110] In one embodiment of the method disclosed herein, the cell population in step b-1) is cultured for a period of about 18 to 54 hours, for example, about 20 to 52 hours, for example, about 22 to 50 hours, for example, about 24 to 48 hours. In one embodiment, the cell population in step b-1) is cultured for a period of about 42 to 54 hours, for example, about 44 to 52 hours, for example, about 46 to 50 hours, for example, about 48 hours. In one embodiment, the cell population in step b-1) is cultured for a period of about 18 to 30 hours, for example, about 20 to 28 hours, for example, about 22 to 26 hours, for example, about 24 hours.

[0111] As described above, "conditions permissive for differentiation" refers to conditions that allow cells to develop / differentiate to exhibit characteristics of the cell type, and may include the combination of cell culture medium, the presence or absence of exogenous factors, and the timing thereof. These factors and their derivatives and agonists have been described in detail in connection with step b) above, and for the sake of brevity, the description will not be repeated here. Thus, in one embodiment, a method is provided in which step b-1) comprises culturing the cell population in a culture medium containing one or more factors selected from KGF and its derivatives and agonists, retinoic acid and its derivatives and agonists, SANT-1 and its derivatives and agonists, PDBu and its derivatives and agonists, and LDN and its derivatives and agonists, e.g., one or more factors selected from KGF, retinoic acid, SANT-1, PDBu, and LDN. In one embodiment, step b-1) comprises culturing the cell population in a culture medium containing one or more factors selected from KGF and its derivatives and agonists, and retinoic acid and its derivatives and agonists, e.g., one or more factors selected from KGF and retinoic acid, e.g., both KGF and retinoic acid. In one embodiment, step b-1) comprises culturing the cell population in a culture medium further containing KGF or a derivative or agonist thereof, retinoic acid or a derivative or agonist thereof, SANT-1 or a derivative or agonist thereof, PDBu or a derivative or agonist thereof, and LDN or a derivative or agonist thereof, e.g., a culture medium containing KGF, retinoic acid, SANT-1, PDBu, and LDN. It will be understood that the culture medium may contain a mixture of factors and their derivatives and / or agonists.

[0112] In one embodiment, the culture medium in step b-1) is a culture medium suitable for culturing primitive intestinal cells under conditions that allow differentiation into posterior foregut cells. Non-limiting examples of the medium for step 3 (S3) are as defined in this Example. Those skilled in the art will understand that other suitable media may also be used. The culture medium in step b-1) may be supplemented with other factors as specified herein.

[0113] In one embodiment, the culture medium in step b-1) contains 25 to 75 ng / mL of KGF or a derivative or agonist thereof, for example, 50 ng / mL of KGF or a derivative or agonist thereof. In one embodiment, the medium in step b-1) contains about 25 to 75 ng / mL of KGF, for example, about 50 ng / mL of KGF. In one embodiment, the medium in step b-1) contains about 1 to 3 μM of RA, for example, about 2 μM of RA.

[0114] In one embodiment, the medium in step b-1) contains about 0.10 to 0.50 μM SANT-1, for example, about 0.25 μM SANT-1.

[0115] In one embodiment, the medium in step b-1) contains about 250 to 750 nM PDBu, for example, about 500 nM PDBu.

[0116] In one embodiment, the medium in step b-1) contains about 100 to 300 nM LDN, for example, 100 to 250 nM LDN, for example, 150 to 250 nM LDN, for example, about 200 nM LDN.

[0117] In one embodiment, step b-1) of the method disclosed herein comprises culturing the cell population in a culture medium comprising about 50 ng / mL KGF, about 2 μM retinoic acid, about 0.25 μM SANT-1, about 500 nM PDBu, and about 200 nM LDN.

[0118] In another embodiment of the method disclosed herein, the method comprises, after steps a) to c), the following steps a+1) to c+1): a+1) providing a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of PDX1 and NKX6.1; b+1) culturing the population of pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells; and c+1) thereby generating a population of endocrine precursor cells characterized by expression of NEUROD1, such as expression of NKX6.1 and NEUROD1.

[0119] In one embodiment, the endocrine precursor cell population is further characterized by the expression of at least one of NKX6.1 and NGN3.Those skilled in the art will understand that different combinations of two markers, such as NKX6.1 and NGN3, NKX6.1 and NEUROD1, or NKX6.1 and NGN3, can also be used on endocrine precursor cells to distinguish these cells from pancreatic precursor cells.For example, the endocrine precursor cell population in step c+1) can be characterized by the expression of PDX1, NKX6.1, and NEUROD1, or PDX1, NKX6.1, NEUROD1, and NGN3, or PDX1, NKX6.1, NEUROD1, and NGN3.PDX1 + / NKX6.1 + It will be understood that expression of NGN3 or NEUROD1 in cells (in other words, cells positive for both PDX1 and NKX6.1) indicates that the pancreatic progenitor cells have committed to an endocrine precursor cell fate. Those skilled in the art will understand that due to experimental limitations in staining protocols, expression of NGN3 or NEUROD1 may be assessed in cells stained for PDX1 or NKX6.1. In one embodiment of the method disclosed herein, the cell population of endocrine precursor cells in step c+1) is characterized by expression of PDX1, NKX6.1 and NEUROD1, or PDX1, NKX6.1 and NGN3, or PDX1, NKX6.1, NEUROD1 and NGN3.

[0120] In one embodiment of the methods disclosed herein, the cell population of pancreatic progenitor cells provided in step a+1) is characterized by expression of PDX1 and NKX6.1, in other words, co-expression of PDX1 and NKX6.1. In another embodiment, the cell population of pancreatic progenitor cells in step a+1) is further characterized by expression of one or more of the markers PTF1A, SOX9, and HNF6, such as two of the markers PTF1A, SOX9, and HNF6, such as all three of the markers PTF1A, SOX9, and HNF6. In another embodiment, the cell population of pancreatic progenitor cells in step a+1) is further characterized by expression of one or both of the markers PTF1A and SOX9.

[0121] In embodiments of the methods disclosed herein, the cell population in step b+1) is cultured for about 3-5 days, e.g., about 3-4 days, or about 4-5 days, e.g., about 4 days. In one embodiment, the cell population in step b+1) is cultured for about 5 days. This culture time is believed to be sufficient to generate the endocrine precursor cell population in step c+1).

[0122] Similar to the steps a) to c), steps a+1) to c+1) are performed in 2D culture on a 2D substrate. Note that the descriptions regarding the 2D substrate related to steps a) to c) are similarly relevant to steps a+1) to c+1) and will not be repeated here for the sake of brevity.

[0123] Thus, in one embodiment disclosed herein, the cells of step b+1) are cultured on a 2D substrate, in one embodiment, said cells are adherent to said 2D substrate. The 2D substrate may comprise one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and Matrigel™, e.g., selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, and Matrigel™, e.g., selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen, gelatin, and Matrigel™, e.g., selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen, gelatin, and Matrigel™.

[0124] In one embodiment, the 2D substrate may comprise or consist of laminin (LN) and fragments thereof, such as recombinantly produced laminin (LN) and fragments thereof.

[0125] In one embodiment of the method disclosed herein, the laminin (LN) and fragments thereof are selected from the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, LN-121 and fragments thereof, and LN-111 and fragments thereof; For example, the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof; For example, the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, and LN-332 and fragments thereof; For example, the laminin and fragments thereof are selected from the group consisting of LN-521 and fragments thereof, or LN-511 and fragments thereof. In one embodiment, the laminin and fragments thereof are selected from the group consisting of LN-521, LN-511, LN-332, LN-421, LN-121, and LN-111, such as the group consisting of LN-521, LN-511, LN-332, LN-421, and LN-121, such as the group consisting of LN-521, LN-511, and LN-332, such as the group consisting of LN-521 and LN-511, for example, the laminin and fragments thereof are LN-521 or the laminin and fragments thereof are LN-511.

[0126] In one embodiment, the fragment(s) are E8 fragment(s). In one embodiment, the laminin and fragments comprise an E8 fragment of laminin, e.g., an E8 fragment selected from the group consisting of an E8 fragment of LN-511, an E8 fragment of LN-521, an E8 fragment of LN-332, an E8 fragment of LN-421, an E8 fragment of LN-121, and an E8 fragment of LN-111, e.g., an E8 fragment of LN-511, an E8 fragment of LN-521, an E8 fragment of LN-332, an E8 fragment of LN-421, and an E8 fragment of LN-121, e.g., an E8 fragment of LN-511, an E8 fragment of LN-521, and an E8 fragment of LN-332, e.g., an E8 fragment of LN-511 and an E8 fragment of LN-521, e.g., an E8 fragment selected from the group consisting of an E8 fragment of LN-511 or an E8 fragment of LN-521.

[0127] As mentioned above, "conditions permissive for differentiation" refers to conditions that allow cells to exhibit characteristics of the cell type, and may include the combination of cell culture medium, the presence or absence of exogenous factors, and the timing thereof. These factors and their derivatives and agonists have been described in detail in connection with steps b) and b-1) above, and for the sake of brevity, the description will not be repeated here.

[0128] In one embodiment, the culture medium in step b+1) is a culture medium suitable for culturing pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells. Non-limiting examples of the medium for step 5 (S5) are as defined in this Example. Those skilled in the art will understand that other suitable media may also be used. The culture medium in step b+1) may be supplemented with other factors as specified herein.

[0129] Thus, in one embodiment, a method is provided in which step b+1) comprises culturing the cell population in a culture medium containing one or more factors selected from BTC and derivatives and agonists thereof, retinoic acid and derivatives and agonists thereof, Alk5 inhibitors (such as Alk5iII) and derivatives and agonists thereof, retinoic acid and derivatives and agonists thereof, γ-secretase inhibitors (such as GSI-XX) and derivatives and agonists thereof, GC-1 and derivatives and agonists thereof, LDN and derivatives and agonists thereof, retinoic acid and derivatives and agonists thereof, and SANT-1 and derivatives and agonists thereof, for example, one or more factors selected from BTC, Alk5 inhibitors (such as Alk5iII), γ-secretase inhibitors (such as GSI-XX), GC-1, LDN, retinoic acid, and SANT-1. Thus, in one embodiment, a method is provided in which step b+1) comprises culturing the cell population in a culture medium comprising BTC or a derivative or agonist thereof, an Alk5 inhibitor (such as Alk5iII) or a derivative or agonist thereof, a γ-secretase inhibitor (such as GSI-XX) or a derivative or agonist thereof, GC-1 or a derivative or agonist thereof, LDN or a derivative or agonist thereof, retinoic acid or a derivative or agonist thereof, and SANT-1 or a derivative or agonist thereof, for example BTC, an Alk5 inhibitor (such as Alk5iII), a γ-secretase inhibitor (such as GSI-XX), GC-1, LDN, retinoic acid, and SANT-1. Thus, in one embodiment, a method is provided, wherein step b+1) comprises culturing the cell population in a culture medium comprising BTC and / or a derivative and / or agonist thereof, an Alk5 inhibitor (such as Alk5iII) and / or a derivative and / or agonist thereof, a γ-secretase inhibitor (such as GSI-XX) and / or a derivative and / or agonist thereof, GC-1 and / or a derivative and / or agonist thereof, LDN and / or a derivative and / or agonist thereof, retinoic acid and / or a derivative and / or agonist thereof, and SANT-1 and / or a derivative and / or agonist thereof.It will be understood that the culture medium may contain a mixture of factors and their derivatives and / or agonists. In one embodiment of the methods disclosed herein, step b+1) comprises culturing the cell population in a culture medium comprising BTC, Alk5iII, GSI-XX, GC-1, LDN, retinoic acid, and SANT-1. In particular, in one embodiment, a method is provided in which step b+1) comprises culturing the cell population in a culture medium comprising at least an Alk5 inhibitor or a derivative or agonist thereof, and a γ-secretase inhibitor and a derivative and agonist thereof, such as Alk5iII and GSI-XX.

[0130] BTC, also known as betacellulin, is a member of the EGF family of growth factors that induces differentiation of beta cells as well as other cell types. As used herein, the term "BTC and its derivatives and agonists" refers to EGFR ligand / EGF family growth factors.

[0131] An example of an ALK5 inhibitor is Alk5iII (ALK5 inhibitor II), which is a cell-permeable, selective inhibitor of the TGF-β type 1 activin-like kinase receptor ALK5. Non-limiting examples of ALK5 inhibitors include Alk5iII, LY2157299, LY364947, RepSox, SB525334, A83-01, GW788388, LY-2109761, SB-505124, and D4476. In one embodiment, the Alk5 inhibitor or its derivative or agonist is selected from the group consisting of Alk5iII, LY2157299, LY364947, RepSox, SB525334, A83-01, GW788388, LY-2109761, SB-505124, and D4476.

[0132] GSI-XX, also known as γ-secretase inhibitor XX, is a cell-permeable dibenzazepine compound that inhibits γ-secretase. γ-Secretase is a multisubunit protease complex, itself a transmembrane protein, cleaving single-pass transmembrane proteins at residues within the transmembrane domain. Non-limiting examples of γ-secretase inhibitors include GSI-XX, DAPT, RO4929097, YO-01027, BMS-906024, Aβ42-IN-2, LY-411575, and MK-0752. Thus, in one embodiment, the γ-secretase inhibitor is selected from GSI-XX, DAPT, RO4929097, YO-01027, BMS-906024, Aβ42-IN-2, LY-411575, and MK-0752.

[0133] GC-1 is a thyroid hormone receptor (TR) agonist and is more potent than the thyroid hormone T3, which is important for β-cell development.

[0134] As used herein, the term "GC-1 and its derivatives and agonists" refers to GC-1, T3, and T4. Thus, in one embodiment, the GC-1 and its derivatives and agonists are selected from the group consisting of GC-1, T3, and T4. In one embodiment, the medium in step b+1) contains about 10 to 30 ng / mL of BTC, for example, about 20 ng / mL of BTC.

[0135] In one embodiment, the medium in step b+1) contains about 5 to 15 μM Alk5iII, for example about 10 μM Alk5iII.

[0136] In one embodiment, the medium in step b+1) contains about 50 to 150 nM GSI-XX, for example about 100 nM GSI-XX.

[0137] In one embodiment, the medium in step b+1) contains about 0.5 to 1.5 μM GC-1, for example about 1 μM GC-1.

[0138] In one embodiment, the medium in step b+1) contains about 50 to 150 nM LDN, for example, 75 to 125 nM LDN, for example, about 100 nM LDN.

[0139] In one embodiment, the medium in step b+1) contains about 25 to 150 nM RA, for example about 100 nM RA.

[0140] In one embodiment, the medium in step b-1) contains about 0.10 to 0.50 μM SANT-1, for example, about 0.25 μM SANT-1.

[0141] In one embodiment, step b+1) of the method disclosed herein comprises culturing a cell population in a culture medium comprising about 20 ng / mL BTC, about 10 μM Alk5iII, about 100 nM GSI-XX, about 1 μM GC-1, about 100 nM LDN, about 100 nM RA, and about 0.25 μM SANT-1.

[0142] As noted above, the present disclosure is based on the surprising realization that short-term culture of posterior foregut (PF) cells under conditions permissive for differentiation into pancreatic progenitor cells (PP) gives rise to more endocrine progenitor cells (EP), although fewer PP cells are obtained than corresponding methods involving longer culture times.

[0143] In one embodiment disclosed herein, a method is provided, wherein in step c+1), more than about 30%, such as more than about 40%, 40%, such as more than about 45%, such as more than about 50% of the total cell population are endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and at least NEUROD1. As explained and exemplified above, other combinations of two or more of the markers PDX1, NKX6.1, NEUROD1, and NGN3 can be used as a characteristic of endocrine precursor cells.

[0144] In one embodiment, a method is provided as disclosed herein, wherein the number of endocrine precursor cells in step c+1) is higher than the number of endocrine precursor cells obtained using a corresponding method in which step b) of culturing a cell population of posterior foregut cells under conditions permissive for differentiation into pancreatic precursor cells is performed for about 24 hours or less and / or about 96 hours or more. As shown in the accompanying Examples, the number of EP cells obtained by the methods disclosed herein is significantly higher than methods using longer or shorter culturing times in step b). This is particularly surprising and unexpected, as the number of PP cells is lower than the number of PP cells obtained when step b) is performed for about 24 hours or less and / or about 96 hours or more. This effect is demonstrated in the accompanying Examples, particularly Figure 4, where the number of endocrine precursor cells is scored by coexpression of NKX6.1 and NEUROD1. Importantly, this effect has been demonstrated in cell cultures of various human stem cell lines, including human ES cell lines and iPSC cell lines.

[0145] In one embodiment, the method results in at least about 10%, such as at least about 15%, for example at least about 20%, such as at least about 30%, for example at least about 40%, such as at least 50%, for example at least 60% more endocrine precursor cells than a corresponding method in which step b) of culturing the cell population of posterior foregut cells under conditions permissive for differentiation into pancreatic precursor cells is for about 24 hours or less and / or for about 96 hours or more.

[0146] It will be appreciated that the endocrine precursor cells obtained in step c+1) can be further differentiated into pancreatic monohormonal beta cells. Thus, in one embodiment of the methods described herein, the method further comprises culturing said endocrine precursor cells under conditions that allow differentiation into pancreatic beta cells, such as monohormonal pancreatic beta cells.

[0147] In one embodiment of the method, the cells are not transferred from culture on a 2D substrate to culture on a 3D substrate before they exhibit expression of markers characteristic of endocrine precursor cells, which are characterized by expression of PDX1, NKX6.1, and at least one of NEUROD1 and NGN3, as described above. In one embodiment, there is provided a method as disclosed herein, said method further comprising, after steps a+1) to c+1), the following steps a+2) to c+2): a+2) transferring a population of endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1, from culture on a 2D substrate to 3D culture conditions; b+2) culturing the endocrine precursor cell population under conditions that allow differentiation into pancreatic monohormonal β cells; and c+2) thereby generating a population of pancreatic monohormonal beta cells, such as pancreatic monohormonal beta cells characterized by the expression of insulin, said monohormonal beta cells further capable of expressing at least one of NKX6.1, PDX1, and NEUROD1.

[0148] In one embodiment, the pancreatic monohormonal beta cell population generated in step c+2) is part of at least one pancreatic islet-like cell aggregate.

[0149] In one embodiment, step a+2) of transferring the endocrine precursor cell population from culture on a 2D substrate to 3D culture conditions comprises i) providing a single cell suspension of the EP cell population, and ii) allowing the EP cell population in the single cell suspension to form a 3D structure. In one embodiment, step a+2 is performed under conditions permissive for differentiation into pancreatic monohormonal β cells, e.g., in a cell culture medium permissive for differentiation of pancreatic monohormonal β cells. In one embodiment, step i) of providing a single cell suspension of the EP cell population is performed 24, 48, 72, or 96 days after transferring the cells from conditions permissive for differentiation into endocrine precursor cells to conditions permissive for differentiation into pancreatic monohormonal β cells, e.g., 24, 48, 72, or 96 days after transferring the cells from a cell culture medium permissive for differentiation into endocrine precursor cells to a cell culture medium permissive for differentiation into pancreatic monohormonal β cells.

[0150] In step ii), the EP cells in single cell suspension are allowed to form 3D structures. In one embodiment, the 3D culture conditions allow the cells to self-aggregate. The aggregation can be forced or induced, but can also be spontaneous. The aggregation leads to the formation of islet-like cell aggregates, as disclosed herein.

[0151] In one embodiment, step ii) is performed in the presence of a ROCK inhibitor. The ROCK inhibitor may be H1152 or any analog or agonist thereof. In one embodiment, the concentration of H1152 is in the range of 0 to 10 μM. As shown in this example, without being bound by theory, the inventors believe that H1152 may promote the survival of S5 EP cells as single cells in suspension. Therefore, in one embodiment of the methods disclosed herein, the ROCK inhibitor is present in the culture medium for about 24 hours during step ii).

[0152] Those skilled in the art will understand that step i) of providing a single-cell suspension of EP cell populations involves dissociating EP cells from adherent cultures on 2D substrates into single cells. Such dissociation may involve the use of dissociation reagents, such as natural enzymes, gentler non-enzymatic alternatives, or may function by chelating calcium to prevent cadherin attachment and release cells from the surface and each other. Dissociation may also be achieved by mechanical means. Non-limiting examples of dissociation reagents include trypsin, collagenase, displase, and dissociation reagents such as Accutase®, Accumax™, and ACS-3010. In one embodiment, step i) of providing a single-cell suspension of EP cell populations involves dissociation of EP cells by enzymatic means, such as using a solution containing enzymes, e.g., proteolytic and / or collagenolytic enzymes. For example, such solutions include Accutase®. Those skilled in the art will recognize suitable methods for dissociating EP cells and providing a single-cell suspension of EP cell populations.

[0153] In one embodiment, the isolation is performed before exposing the cells to conditions permissive for differentiation into pancreatic monohormonal β cells, e.g., before culturing the cells in a medium permissive for differentiation into pancreatic monohormonal β cells. In one embodiment, the isolation is performed within 96 hours, e.g., within 72 hours, e.g., within 48 hours, e.g., within 24 hours, after changing the culture medium from a medium permissive for differentiation into endocrine precursor cells to a medium permissive for differentiation into pancreatic monohormonal β cells. For example, as described in the accompanying Examples, this may be within 96 hours, 72 hours, 48 hours, or 24 hours, e.g., within 24 to 48 hours, after changing from S5 medium to S6 medium. In one embodiment, the isolation is performed within 24 to 48 hours after changing from conditions permissive for differentiation into endocrine precursor cells to conditions permissive for differentiation into pancreatic monohormonal β cells.

[0154] In step ii), the EP cells in single cell suspension are allowed to form 3D structures. In one embodiment, the 3D culture conditions allow the cells to self-aggregate. The aggregation can be forced or induced, but can also be spontaneous.

[0155] In one embodiment, the culture medium in step b+2) is a culture medium suitable for culturing endocrine precursor cells under conditions that allow differentiation into monohormonal beta cells. Non-limiting examples of culture media for step 6 (S6) are as defined in this Example. Those skilled in the art will understand that other suitable media may also be used. The culture medium in step iv) may be supplemented with other factors as specified herein.

[0156] Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is a water-soluble analog of vitamin E with potent antioxidant properties. Trolox is also a potent inhibitor of membrane damage. In one embodiment, the medium in step iv) contains about 5-15 μM Trolox, for example, about 10 μM Trolox. As will be appreciated by those skilled in the art, Trolox can be substituted with its derivatives or agonists.

[0157] N-acetyl-L-cysteine is a cell culture component, such as intestinal basal medium for culturing mouse intestinal stem cells, and is also used as a component of growth medium. In one embodiment, the medium in step iv) contains about 0.5 to 3 mM N-acetyl-L-cysteine, for example, about 1 mM N-acetyl-L-cysteine. As will be appreciated by those skilled in the art, N-acetyl-L-cysteine can be substituted with a derivative or agonist thereof.

[0158] H1152 is a Rho kinase inhibitor and is a cell-permeable, highly specific, reversible, potent, and ATP-competitive inhibitor of Rho-associated kinase (ROCK). As will be appreciated by those skilled in the art, H1152 can be substituted with derivatives or agonists thereof.

[0159] GC-1 is a thyroid hormone receptor (TR) agonist and is more potent than the thyroid hormone T3, which is important for β-cell development and is described in more detail below. In one embodiment, the medium in step iv) contains about 0.5 μM to 3 μM GC-1, for example, about 1 μM GC-1. As will be appreciated by those skilled in the art, GC-1 can be replaced with its derivatives or agonists.

[0160] In one embodiment, step b+2) of the method disclosed herein comprises culturing the EP cell population in a culture medium comprising H1152, GC-1, Trolox, and N-acetyl-L-cysteine. In one embodiment, step iv) of the method disclosed herein comprises culturing the EP cell population in a culture medium comprising H1152, GC-1, Trolox, and N-acetyl-L-cysteine for about 3 weeks, followed by culturing the EP cell population in a medium comprising GC-1, Trolox, and N-acetyl-L-cysteine but without H1152 for about 3 weeks.

[0161] In one embodiment, step b+2) of the method disclosed herein comprises culturing the EP cell population in a culture medium comprising about 10 μM H1152, about 1 μM GC-1, about 10 μM Trolox, and about 1 mM N-acetyl-L-cysteine. In one embodiment, step iv) of the method disclosed herein comprises culturing the EP cell population in a culture medium comprising about 10 μM H1152, about 1 μM GC-1, about 10 μM Trolox, and about 1 mM N-acetyl-L-cysteine for about 3 weeks, followed by culturing in a medium lacking H1152 but comprising about 1 μM GC-1, about 10 μM Trolox, and about 1 mM N-acetyl-L-cysteine.

[0162] In one embodiment, the culturing in step b+2) is carried out on a shaker, such as an orbital shaker.

[0163] In one embodiment, the 3D culture conditions allow cells to self-aggregate. Without being bound by theory, it is believed that self-aggregation allows for selective enrichment of endocrine precursor cells. Furthermore, it is believed that the selective enrichment increases the production of pancreatic monohormonal beta cells in the culture. In one embodiment, the pancreatic monohormonal beta cells are produced as part of cell aggregates. In one embodiment, the aggregates comprise monohormonal beta cells. In one embodiment, the aggregates further comprise pancreatic monohormonal alpha cells and / or delta cells.

[0164] In one embodiment, the endocrine precursor cell population in step a+2) is characterized by the expression of NKX6.1, PDX1, and NEUROD1, or is characterized by the expression of NKX6.1, PDX1, and NGN3, or is characterized by the expression of PDX1, NKX6.1, NEUROD1, and NGN3.

[0165] In one embodiment, the monohormonal beta cell population is characterized by expression of insulin and PDX1. In one embodiment, the monohormonal beta cell population is characterized by expression of insulin and NKX6.1. In one embodiment, the monohormonal beta cell population is characterized by expression of insulin and NEUROD1. In one embodiment, the monohormonal beta cell population is characterized by expression of insulin and two of NKX6.1, PDX1, and NEUROD1, such as insulin and NKX6.1 and PDX1, or insulin and NKX6.1 and NEUROD1, or insulin and PDX1 and NEUROD1. In one embodiment, the monohormonal beta cell population is characterized by expression of insulin, PDX1, NKX.1, and NEUROD1.

[0166] In one embodiment, steps a+2) to c+2) follow steps a+1) to c+1), such as immediately after steps a+1) to c+1). It will be appreciated that the endocrine precursor cells can be cryopreserved for a desired period of time before steps a+2) to c+2) are performed. In this case, steps a+2) to c+2) follow a cryopreservation step followed by recovery of the cryopreserved cells. Thus, steps a+1) to c+1) can be followed by cryopreservation, e.g., a cryopreservation step followed by recovery of the cryopreserved cells, which is followed by steps a+2) to c+2). Those skilled in the art are familiar with the cryopreserved cell recovery process, which typically involves rapidly thawing the cells in a 37°C water bath, removing the cells from the freezing medium by gentle centrifugation and / or dilution with growth medium, and seeding the cells into a culture vessel containing complete growth medium. Thus, in one embodiment, steps a+2) to c+2) follow the cryopreservation and subsequent recovery of the EP cells obtained in c+1). As shown in the accompanying examples, cryopreservation does not adversely affect the generation of the islet-like cell aggregates or monohormonal beta cells.

[0167] In one embodiment, step b+2) comprises culturing the endocrine precursor cell population for about 3 weeks or more, for example, about 3 to 5 weeks, for example, about 4 weeks.

[0168] As used herein, the term "monohormonal" refers to cells that express only one type of hormone, such as monohormonal beta cells that express only insulin and not other hormones expressed by pancreatic islet cells, such as glucagon or somatostatin. As used herein, the term "polyhormonal" refers to cells that express at least two different hormones.

[0169] Advantageously, monohormonal β cells in vivo are monohormonal cells, and the population obtained by the methods of the invention exhibits characteristics of natural in vivo β cells or endogenous in vivo β cells, e.g., characteristics of healthy natural in vivo β cells or healthy endogenous in vivo β cells.

[0170] Thus, in one embodiment of the method of the invention disclosed herein, the beta cell population generated in step c+2) is monohormonal. In particular, the monohormonal beta cell population generated in step c+2) does not express glucagon or somatostatin. In particular, the monohormonal beta cell population generated in step c+2) does not express glucagon and somatostatin.

[0171] It will be appreciated that the increased proportion and / or number of EP cells obtained by the methods of the present invention compared to prior art methods comprising step b) culturing a population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for at least about 96 hours may result in an increased proportion of monohormonal β cells as defined herein, e.g., an increased proportion of monohormonal β cells in the islet-like cell aggregates.

[0172] Thus, in one embodiment of the methods disclosed herein, step c+2) provides a method in which greater than about 40%, e.g., about 40-50%, e.g., about 40-60%, e.g., about 40-70%, of the total cell population are monohormonal β cells, e.g., monohormonal β cells characterized by insulin expression. In one embodiment, the method results in at least 30%, e.g., at least about 35%, e.g., at least about 40% more monohormonal β cells, e.g., monohormonal β cells characterized by insulin expression, than a corresponding method in step b) culturing the cell population of posterior foregut cells for about 96 hours under conditions permissive for differentiation into pancreatic progenitor cells. The monohormonal β cells may be characterized by at least one of insulin and NKX6.1, PDX1, and NEUROD1, e.g., expression of insulin and NKX6.1.

[0173] In one embodiment, the whole cell population of monohormonal beta cells expresses insulin and is further characterized by expression of NKX6.1, PDX1, and / or NEUROD1. In one embodiment, the whole cell population of monohormonal beta cells is characterized by expression of insulin and NKX6.1, insulin and PDX1, or insulin and NEUROD1.

[0174] In one embodiment, the entire population of monohormonal beta cells is characterized by expression of insulin and two of NKX6.1, PDX1, and NEUROD1, such as insulin and NKX6.1 and PDX1, or insulin and NKX6.1 and NEUROD1, or insulin and PDX1 and NEUROD1. In one embodiment, the monohormonal beta cell population is characterized by expression of insulin, PDX1, NKX6.1, and NEUROD1.

[0175] In one particular embodiment, the method results in at least two times more monohormonal β cells, such as monohormonal pancreatic β cells characterized by the expression of insulin, than monohormonal pancreatic α cells characterized by the expression of glucagon.

[0176] Those skilled in the art understand that monohormonal pancreatic β cells not only express insulin along with markers characteristic of mature pancreatic β cells, but also that the monohormonal β cells are functional pancreatic β cells and therefore capable of responding to glucose stimulation. The outcome of glucose stimulation may be scored by insulin production and / or C-peptide expression. C-peptide is released simultaneously with insulin, and for every insulin molecule produced, there is a C-peptide molecule produced by the β cell. While C-peptide itself does not affect blood glucose levels, C-peptide tends to remain in the blood longer than insulin, making it a useful marker of insulin production.

[0177] As mentioned above, the pancreatic monohormonal beta cell population obtained in step c+2) may be part of at least one pancreatic islet-like cell aggregate.

[0178] Thus, in one embodiment, the islet-like cell aggregates comprise at least about 25%, such as at least about 30%, for example, at least about 35%, for example, at least about 40%, for example, at least about 45%, for example, at least about 50%, for example, at least about 55%, for example, at least about 60%, for example, at least about 65%, for example, at least about 70% monohormonal beta cells. In one embodiment, the islet-like cell aggregates comprise about 25-70%, for example, 30-70%, for example, 30-70% monohormonal beta cells, for example, 35-70%, for example, 35-70%, for example, 40-70%, for example, 45-70%, for example, 45-65%, for example, 45-60%, for example, 45-55%, for example, about 50% monohormonal beta cells. In one embodiment, the islet-like cell aggregates comprise about 35-65%, for example, 40-65%, for example, 40-60% monohormonal beta cells. In one embodiment, the islet-like cell aggregates comprise about 7-25%, such as 7-20%, 10-20%, such as 15-20%, for example, about 20% monohormonal alpha cells. In one embodiment, the islet-like cell aggregates comprise up to about 10%, such as up to about 7%, for example, up to about 6%, such as up to about 5%, for example, up to about 4%, such as up to about 3%, for example, up to about 2%, for example, up to about 0.5% monohormonal alpha cells. In one embodiment, the islet-like cell aggregates comprise up to about 10%, such as up to about 7%, for example, up to about 6%, such as up to about 5%, for example, up to about 4%, such as up to about 3%, for example, up to about 2%, for example, up to about 0.5%, such as up to about 0.3%, for example, up to about 0.1% monohormonal beta cells, preferably up to about 0.3%, for example, up to about 0.1% monohormonal alpha cells. In one embodiment, said islet-like cell aggregates comprise less than 5%, such as less than 4%, such as less than 3%, such as less than 1% delta cells.

[0179] In one embodiment, the islet-like cell aggregates comprise up to about 5%, such as up to about 4%, for example up to about 3%, such as up to about 2%, for example up to about 1%, for example up to about 0.5%, for example up to about 0.1% proliferating cells, e.g., proliferating cells expressing Ki-67.

[0180] In one embodiment, the islet-like cell aggregates comprise at least 40%, e.g., at least 50%, monohormonal beta cells, about 15-20%, e.g., about 20%, monohormonal alpha cells, and less than about 2%, e.g., less than about 1%, proliferating cells.

[0181] In one embodiment, the composition of the islet-like cell aggregates is examined after 38-42 days of culture, e.g., day 38, day 39, day 40, day 41, or day 42. That is, the composition of the islet-like cell aggregates is examined at the end of stage 6.

[0182] Thus, in one embodiment, there is provided a method described herein, wherein said monohormonal beta cells are functional pancreatic beta cells, such as functional pancreatic beta cells as assessed by expression of C-peptide upon glucose stimulation.

[0183] An object of the present invention is to provide mammalian cells of the pancreatic β cell lineage, e.g., human cells of the pancreatic β cell lineage, by in vitro differentiation. The source cells may be derived from pluripotent cells, e.g., embryonic stem cells or induced pluripotent stem cell cell lines. Thus, the cells may be derived from established cell lines or primary cells obtained directly from a patient, e.g., patient-specific cells. Thus, in one embodiment of the method disclosed herein, the cell population in step a) or a) or a-1) is derived from a culture of pluripotent stem cells, such as a culture of induced pluripotent stem (iPS) cells or a culture of embryonic stem (ES) cells. In one embodiment, the cell population in step a) or a-1) is a primary cell population derived directly from a patient. Alternatively, the cell population in step a) or a-1) may be derived from a culture of pluripotent cells restricted to the endodermal lineage, e.g., a cell line. The cells may be human cells.

[0184] In one embodiment, said cell population in step a) or a-1) is a mammalian cell population, such as a human cell population.

[0185] Stem cells are known in the art to be undifferentiated cells defined by the ability to both self-renew and differentiate at the single-cell level. Stem cells can generate progeny cells, including self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm). Stem cells generate tissues of multiple germ layers after implantation and significantly contribute to most (if not all) tissues after injection into blastocysts.

[0186] Stem cells are classified according to their developmental potential: (1) totipotent, i.e., capable of giving rise to all embryonic and extraembryonic cell types; (2) pluripotent, i.e., capable of giving rise to all embryonic cell types; (3) pluripotent, i.e., capable of giving rise to a subset of cell lineages within a particular tissue, organ, or physiological system; (4) oligopotent, i.e., capable of giving rise to a more restricted subset of cell lineages than pluripotent stem cells; and (5) unipotent, i.e., capable of giving rise to a single cell lineage.

[0187] As noted above, differentiation is the process by which a non-specialized ("non-committed") or less specialized cell acquires the characteristics of a specialized cell, such as a nerve cell or muscle cell. A differentiated or differentiation-induced cell is a cell that occupies a more specialized ("committed") position within its lineage. When applied to the process of differentiation, the term "commitment" refers to a cell that has progressed in the differentiation pathway to a point where, under normal circumstances, it would continue to differentiate into a particular cell type or subset of cell types, but is unable under normal circumstances to differentiate into a different cell type or revert to a less differentiated cell type. "Dedifferentiation" refers to the process by which a cell reverts to a less specialized (or non-committed) position within a cell lineage.

[0188] It should be noted that the technical teachings of the present invention can be practiced using any human pluripotent embryonic stem cells, including human pluripotent embryonic stem cells obtained without destroying a human embryo, such as parthenogenetically activated oocytes. In a specific embodiment, the cell population in step a) or step a-1) is derived from a human embryonic stem cell population. In one embodiment, the cell population in step a) or step a-1) is derived from a human embryonic stem cell population obtained without destroying a human embryo.

[0189] In one specific embodiment, the cell population in step a) or step a-1) is derived from a human embryonic stem cell population, such as a human embryonic stem cell population selected from the group of embryonic stem cell lines consisting of HS980 cells, H1 cells, and H9 cells, for example, the group of embryonic stem cell lines consisting of H1 cells and HS980 cells, or the group of embryonic stem cell lines consisting of H1 cells and H9 cells, or the group of embryonic stem cell lines consisting of HS980 cells and H9 cells. In one embodiment, the cells are H1 cells. In one embodiment, the cells are H9 cells. In one embodiment, the cells are HS980 cells. In one embodiment, the human embryonic stem cell population is a population obtained without destroying an embryo.

[0190] For purposes of compliance with European patent practice only, the above embodiments relating to HS980 cells, H1 cells, and / or H9 cells are to be regarded as reference examples for European jurisdictions. In one particular embodiment, the cell population in step a), a-1), a+1), or i) is derived from iPS cells, such as human iPS cells.

[0191] In a specific embodiment, the iPS cells are selected from the group consisting of patient-derived iPS cells and iPS cell lines. In a specific embodiment, such iPS cell line is CTRL-7-II (C7). C7 is described in Kele M et al. 2016.

[0192] Those skilled in the art will understand that the term pancreatic β cell lineage can refer to pancreatic progenitor cells, endocrine precursor cells, or pancreatic β cells. In other words, the methods disclosed herein can be for the generation of pancreatic progenitor cells or endocrine precursor cells or pancreatic β cells.

[0193] Thus, in one embodiment there is provided a method as described herein, wherein said cell of a pancreatic beta cell lineage is a pancreatic progenitor cell. In one embodiment there is provided a method as described herein, wherein the cell of a pancreatic beta cell lineage is an endocrine precursor cell. In one embodiment, the cell of a pancreatic beta cell lineage is a pancreatic beta cell.

[0194] As mentioned above, the method may further comprise a step of cryopreserving cells, and may be particularly suitable for cryopreserving endocrine precursor cells. Thus, in one embodiment, a method is provided comprising a step of cryopreserving endocrine precursor cells. In one embodiment, the cryopreservation of the endocrine precursor cells is the cryopreservation of the cells generated in step c+1).

[0195] In a second aspect of the present disclosure, an isolated population of pancreatic β cells or cells of a pancreatic β cell lineage obtained by the method is provided. It will be appreciated that the isolated population of pancreatic β cells or cells of a pancreatic β cell lineage may be useful in therapies such as cell replacement therapy, as well as in drug development or other research applications. In particular, it will be appreciated that it would be advantageous to provide a population that exhibits a high percentage or ratio of a desired cell type without the need for additional selection or sorting of the cells. For clarity, the term "isolated" with respect to a pancreatic β cell population or cells of a pancreatic β cell lineage refers to cells that are removed from their natural environment, such as the in vivo environment (i.e., isolated). It will be appreciated that the pancreatic β cells or cell populations of a pancreatic β cell lineage disclosed herein can be part of a cell aggregate or can constitute the entire cell aggregate formed during cell culture, such as the isolated islet-like cell aggregates disclosed herein. The isolated islet-like cell aggregates can include, for example, pancreatic alpha cells and / or delta cells, or cells of the pancreatic alpha and / or delta cell lineages, in addition to the pancreatic beta cell population or cells of the pancreatic beta cell lineage. The isolated islet-like cell aggregates of the present invention include the cell types disclosed above.

[0196] Thus, in one embodiment of an isolated population of pancreatic beta cells, isolated cells of a pancreatic beta cell lineage, or isolated islet-like cell aggregates, a cell population is provided, wherein the cell population has not been enriched for a desired phenotype, e.g., has not been subjected to selection for a desired phenotype, such as selection based on expression of a desired marker or FACS-based selection for a desired phenotype. For clarity, the term "enriched" as used herein refers to enrichment of cells by intervention, such as manual, or automated intervention or selection, e.g., sorting of cells based on cell characteristics, and not to processes that occur naturally in cell culture. In one embodiment, the cells are not enriched prior to forming the 3D structure in step a+2).

[0197] The populations of the invention are characterized by a high percentage of cells with desirable properties obtained by such differentiation methods. Accordingly, in one embodiment of this aspect of the invention, an isolated population of pancreatic beta cells is provided, wherein greater than about 40%, e.g., about 40-60%, e.g., about 40-50%, of the total cell population are monohormonal beta cells, such as monohormonal beta cells characterized by insulin expression. In one embodiment, the population contains at least two times more monohormonal pancreatic beta cells characterized by insulin expression than monohormonal pancreatic alpha cells characterized by glucagon expression.

[0198] The monohormonal beta cells may be characterized as described above, for example, by expression of insulin and at least one or more, e.g., two, of the markers NKX6.1, PDX1, and NEUROD1. The monohormonal beta cells may be characterized by expression of insulin and NKX6.1, e.g., by expression of insulin, NKX6.1, and at least one of PDX1 and NEUROD1. In one embodiment, the monohormonal beta cells may be characterized by expression of insulin, NKX6.1, PDX1, and NEUROD1.

[0199] In one embodiment, the monohormonal beta cells may comprise part of a cell aggregate, such as an isolated islet-like cell aggregate that further comprises pancreatic monohormonal alpha cells and / or delta cells. One skilled in the art will appreciate that isolated islet-like cell aggregates and isolated cell populations derived therefrom are expected to exhibit a similar distribution of cell types.

[0200] In one embodiment, said isolated islet-like cell aggregates, or isolated cell populations derived therefrom, comprise at least about 25%, such as at least about 25%, for example at least about 30%, such as at least about 35%, for example at least about 40%, such as at least about 45%, for example at least about 50%, such as at least about 55%, for example at least about 60%, such as at least about 65%, for example at least about 70% monohormonal beta cells. In one embodiment, said islet-like cell aggregates, or isolated cell populations derived therefrom, comprise about 25-70%, such as about 30-70%, for example about 40-70%, such as about 40-60% monohormonal beta cells.

[0201] In one embodiment, said pancreatic islet-like cell aggregates or isolated cell populations derived therefrom comprise at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70% beta cells, monohormonal beta cells, in one embodiment said monohormonal beta cells are characterized by insulin expression.

[0202] In one embodiment, said pancreatic islet-like cell aggregates, or isolated cell populations derived therefrom, comprise up to about 20%, such as up to about 18%, for example up to about 16%, such as up to about 13%, for example up to about 10% monohormonal alpha cells, hi one embodiment, said monohormonal alpha cells are characterized by glucagon expression.

[0203] In one embodiment, the islet-like cell aggregates, or isolated cell populations derived therefrom, comprise monohormonal beta cells and alpha cells, and comprise less than about 5%, e.g., less than about 4, 3, 2, or 1%, of cells selected from the group consisting of delta cells, acinar cells, duct cells, and activated stellate cells. In one embodiment of the method, the islet-like cell aggregates, or isolated cell populations derived therefrom, comprise at most about 5%, e.g., at most about 4, 3, 2, or 1%, of polyhormonal cells. In one embodiment of the method, the islet-like cell aggregates, or isolated cell populations derived therefrom, comprise at most about 5%, e.g., at most about 4, 3, 2, or 1%, of non-endocrine cells.

[0204] In one embodiment, the in vitro islet-like cell aggregates or isolated cell populations derived therefrom are scored at the end of S6, e.g., day 38-42, e.g., day 38, 39, 40, 41, 42, or later, of culture as described herein.

[0205] As mentioned above, the pancreatic monohormonal beta cell population obtained in step c+2) may be part of at least one pancreatic islet-like cell aggregate.

[0206] In one embodiment, the islet-like cell aggregates or isolated cell populations derived therefrom comprise about 25-70%, for example, 30-70%, for example, 30-70%, monohormonal β cells, for example, 35-70%, for example, 35-70%, for example, 40-70%, for example, 45-70%, for example, 45-65%, for example, 45-60%, for example, 45-55%, for example, about 50% monohormonal β cells. In one embodiment, the islet-like cell aggregates or isolated cell populations derived therefrom comprise about 35-65%, for example, 40-65%, for example, 40-60% monohormonal β cells. In one embodiment, the islet-like cell aggregates or isolated cell populations derived therefrom comprise about 7-25%, for example, 7-20%, 10-20%, for example, 15-20%, for example, about 20% monohormonal α cells. In one embodiment, said pancreatic islet-like cell aggregates or isolated cell populations derived therefrom comprise up to about 10%, such as up to about 7%, for example up to about 6%, such as up to about 5%, for example up to about 4%, such as up to about 3%, for example up to about 2%, such as up to about 0.5%, 0.3%, for example up to about 0.1% polyhormonal alpha cells.

[0207] In one embodiment, said pancreatic islet-like cell aggregates or isolated cell populations derived therefrom comprise up to about 10%, such as up to about 7%, for example up to about 6%, such as up to about 5%, for example up to about 4%, such as up to about 3%, for example up to about 2%, such as up to about 0.5%, for example up to about 0.3%, such as up to about 0.1% polyhormonal beta cells.

[0208] In one embodiment, said islet-like cell aggregates or isolated cell populations derived therefrom comprise less than 5%, such as less than 4%, such as less than 3%, such as less than 1% delta cells.

[0209] In one embodiment, the pancreatic islet-like cell aggregates or isolated cell populations derived therefrom comprise up to about 5%, such as up to about 4%, for example up to about 3%, such as up to about 2%, for example up to about 1%, such as up to about 0.5%, for example up to about 0.1% proliferating cells, e.g., proliferating cells expressing Ki-67.

[0210] In one embodiment, the islet-like cell aggregates or isolated cell populations derived therefrom comprise at least 40%, e.g., at least 50%, monohormonal beta cells, about 15-20%, e.g., about 20%, monohormonal alpha cells, and less than about 2%, e.g., less than about 1%, proliferating cells.

[0211] In one embodiment, the composition of the islet-like cell aggregates is examined after 38-42 days of culture, e.g., day 38, day 39, day 40, day 41, or day 42. That is, the composition of the islet-like cell aggregates is examined at the end of stage 6.

[0212] In one embodiment of any aspect of the invention, there is provided an isolated cell population of a pancreatic beta cell lineage as described herein, said population comprising pancreatic progenitor cells, hi one embodiment, said pancreatic progenitor cells are characterized by expression of PDX1 and NKX6.1, or characterized by expression of PDX1, NKX6.1 and PTF1A, or characterized by expression of PDX1, NKX6.1 and SOX9, or characterized by expression of PDX1, NKX6.1, PTF1A and SOX9.

[0213] In another embodiment of the present invention, there is provided an isolated cell population of the pancreatic β-cell lineage described herein, wherein the population comprises endocrine precursor cells. The endocrine precursor cells may be characterized by expression of NKX6.1 and NEUROD1, or may be characterized by expression of PDX1, NKX6.1, Ngn3, and / or NEUROD1. In one embodiment, at least 30%, such as at least 40%, such as at least 50%, such as at least 60% of all cells are characterized by expression of NKX6.1 and NEUROD1 or NGN3. The endocrine precursor cells are characterized by expression of NKX6.1 and NEUROD1, expression of NKX6.1 and NGN3, expression of at least one of PDX1, NKX6.1, and NGN3 and NEUROD1, or expression of PDX1, NKX6.1, NGN3, and NEUROD1.

[0214] As noted above, the isolated populations of pancreatic β cells or cells of the pancreatic β cell lineage disclosed herein are believed to be useful in the treatment and / or prevention of diabetes.

[0215] Thus, in a third aspect of the present disclosure, an isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage as disclosed herein is provided for use in therapy, i.e., as a pharmaceutical. It will be understood that the cells or islet-like cell aggregates can be transplanted into patients in need thereof for cell replacement therapy. The cell replacement therapy may be performed to provide pancreatic beta cells or cells of the pancreatic beta cell lineage to patients who have no endogenous beta cells or only non-functional beta cells, or to patients who require more pancreatic beta cells or cells of the pancreatic beta cell lineage because their endogenous beta cell population is reduced or functional below the level required for a healthy patient. The cells or islet-like cell aggregates may be donor-derived and thus allogeneic. For example, the cells may be derived from a related or unrelated donor, or from an established cell line, such as a stem cell line, capable of developing along the pancreatic beta cell lineage. Non-limiting examples include hES cell lines, non-embryonic stem cell lines with the potential to develop along endocrine lineages (e.g., pluripotent, oligopotent, or unipotent cell lines), iPS cell lines, and iPS cell-derived cell lines with the potential to develop along a pancreatic β cell lineage. The cells may be endogenous to the patient, e.g., derived from iPS cells obtained from the patient or from cells from another patient with the potential to develop along a pancreatic β cell lineage. The populations or islet-like cell aggregates obtained by the methods disclosed herein are believed to be beneficial for therapeutic use because they contain a higher percentage of total cells of the desired cell type than populations obtained by previously described culture methods. Thus, the cell populations or islet-like aggregates obtained by the disclosed methods may not need to be subjected to cell sorting or similar stressful and potentially damaging techniques, at least to a lesser extent, or even at all, to obtain a homogenous population containing a high percentage of the desired cell type. Therefore, it is expected that treatments such as transplants can be performed with healthier, more viable cell populations with a lower proportion of damaged or unhealthy cells, which would be beneficial for patients in terms of fewer potential side effects and better clinical outcomes.

[0216] In a fourth aspect, there is provided a population of isolated pancreatic beta cells or pancreatic beta cell lineage cells or isolated pancreatic islet-like cell aggregates disclosed herein for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes. In one embodiment, the diabetes is type 1 diabetes. In one embodiment, the type 2 diabetes is insulin-deficient type 2 diabetes.

[0217] In one embodiment, there is provided an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage, or isolated islet-like cell aggregates, for use in therapeutic treatment, wherein said cell population or isolated islet-like cell aggregates have been produced by a method as defined herein.

[0218] In one embodiment, there is provided an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage or isolated islet-like cell aggregates for use in therapy, i.e., for use as a medicament, said use comprising the steps of: generating cells of pancreatic beta cells or cells of a pancreatic beta cell lineage or isolated islet-like cell aggregates according to the methods defined herein; and administering a therapeutically effective amount of the cells or isolated islet-like cell aggregates to the patient.

[0219] In one embodiment, said use further comprises isolating cells from said patient, such as cells or stem cells for the generation of iPS cells from said patient, and using said cells to generate cells of pancreatic beta cells or cells of a pancreatic beta cell lineage or isolated pancreatic islet-like cell aggregates according to a method defined herein.

[0220] In one embodiment, there is provided an isolated population of pancreatic monohormonal beta cells or cells of the pancreatic beta cell lineage, or isolated pancreatic islet-like cell aggregates, for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes, wherein said cell population or isolated pancreatic islet-like cell aggregates have been produced by the methods defined herein.

[0221] In another embodiment, there is provided an isolated population of pancreatic monohormonal beta cells or cells of the pancreatic beta cell lineage or isolated pancreatic islet-like cell aggregates for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes, said use comprising the steps of: generating cells of pancreatic beta cells or cells of a pancreatic beta cell lineage or isolated islet-like cell aggregates according to the methods defined herein; and and administering a therapeutically effective amount of the cells or isolated islet-like cell aggregates to the patient. In one embodiment, the pancreatic beta cells may be administered in the form of cell aggregates as defined above. In one embodiment, the use further comprises isolating cells from the patient, such as cells or stem cells for the generation of iPS cells from the patient, and using the cells to generate pancreatic beta cells or cells of a pancreatic beta cell lineage according to the methods defined herein. As with what is described in relation to the third aspect, other cell types, such as allogeneic and endogenous cells, are also contemplated as useful in this regard.

[0222] It will be appreciated that cell replacement therapy can include transplantation of pancreatic beta cells, such as monohormonal pancreatic beta cells, which have the ability to produce insulin in the patient's body and respond appropriately to glucose stimulation in the patient. It will be appreciated that cell replacement therapy can include transplantation of pancreatic islet-like cell aggregates (cell aggregates) or cells derived therefrom.

[0223] Also contemplated are cells at earlier stages of development along the pancreatic β cell lineage, such as PP cells and EP cells, which continue to differentiate along the pancreatic β cell lineage pathway in the patient to generate monohormonal pancreatic β cells capable of functioning in the patient, producing insulin in the patient, and responding appropriately to glucose stimulation in the patient.

[0224] Thus, in one embodiment, the use comprises transplanting the cell population into a patient in need thereof. In one embodiment, the use comprises transplanting monohormonal pancreatic beta cells into a patient in need thereof. In one embodiment, the use comprises transplanting endocrine precursor cells into a patient in need thereof. In another embodiment, the use comprises transplanting pancreatic progenitor cells into a patient in need thereof.

[0225] The isolated populations of pancreatic beta cells or cells of the pancreatic beta cell lineage are believed to be useful as pharmaceutical compositions.

[0226] Accordingly, in a related fifth aspect of the present disclosure, there is provided a pharmaceutical composition comprising an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage as disclosed herein and at least one pharmaceutically acceptable excipient or carrier.

[0227] It is also envisioned that it may be beneficial to administer the pancreatic beta cells or isolated populations of cells of a pancreatic beta cell lineage disclosed herein, or the pharmaceutical compositions disclosed herein, together with a carrier substrate that promotes the growth, proliferation, and / or, optionally, differentiation of the cell population. Accordingly, in a sixth aspect of the present disclosure, a kit of parts is provided comprising the pancreatic beta cells or isolated populations of cells of a pancreatic beta cell lineage disclosed herein, or the pharmaceutical compositions disclosed herein, and a suitable carrier substrate. The suitable carrier substrate may be any suitable substrate, for example, a 3D scaffold or a 2D scaffold, including the 2D substrate described in connection with the first aspect disclosed herein. For the sake of brevity, the 2D substrate will only be briefly described below. Thus, the 2D substrate can be one or more of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and Matrigel™, such as LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, LN-121 and fragments thereof, and LN-111 and fragments thereof. For example, the fragment can be an E8 fragment.

[0228] In one particular embodiment, a kit of parts is provided, wherein said carrier substrate is a 2D substrate as defined herein and said cells of the pancreatic beta cell lineage are endocrine precursor cells, hi another embodiment, a kit of parts is provided, wherein said carrier substrate is a 3D substrate and said cells are monohormonal beta cells.

[0229] It will be appreciated that the isolated pancreatic β cell populations, cells of a pancreatic β cell lineage, or isolated islet-like cell aggregates described herein may have many applications in biological research, such as drug screening, for example, in vitro drug screening. It will be appreciated that the populations obtained by the methods disclosed herein are advantageous because they contain a higher percentage of total cells of the desired cell type than populations obtained by the aforementioned culture methods. Thus, the cell cultures of the present invention do not need to be subjected to cell sorting or similar stressful and potentially damaging techniques to obtain a homogenous population containing a high percentage of the desired cell type, at least to a lesser extent, or even at all. A homogenous population containing a high percentage of the desired cell type is more likely to generate data in drug screening assays that are less or not hindered by potential responses from other unrelated or contaminating cell types. Thus, in a seventh aspect of the present disclosure, there is provided the use of the isolated pancreatic β cell populations, cells of a pancreatic β cell lineage, or isolated islet-like cell aggregates disclosed herein in drug screening, such as in vitro drug screening.

[0230] In this regard, an in vitro drug screening method is provided, comprising generating cells of the pancreatic endocrine lineage, such as a pancreatic β cell lineage, according to the methods defined herein and exposing the cells to at least one candidate drug compound. Similarly, the method may comprise generating isolated pancreatic islet-like cell aggregates and exposing the aggregates to at least one candidate drug compound. The method may further comprise evaluating the response of the pancreatic endocrine lineage cells or aggregates to the candidate drug compound. Depending on the purpose of the drug screening, the cells generated in the first step of the method may be pancreatic progenitor cells, endocrine precursor cells, or monohormonal β cells. Furthermore, an in vitro drug screening method is provided, comprising providing cells of the pancreatic endocrine lineage obtained by the methods described herein and exposing the cells to at least one candidate drug compound.

[0231] In an eighth aspect, there is provided a method of treating a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of pancreatic beta cells or cells of a pancreatic beta cell lineage as disclosed herein. Further provided is a method of treating diabetes in a patient in need thereof, comprising generating cells of the pancreatic endocrine lineage, such as a pancreatic beta cell lineage, according to a method defined herein; Administering to said patient a therapeutically effective amount of said pancreatic beta cells or cells of a pancreatic beta cell lineage, in one embodiment said cells are allogeneic, and in another embodiment said cells are endogenous to the patient.

[0232] In one embodiment, the method is for the treatment of diabetes, such as type 1 diabetes or type 2 diabetes.

[0233] Accordingly, there is provided a method of treating diabetes in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of pancreatic beta cells, cells of a pancreatic beta cell lineage, or isolated pancreatic islet-like cell aggregates as disclosed herein. Also provided is a method of treating a patient in need thereof, comprising generating cells of the pancreatic endocrine lineage, such as a pancreatic beta cell lineage, according to the methods defined herein; Methods are provided that include administering a therapeutically effective amount of the pancreatic beta cells or cells of a pancreatic beta cell lineage to the patient. The methods further include isolating cells from the patient, such as cells or stem cells for the generation of iPS cells from the patient, and using the cells to generate pancreatic beta cells or cells of a pancreatic beta cell lineage according to the methods defined herein. As described above, cells of a pancreatic beta cell lineage can be pancreatic progenitor cells, endocrine precursor cells, or monohormonal beta cells. The cells can be in the form of aggregates, such as the isolated islet-like cell aggregates disclosed herein. The cells can be administered by transplantation. In one embodiment, the cells are endogenous to the patient, i.e., patient-specific cells. In one embodiment, the cells are allogeneic cells.

[0234] In a related ninth aspect, there is provided a use of an isolated population of pancreatic beta cells, cells of a pancreatic beta cell lineage, or isolated pancreatic islet-like cell aggregates described herein for the manufacture of a medicament for treating diabetes in a patient in need thereof. In one embodiment, the manufacture of the medicament comprises generating pancreatic beta cells or cells of a pancreatic beta cell lineage by a method defined herein. It will be understood that the cells may be pancreatic progenitor cells, endocrine precursor cells, or monohormonal beta cells, as disclosed herein. The cells may be in the form of aggregates, such as isolated islet-like cell aggregates disclosed herein. In one embodiment, the cells are endogenous to the patient, i.e., patient-specific cells. In one embodiment, the cells are allogeneic cells.

[0235] It will be understood that any discussion relating to the third and fourth aspects of the present disclosure and embodiments thereof also relates to this eighth aspect and related ninth aspect and will not be repeated here for the sake of brevity. In one embodiment, there is provided a method of treating diabetes in a patient suffering from type 1 or type 2 diabetes, as disclosed herein. In one embodiment, said administering comprises transplanting said population into said patient. In one embodiment, said administering comprises transplanting said pancreatic beta cell population, cells of a pancreatic beta cell lineage, or isolated pancreatic islet-like cell aggregates into said patient.

[0236] In yet additional related aspects, methods for generating pancreatic progenitor cells, endocrine precursor cells, and monohormonal β cells are provided, as described below. The monohormonal β cells can be part of pancreatic islet-like cell aggregates. Those skilled in the art will understand that any embodiment disclosed in connection with the first aspect of the present disclosure also relates to the methods of these additional aspects, if applicable. Thus, for example, any embodiment specifying the features of steps a) through c) relates to any of the methods for generating pancreatic progenitor cells, endocrine precursor cells, and monohormonal β cells; any embodiment specifying the features of steps a+1) through c+1) relates to any of the methods for generating endocrine precursor cells and monohormonal β cells; and any embodiment specifying the features of steps a+2) through c+2) relates to any of the methods for generating monohormonal β cells. Similarly, the same principles apply to all embodiments specifying the features of steps a-1) through c-1) above.

[0237] In one aspect, a method for generating pancreatic progenitor cells is provided, comprising the steps of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for no more than about 78 hours; and c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1.

[0238] In one aspect, a method for generating endocrine precursor cells is provided, comprising the steps of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for no more than about 78 hours; c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1; a+1) providing a cell population of pancreatic progenitor cells; b+1) culturing the population of pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells; and c+1) thereby generating a population of endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1.

[0239] In another aspect, a method for generating monohormonal beta cells is provided, comprising the steps of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for no more than about 78 hours; c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1; a+1) providing a cell population of pancreatic progenitor cells; b+1) culturing the population of pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells; c+1) thereby generating a population of endocrine precursor cells, such as endocrine precursor cells, characterized by expression of NKX6.1 and NEUROD1; a+2) transferring the endocrine precursor cell population from culture on a 2D substrate to 3D culture conditions; b+2) culturing the endocrine precursor cell population under conditions that allow differentiation into pancreatic monohormonal beta cells; and c+2) thereby generating a population of monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin.

[0240] In embodiments of the method for generating pancreatic progenitor cells, the method for generating endocrine precursor cells, and the method for generating monohormonal β cells disclosed herein, the method further comprises, prior to steps a) to c), the following steps a-1) to c-1): a-1) providing a cell population of primitive intestinal cells, such as primitive intestinal cells, characterized by the expression of HNF1β and HNF4α; b-1) culturing the population of primitive intestinal cells for about 54 hours or less under conditions that allow differentiation into posterior foregut cells; and c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1.

[0241] As used herein, the term "population" when used in reference to a particular type of cell is to be interpreted as a population containing said cells. For example, the phrase "EP cell population" is to be understood as a population containing EP cells. The population may further include other cells, such as, but not limited to, cells at an early developmental stage, such as PP cells.

[0242] As used herein, when the term "about" is used in relation to a numerical value, it is to be interpreted as being within ±10%, such as ±9%, for example ±8%, such as ±7%, for example ±6%, such as ±5%, for example ±4%, for example ±3%, for example ±2%, such as ±1%. For example, if a value is stated to be about 10, this means that the value is actually in the range of 9-11, such as in the range of 9.9-10.9, for example in the range of 9.8-10.8, such as in the range of 9.7-10.7, for example in the range of 9.6-10.6, such as in the range of 9.5-10.5, for example in the range of 9.4-10.4, such as in the range of 9.3-10.3, for example in the range of 9.2-10.2, for example in the range of 9.1-10.1.

[0243] Those skilled in the art know that numerical values relating to measurements are subject to measurement error and have limited accuracy. Therefore, the usual convention in scientific and technical literature applies: the last decimal point of a numerical value indicates its degree of accuracy. Unless another error range is specified, the rounding rule is applied to the last decimal point to determine the maximum range. For example, if a measurement is 3.5 cm, the error range is 3.45 to 3.54. Those skilled in the art should proceed by the same standard when interpreting ranges of values in patent specifications.

[0244] While the present invention has been described with reference to various exemplary aspects and embodiments, those skilled in the art will recognize that various changes can be made and equivalents substituted for elements thereof without departing from the scope of the invention. Furthermore, many modifications can be made to adapt a particular situation, culture conditions, or cell population to the teachings of the present invention without departing from the essential scope of the invention. Therefore, the present invention is not limited to the particular embodiments envisioned, but is intended to encompass all embodiments falling within the scope of the appended claims. [Brief explanation of the drawings]

[0245] [Figure 1A]Figure 1 is a schematic diagram showing the developmental stages of the pancreatic β-cell lineage and the expression of markers characteristic of each developmental stage. (A) Pancreatic endocrine cell types can be generated from human pluripotent embryonic stem cells (hES) or iPS cells by recapitulating embryonic pancreatic development in a Petri dish. Pancreatic differentiation is divided into multiple stages, including the definitive endoderm (DE), primitive gut (PGT), posterior foregut (PF), multipotent pancreatic progenitor cells (PP), endocrine precursor cells (EP), and pancreatic islets (ISL). Key markers expressed at each stage are shown in the diagram. (B) Figure 1B is a schematic diagram of a conventional long-term differentiation protocol and the short-term differentiation protocol disclosed herein. The effects of long-term and short-term durations of stages 3 and 4 (S3+S4) on subsequent endocrine differentiation were analyzed. Expression of stage-specific markers was examined at the end of stage 2 (S2), stage 3 (S3), and stage 4 (S4), at day 4 of stage 5 (S5), and at stage 6 (S6). 2DLN-521 is an example of a 2D substrate and may be substituted with other 2D substrates disclosed herein. [Figure 1B] Figure 1 is a schematic diagram showing the developmental stages of the pancreatic β-cell lineage and the expression of markers characteristic of each developmental stage. (A) Pancreatic endocrine cell types can be generated from human pluripotent embryonic stem cells (hES) or iPS cells by recapitulating embryonic pancreatic development in a Petri dish. Pancreatic differentiation is divided into multiple stages, including the definitive endoderm (DE), primitive gut (PGT), posterior foregut (PF), multipotent pancreatic progenitor cells (PP), endocrine precursor cells (EP), and pancreatic islets (ISL). Key markers expressed at each stage are shown in the diagram. (B) Figure 1B is a schematic diagram of a conventional long-term differentiation protocol and the short-term differentiation protocol disclosed herein. The effects of long-term and short-term durations of stages 3 and 4 (S3+S4) on subsequent endocrine differentiation were analyzed. Expression of stage-specific markers was examined at the end of stage 2 (S2), stage 3 (S3), and stage 4 (S4), at day 4 of stage 5 (S5), and at stage 6 (S6). 2DLN-521 is an example of a 2D substrate and may be substituted with other 2D substrates disclosed herein. [Figure 2A]Figure 2 shows a comparison of pancreatic differentiation on different coated substrates. HS980 and H1 cells were differentiated on Matrigel-, LN-511-, and LN-521-coated plates using a long-term protocol. Expression of key progenitor markers, PDX1, NKX6.1, and NEUROD1, was examined by flow cytometry at the end of S4 or on day 4 of S5. Representative dot plots for (A) stage 4 and (B and C) stage 5 HS980 cells are shown. Bar graphs representing the results for both HS980 and H1 cells are shown. Results were calculated from three independent samples. To compare the percentages of (A and B) PDX1+ / NKX6.1+ cells and (C) NKX6.1+ / NEUROD1+ cells among the three substrates, unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 2B] Figure 2 shows a comparison of pancreatic differentiation on different coated substrates. HS980 and H1 cells were differentiated on Matrigel-, LN-511-, and LN-521-coated plates using a long-term protocol. Expression of key progenitor markers, PDX1, NKX6.1, and NEUROD1, was examined by flow cytometry at the end of S4 or on day 4 of S5. Representative dot plots for (A) stage 4 and (B and C) stage 5 HS980 cells are shown. Bar graphs representing the results for both HS980 and H1 cells are shown. Results were calculated from three independent samples. To compare the percentages of (A and B) PDX1+ / NKX6.1+ cells and (C) NKX6.1+ / NEUROD1+ cells among the three substrates, unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 2C]Figure 2 shows a comparison of pancreatic differentiation on different coated substrates. HS980 and H1 cells were differentiated on Matrigel-, LN-511-, and LN-521-coated plates using a long-term protocol. Expression of key progenitor markers, PDX1, NKX6.1, and NEUROD1, was examined by flow cytometry at the end of S4 or on day 4 of S5. Representative dot plots for (A) stage 4 and (B and C) stage 5 HS980 cells are shown. Bar graphs representing the results for both HS980 and H1 cells are shown. Results were calculated from three independent samples. To compare the percentages of (A and B) PDX1+ / NKX6.1+ cells and (C) NKX6.1+ / NEUROD1+ cells among the three substrates, unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 3A] Figure 3 shows a comparison of primitive gut (PGT) cells and posterior foregut (PF) cells at various culture periods. HS980 and H1 cells were differentiated to S3 on LN-521-coated plates. Expression of the PF marker PDX1 was analyzed by immunocytochemistry (ICC) and flow cytometry at the end of S2 or on day 1 or 2 after the onset of S3. Representative confocal micrographs and dot plots of HS980 cells are shown. (A) HS980 cells. (B) H1 cells. [Figure 3B] Figure 3 shows a comparison of primitive gut (PGT) cells and posterior foregut (PF) cells at various culture periods. HS980 and H1 cells were differentiated to S3 on LN-521-coated plates. Expression of the PF marker PDX1 was analyzed by immunocytochemistry (ICC) and flow cytometry at the end of S2 or on day 1 or 2 after the onset of S3. Representative confocal micrographs and dot plots of HS980 cells are shown. (A) HS980 cells. (B) H1 cells. [Figure 4A]Figure 4 shows the results of evaluating the effect of S4 duration on endocrine differentiation. HS980, H1, and H9 cells were differentiated on LN-521-coated plates. The S4 duration was 1, 2, 3, 4, or 5 days. The expression of progenitor markers NKX6.1 and NEUROD1 was examined by flow cytometry at the end of S4 or on day 4 of S5. The expression of endocrine markers insulin (INS) and glucagon (GCG) was examined at the end of S6. Results were calculated from multiple independent experiments. To compare the proportions of different cell populations between different S4 durations, unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Schematic diagram of different S4 durations. (B) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 at S4 and S5 in HS980 cells. (C) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 in H1 and H9 cells at S5. (D) Bar graphs showing the calculated percentages of NKX6.1+ / NEUROD1+ cells and NKX6.1+ / NEUROD1- cells. Results show the percentages of H980, H1, and H9 cells. (E) Bar graphs showing the calculated percentages of NEUROD1+ and NKX6.1+ cells at S5. Results show the percentages of H980, H1, and H9 cells. (F) Representative dot plots from the expression analysis of INS and GCC in HS980, H1, and H9 cells at S6. (G) Bar graphs showing the calculated percentages of INS+ / GCG- monohormonal β cells and GCG+ / INS- monohormonal α cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 4B]Figure 4 shows the results of evaluating the effect of S4 duration on endocrine differentiation. HS980, H1, and H9 cells were differentiated on LN-521-coated plates. The S4 duration was 1, 2, 3, 4, or 5 days. The expression of progenitor markers NKX6.1 and NEUROD1 was examined by flow cytometry at the end of S4 or on day 4 of S5. The expression of endocrine markers insulin (INS) and glucagon (GCG) was examined at the end of S6. Results were calculated from multiple independent experiments. To compare the proportions of different cell populations between different S4 durations, unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Schematic diagram of different S4 durations. (B) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 at S4 and S5 in HS980 cells. (C) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 in H1 and H9 cells at S5. (D) Bar graphs showing the calculated percentages of NKX6.1+ / NEUROD1+ cells and NKX6.1+ / NEUROD1- cells. Results show the percentages of H980, H1, and H9 cells. (E) Bar graphs showing the calculated percentages of NEUROD1+ and NKX6.1+ cells at S5. Results show the percentages of H980, H1, and H9 cells. (F) Representative dot plots from the expression analysis of INS and GCC in HS980, H1, and H9 cells at S6. (G) Bar graphs showing the calculated percentages of INS+ / GCG- monohormonal β cells and GCG+ / INS- monohormonal α cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 4C]Figure 4 shows the results of evaluating the effect of S4 duration on endocrine differentiation. HS980, H1, and H9 cells were differentiated on LN-521-coated plates. The S4 duration was 1, 2, 3, 4, or 5 days. The expression of progenitor markers NKX6.1 and NEUROD1 was examined by flow cytometry at the end of S4 or on day 4 of S5. The expression of endocrine markers insulin (INS) and glucagon (GCG) was examined at the end of S6. Results were calculated from multiple independent experiments. To compare the proportions of different cell populations between different S4 durations, unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Schematic diagram of different S4 durations. (B) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 at S4 and S5 in HS980 cells. (C) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 in H1 and H9 cells at S5. (D) Bar graphs showing the calculated percentages of NKX6.1+ / NEUROD1+ cells and NKX6.1+ / NEUROD1- cells. Results show the percentages of H980, H1, and H9 cells. (E) Bar graphs showing the calculated percentages of NEUROD1+ and NKX6.1+ cells at S5. Results show the percentages of H980, H1, and H9 cells. (F) Representative dot plots from the expression analysis of INS and GCC in HS980, H1, and H9 cells at S6. (G) Bar graphs showing the calculated percentages of INS+ / GCG- monohormonal β cells and GCG+ / INS- monohormonal α cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 4D]Figure 4 shows the results of evaluating the effect of S4 duration on endocrine differentiation. HS980, H1, and H9 cells were differentiated on LN-521-coated plates. The S4 duration was 1, 2, 3, 4, or 5 days. The expression of progenitor markers NKX6.1 and NEUROD1 was examined by flow cytometry at the end of S4 or on day 4 of S5. The expression of endocrine markers insulin (INS) and glucagon (GCG) was examined at the end of S6. Results were calculated from multiple independent experiments. To compare the proportions of different cell populations between different S4 durations, unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Schematic diagram of different S4 durations. (B) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 at S4 and S5 in HS980 cells. (C) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 in H1 and H9 cells at S5. (D) Bar graphs showing the calculated percentages of NKX6.1+ / NEUROD1+ cells and NKX6.1+ / NEUROD1- cells. Results show the percentages of H980, H1, and H9 cells. (E) Bar graphs showing the calculated percentages of NEUROD1+ and NKX6.1+ cells at S5. Results show the percentages of H980, H1, and H9 cells. (F) Representative dot plots from the expression analysis of INS and GCC in HS980, H1, and H9 cells at S6. (G) Bar graphs showing the calculated percentages of INS+ / GCG- monohormonal β cells and GCG+ / INS- monohormonal α cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 4E]Figure 4 shows the results of evaluating the effect of S4 duration on endocrine differentiation. HS980, H1, and H9 cells were differentiated on LN-521-coated plates. The S4 duration was 1, 2, 3, 4, or 5 days. The expression of progenitor markers NKX6.1 and NEUROD1 was examined by flow cytometry at the end of S4 or on day 4 of S5. The expression of endocrine markers insulin (INS) and glucagon (GCG) was examined at the end of S6. Results were calculated from multiple independent experiments. To compare the proportions of different cell populations between different S4 durations, unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Schematic diagram of different S4 durations. (B) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 at S4 and S5 in HS980 cells. (C) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 in H1 and H9 cells at S5. (D) Bar graphs showing the calculated percentages of NKX6.1+ / NEUROD1+ cells and NKX6.1+ / NEUROD1- cells. Results show the percentages of H980, H1, and H9 cells. (E) Bar graphs showing the calculated percentages of NEUROD1+ and NKX6.1+ cells at S5. Results show the percentages of H980, H1, and H9 cells. (F) Representative dot plots from the expression analysis of INS and GCC in HS980, H1, and H9 cells at S6. (G) Bar graphs showing the calculated percentages of INS+ / GCG- monohormonal β cells and GCG+ / INS- monohormonal α cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 4F]Figure 4 shows the results of evaluating the effect of S4 duration on endocrine differentiation. HS980, H1, and H9 cells were differentiated on LN-521-coated plates. The S4 duration was 1, 2, 3, 4, or 5 days. The expression of progenitor markers NKX6.1 and NEUROD1 was examined by flow cytometry at the end of S4 or on day 4 of S5. The expression of endocrine markers insulin (INS) and glucagon (GCG) was examined at the end of S6. Results were calculated from multiple independent experiments. To compare the proportions of different cell populations between different S4 durations, unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Schematic diagram of different S4 durations. (B) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 at S4 and S5 in HS980 cells. (C) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 in H1 and H9 cells at S5. (D) Bar graphs showing the calculated percentages of NKX6.1+ / NEUROD1+ cells and NKX6.1+ / NEUROD1- cells. Results show the percentages of H980, H1, and H9 cells. (E) Bar graphs showing the calculated percentages of NEUROD1+ and NKX6.1+ cells at S5. Results show the percentages of H980, H1, and H9 cells. (F) Representative dot plots from the expression analysis of INS and GCC in HS980, H1, and H9 cells at S6. (G) Bar graphs showing the calculated percentages of INS+ / GCG- monohormonal β cells and GCG+ / INS- monohormonal α cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 4G]Figure 4 shows the results of evaluating the effect of S4 duration on endocrine differentiation. HS980, H1, and H9 cells were differentiated on LN-521-coated plates. The S4 duration was 1, 2, 3, 4, or 5 days. The expression of progenitor markers NKX6.1 and NEUROD1 was examined by flow cytometry at the end of S4 or on day 4 of S5. The expression of endocrine markers insulin (INS) and glucagon (GCG) was examined at the end of S6. Results were calculated from multiple independent experiments. To compare the proportions of different cell populations between different S4 durations, unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Schematic diagram of different S4 durations. (B) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 at S4 and S5 in HS980 cells. (C) Representative dot plots from the expression analysis of progenitor markers NKX6.1 and NEUROD1 in H1 and H9 cells at S5. (D) Bar graphs showing the calculated percentages of NKX6.1+ / NEUROD1+ cells and NKX6.1+ / NEUROD1- cells. Results show the percentages of H980, H1, and H9 cells. (E) Bar graphs showing the calculated percentages of NEUROD1+ and NKX6.1+ cells at S5. Results show the percentages of H980, H1, and H9 cells. (F) Representative dot plots from the expression analysis of INS and GCC in HS980, H1, and H9 cells at S6. (G) Bar graphs showing the calculated percentages of INS+ / GCG- monohormonal β cells and GCG+ / INS- monohormonal α cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 5A-1]Figure 5 shows the evaluation of pancreatic differentiation on different coated substrates. HS980, H1, and H9 cells were differentiated into S5 EP cells on plates coated with various recombinant human laminin isoforms (LN) or Matrigel using a short-term protocol. H1 cells were also differentiated on plates coated with fibronectin (FN) or vitronectin (VTN). Expression of NKX6.1 and NEUROD1 was measured by flow cytometry on day 4 after the initiation of S5. HS980 cells on LN-332, LN-511, LN-521, and Matrigel were further differentiated into S6 islet cells in 3D suspension. Expression of INS and GCG was measured by flow cytometry at the end of S6. Representative dot plots for S5 EP cells are shown in Figures 5A-1 and 5A-2, and for S6 islet cells in Figure 5C. To compare the percentage of NKX6.1+ / NEUROD1+ cells between laminin isoforms and Matrigel, a paired two-tailed t-test was performed in Microsoft Excel. (B) Percentage of NKX6.1+ / NEUROD1+ cells. *p<0.05, **p<0.01. [Figure 5A-2]Figure 5 shows the evaluation of pancreatic differentiation on different coated substrates. HS980, H1, and H9 cells were differentiated into S5 EP cells on plates coated with various recombinant human laminin isoforms (LN) or Matrigel using a short-term protocol. H1 cells were also differentiated on plates coated with fibronectin (FN) or vitronectin (VTN). Expression of NKX6.1 and NEUROD1 was measured by flow cytometry on day 4 after the initiation of S5. HS980 cells on LN-332, LN-511, LN-521, and Matrigel were further differentiated into S6 islet cells in 3D suspension. Expression of INS and GCG was measured by flow cytometry at the end of S6. Representative dot plots for S5 EP cells are shown in Figures 5A-1 and 5A-2, and for S6 islet cells in Figure 5C. To compare the percentage of NKX6.1+ / NEUROD1+ cells between laminin isoforms and Matrigel, a paired two-tailed t-test was performed in Microsoft Excel. (B) Percentage of NKX6.1+ / NEUROD1+ cells. *p<0.05, **p<0.01. [Figure 5B]Figure 5 shows the evaluation of pancreatic differentiation on different coated substrates. HS980, H1, and H9 cells were differentiated into S5 EP cells on plates coated with various recombinant human laminin isoforms (LN) or Matrigel using a short-term protocol. H1 cells were also differentiated on plates coated with fibronectin (FN) or vitronectin (VTN). Expression of NKX6.1 and NEUROD1 was measured by flow cytometry on day 4 after the initiation of S5. HS980 cells on LN-332, LN-511, LN-521, and Matrigel were further differentiated into S6 islet cells in 3D suspension. Expression of INS and GCG was measured by flow cytometry at the end of S6. Representative dot plots for S5 EP cells are shown in Figures 5A-1 and 5A-2, and for S6 islet cells in Figure 5C. To compare the percentage of NKX6.1+ / NEUROD1+ cells between laminin isoforms and Matrigel, a paired two-tailed t-test was performed in Microsoft Excel. (B) Percentage of NKX6.1+ / NEUROD1+ cells. *p<0.05, **p<0.01. [Figure 5C]Figure 5 shows the evaluation of pancreatic differentiation on different coated substrates. HS980, H1, and H9 cells were differentiated into S5 EP cells on plates coated with various recombinant human laminin isoforms (LN) or Matrigel using a short-term protocol. H1 cells were also differentiated on plates coated with fibronectin (FN) or vitronectin (VTN). Expression of NKX6.1 and NEUROD1 was measured by flow cytometry on day 4 after the initiation of S5. HS980 cells on LN-332, LN-511, LN-521, and Matrigel were further differentiated into S6 islet cells in 3D suspension. Expression of INS and GCG was measured by flow cytometry at the end of S6. Representative dot plots for S5 EP cells are shown in Figures 5A-1 and 5A-2, and for S6 islet cells in Figure 5C. To compare the percentage of NKX6.1+ / NEUROD1+ cells between laminin isoforms and Matrigel, a paired two-tailed t-test was performed in Microsoft Excel. (B) Percentage of NKX6.1+ / NEUROD1+ cells. *p<0.05, **p<0.01. [Figure 6A] Figure 6 shows the effect of 3D suspension culture of S4 and S5 progenitor cells on islet formation. (A) Schematic diagram showing the timeline of differentiation of HS980 cells on LN-521 using the short-term protocol. Cells were dissociated into single cells at the end of S4 (top timeline) or on days 4 or 6 of S5 (bottom timeline). 4 x 106 S4 and S5 cells per well were maintained in 3D suspension and further differentiated into S6 islet cells for analysis. (B) At the end of S6, islet-like aggregates were counted under a microscope and then dissociated into single cells for cell counting. INS and GCG expression were measured by flow cytometry. Representative dot plots are shown. The percentage of cell population and the number of aggregates / cells per well are shown in bar graphs and were calculated from three independent samples. Unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 6B]Figure 6 shows the effect of 3D suspension culture of S4 and S5 progenitor cells on islet formation. (A) Schematic diagram showing the timeline of differentiation of HS980 cells on LN-521 using the short-term protocol. Cells were dissociated into single cells at the end of S4 (top timeline) or on days 4 or 6 of S5 (bottom timeline). 4 x 106 S4 and S5 cells per well were maintained in 3D suspension and further differentiated into S6 islet cells for analysis. (B) At the end of S6, islet-like aggregates were counted under a microscope and then dissociated into single cells for cell counting. INS and GCG expression were measured by flow cytometry. Representative dot plots are shown. The percentage of cell population and the number of aggregates / cells per well are shown in bar graphs and were calculated from three independent samples. Unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 7A] Figure 7 shows the enrichment of S5 EP cells during aggregate formation in suspension. HS980 and H1 cells were differentiated to S5 on a 2D surface coated with LN-521. Cells were dissociated into single cells on day 4 of S5 and then maintained in suspension for 1 day to generate 3D aggregates. Expression of NKX6.1, NEUROD1, and Ki-67 was examined by flow cytometry before and after aggregate formation. To examine the effect of the ROCK inhibitor H1152 on aggregate formation, different concentrations of H1152, ranging from 0 to 10 μM, were added to the single-cell suspension as indicated. The following day, marker expression and islet cell counts were measured. Representative dot plots are shown in (A), (B), and (C). The percentages of NKX6.1+ / NEUROD1+ cells and NEUROD1+ cells were calculated from three independent experiments and are shown in the bar graph in (A). The number of islet cells generated from 106 single cells in 3D suspension was calculated from three independent samples and is shown in the bar graph in (D). Unpaired two-tailed t-test was performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 7B]Figure 7 shows the enrichment of S5 EP cells during aggregate formation in suspension. HS980 and H1 cells were differentiated to S5 on a 2D surface coated with LN-521. Cells were dissociated into single cells on day 4 of S5 and then maintained in suspension for 1 day to generate 3D aggregates. Expression of NKX6.1, NEUROD1, and Ki-67 was examined by flow cytometry before and after aggregate formation. To examine the effect of the ROCK inhibitor H1152 on aggregate formation, different concentrations of H1152, ranging from 0 to 10 μM, were added to the single-cell suspension as indicated. The following day, marker expression and islet cell counts were measured. Representative dot plots are shown in (A), (B), and (C). The percentages of NKX6.1+ / NEUROD1+ cells and NEUROD1+ cells were calculated from three independent experiments and are shown in the bar graph in (A). The number of islet cells generated from 106 single cells in 3D suspension was calculated from three independent samples and is shown in the bar graph in (D). Unpaired two-tailed t-test was performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 7C]Figure 7 shows the enrichment of S5 EP cells during aggregate formation in suspension. HS980 and H1 cells were differentiated to S5 on a 2D surface coated with LN-521. Cells were dissociated into single cells on day 4 of S5 and then maintained in suspension for 1 day to generate 3D aggregates. Expression of NKX6.1, NEUROD1, and Ki-67 was examined by flow cytometry before and after aggregate formation. To examine the effect of the ROCK inhibitor H1152 on aggregate formation, different concentrations of H1152, ranging from 0 to 10 μM, were added to the single-cell suspension as indicated. The following day, marker expression and islet cell counts were measured. Representative dot plots are shown in (A), (B), and (C). The percentages of NKX6.1+ / NEUROD1+ cells and NEUROD1+ cells were calculated from three independent experiments and are shown in the bar graph in (A). The number of islet cells generated from 106 single cells in 3D suspension was calculated from three independent samples and is shown in the bar graph in (D). Unpaired two-tailed t-test was performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 7D]Figure 7 shows the enrichment of S5 EP cells during aggregate formation in suspension. HS980 and H1 cells were differentiated to S5 on a 2D surface coated with LN-521. Cells were dissociated into single cells on day 4 of S5 and then maintained in suspension for 1 day to generate 3D aggregates. Expression of NKX6.1, NEUROD1, and Ki-67 was examined by flow cytometry before and after aggregate formation. To examine the effect of the ROCK inhibitor H1152 on aggregate formation, different concentrations of H1152, ranging from 0 to 10 μM, were added to the single-cell suspension as indicated. The following day, marker expression and islet cell counts were measured. Representative dot plots are shown in (A), (B), and (C). The percentages of NKX6.1+ / NEUROD1+ cells and NEUROD1+ cells were calculated from three independent experiments and are shown in the bar graph in (A). The number of islet cells generated from 106 single cells in 3D suspension was calculated from three independent samples and is shown in the bar graph in (D). Unpaired two-tailed t-test was performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 8A]Figure 8 shows the comparative results of short-term and long-term pancreatic differentiation protocols. HS980 and H1 cells were differentiated into S5 EP cells on LN-521-coated plates using the short-term protocol disclosed herein and a comparative long-term protocol. On day 4 of S5, cells were dissociated into single cells and further matured into islet-like cell aggregates in 3D suspension. Expression of key markers was examined by flow cytometry at each stage (S3-S6) as indicated. At the end of S6, in vitro glucose-stimulated insulin C-peptide secretion was examined. Islet-like aggregates were counted under a microscope and then dissociated into single cells for cell counting. Results were calculated from multiple independent samples. Unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Representative dot plots of NKX6.1 and NEUROD1 expression in HS980 cells during S3-S5. (B) Bar graph showing quantification of NKX6.1+ / NEUROD1+ and NKX6.1+ / NEUROD1- obtained using the short-term and long-term protocols. (C) Representative dot plots and bar graph showing quantification of INS and GCG expression in HS980 and H1 cells at S6 obtained using the short-term and long-term protocols. (D) Bar graph showing comparison of in vitro glucose-stimulated insulin secretion results in cells obtained using the short-term and long-term protocols, and bar graph showing the number of islet-like aggregates and islet cells per well in cultures using the short-term and long-term protocols. HS980 cell aggregates were analyzed at S6. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 8B]Figure 8 shows the comparative results of short-term and long-term pancreatic differentiation protocols. HS980 and H1 cells were differentiated into S5 EP cells on LN-521-coated plates using the short-term protocol disclosed herein and a comparative long-term protocol. On day 4 of S5, cells were dissociated into single cells and further matured into islet-like cell aggregates in 3D suspension. Expression of key markers was examined by flow cytometry at each stage (S3-S6) as indicated. At the end of S6, in vitro glucose-stimulated insulin C-peptide secretion was examined. Islet-like aggregates were counted under a microscope and then dissociated into single cells for cell counting. Results were calculated from multiple independent samples. Unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Representative dot plots of NKX6.1 and NEUROD1 expression in HS980 cells during S3-S5. (B) Bar graph showing quantification of NKX6.1+ / NEUROD1+ and NKX6.1+ / NEUROD1- obtained using the short-term and long-term protocols. (C) Representative dot plots and bar graph showing quantification of INS and GCG expression in HS980 and H1 cells at S6 obtained using the short-term and long-term protocols. (D) Bar graph showing comparison of in vitro glucose-stimulated insulin secretion results in cells obtained using the short-term and long-term protocols, and bar graph showing the number of islet-like aggregates and islet cells per well in cultures using the short-term and long-term protocols. HS980 cell aggregates were analyzed at S6. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 8C]Figure 8 shows the comparative results of short-term and long-term pancreatic differentiation protocols. HS980 and H1 cells were differentiated into S5 EP cells on LN-521-coated plates using the short-term protocol disclosed herein and a comparative long-term protocol. On day 4 of S5, cells were dissociated into single cells and further matured into islet-like cell aggregates in 3D suspension. Expression of key markers was examined by flow cytometry at each stage (S3-S6) as indicated. At the end of S6, in vitro glucose-stimulated insulin C-peptide secretion was examined. Islet-like aggregates were counted under a microscope and then dissociated into single cells for cell counting. Results were calculated from multiple independent samples. Unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Representative dot plots of NKX6.1 and NEUROD1 expression in HS980 cells during S3-S5. (B) Bar graph showing quantification of NKX6.1+ / NEUROD1+ and NKX6.1+ / NEUROD1- obtained using the short-term and long-term protocols. (C) Representative dot plots and bar graph showing quantification of INS and GCG expression in HS980 and H1 cells at S6 obtained using the short-term and long-term protocols. (D) Bar graph showing comparison of in vitro glucose-stimulated insulin secretion results in cells obtained using the short-term and long-term protocols, and bar graph showing the number of islet-like aggregates and islet cells per well in cultures using the short-term and long-term protocols. HS980 cell aggregates were analyzed at S6. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 8D]Figure 8 shows the comparative results of short-term and long-term pancreatic differentiation protocols. HS980 and H1 cells were differentiated into S5 EP cells on LN-521-coated plates using the short-term protocol disclosed herein and a comparative long-term protocol. On day 4 of S5, cells were dissociated into single cells and further matured into islet-like cell aggregates in 3D suspension. Expression of key markers was examined by flow cytometry at each stage (S3-S6) as indicated. At the end of S6, in vitro glucose-stimulated insulin C-peptide secretion was examined. Islet-like aggregates were counted under a microscope and then dissociated into single cells for cell counting. Results were calculated from multiple independent samples. Unpaired two-tailed t-tests were performed in Microsoft Excel. (A) Representative dot plots of NKX6.1 and NEUROD1 expression in HS980 cells during S3-S5. (B) Bar graph showing quantification of NKX6.1+ / NEUROD1+ and NKX6.1+ / NEUROD1- obtained using the short-term and long-term protocols. (C) Representative dot plots and bar graph showing quantification of INS and GCG expression in HS980 and H1 cells at S6 obtained using the short-term and long-term protocols. (D) Bar graph showing comparison of in vitro glucose-stimulated insulin secretion results in cells obtained using the short-term and long-term protocols, and bar graph showing the number of islet-like aggregates and islet cells per well in cultures using the short-term and long-term protocols. HS980 cell aggregates were analyzed at S6. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. [Figure 9A]Figure 9 shows immunocytochemical analysis of the expression of stage-specific markers in cell culture. HS980 cells differentiated using short-term and long-term protocols were analyzed using antibodies specific for key markers expressed during S4 to S6. (A) Expression of PDX1, NKX6.1, and NEUROD1 in S4 cells. (B) Expression of PDX1, NKX6.1, and NEUROD1 in S5 cells. (C) Expression of insulin C-peptide (CPEP), glucagon (GCG), and somatostatin (SST) in S6 cells. DAPI staining was used to visualize the cells. [Figure 9B] Figure 9 shows immunocytochemical analysis of the expression of stage-specific markers in cell culture. HS980 cells differentiated using short-term and long-term protocols were analyzed using antibodies specific for key markers expressed during S4 to S6. (A) Expression of PDX1, NKX6.1, and NEUROD1 in S4 cells. (B) Expression of PDX1, NKX6.1, and NEUROD1 in S5 cells. (C) Expression of insulin C-peptide (CPEP), glucagon (GCG), and somatostatin (SST) in S6 cells. DAPI staining was used to visualize the cells. [Figure 9C] Figure 9 shows immunocytochemical analysis of the expression of stage-specific markers in cell culture. HS980 cells differentiated using short-term and long-term protocols were analyzed using antibodies specific for key markers expressed during S4 to S6. (A) Expression of PDX1, NKX6.1, and NEUROD1 in S4 cells. (B) Expression of PDX1, NKX6.1, and NEUROD1 in S5 cells. (C) Expression of insulin C-peptide (CPEP), glucagon (GCG), and somatostatin (SST) in S6 cells. DAPI staining was used to visualize the cells. [Figure 10A]Figure 10 shows the comparison of spontaneous aggregation in 3D suspension and forced aggregation using microwell plates. H1 cells were differentiated to S5 on 2D surfaces coated with LN-521. Cells were dissociated into single cells on day 4 of S5 and then maintained in 3D suspension (free), 96-well plates (96-well), or AggreWell plates (AggreWell). NKX6.1, NEUROD1, and Ki-67 expression were examined by flow cytometry before and 1 day after S5 aggregate formation. Representative dot plots are shown in (A). The percentages of S5 NKX6.1+ / NEUROD1+ EP cells and Ki-67+ proliferating cells were calculated from multiple independent samples and are shown as bar graphs in (B). Differentiation to S6 islet cells was performed in suspension and AggreWell plates. At the end of S6, INS, GCG, and Ki-67 expression were examined by flow cytometry. Representative dot plots are shown in (C). Unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01. [Figure 10B] Figure 10 shows the comparison of spontaneous aggregation in 3D suspension and forced aggregation using microwell plates. H1 cells were differentiated to S5 on 2D surfaces coated with LN-521. Cells were dissociated into single cells on day 4 of S5 and then maintained in 3D suspension (free), 96-well plates (96-well), or AggreWell plates (AggreWell). NKX6.1, NEUROD1, and Ki-67 expression were examined by flow cytometry before and 1 day after S5 aggregate formation. Representative dot plots are shown in (A). The percentages of S5 NKX6.1+ / NEUROD1+ EP cells and Ki-67+ proliferating cells were calculated from multiple independent samples and are shown as bar graphs in (B). Differentiation to S6 islet cells was performed in suspension and AggreWell plates. At the end of S6, INS, GCG, and Ki-67 expression were examined by flow cytometry. Representative dot plots are shown in (C). Unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01. [Figure 10C]Figure 10 shows the comparison of spontaneous aggregation in 3D suspension and forced aggregation using microwell plates. H1 cells were differentiated to S5 on 2D surfaces coated with LN-521. Cells were dissociated into single cells on day 4 of S5 and then maintained in 3D suspension (free), 96-well plates (96-well), or AggreWell plates (AggreWell). NKX6.1, NEUROD1, and Ki-67 expression were examined by flow cytometry before and 1 day after S5 aggregate formation. Representative dot plots are shown in (A). The percentages of S5 NKX6.1+ / NEUROD1+ EP cells and Ki-67+ proliferating cells were calculated from multiple independent samples and are shown as bar graphs in (B). Differentiation to S6 islet cells was performed in suspension and AggreWell plates. At the end of S6, INS, GCG, and Ki-67 expression were examined by flow cytometry. Representative dot plots are shown in (C). Unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01. [Figure 11] Figure 11 is a schematic diagram of the in vitro differentiation protocol disclosed herein. The factors and duration of each stage are as follows: Cells on LN-521 coated surfaces were dissociated into single cells on day 14 and then maintained in 3D suspension. [Figure 12A]Figure 12 shows pancreatic differentiation of human ESC and iPSC lines. Cells were differentiated using a short-term protocol. Expression of key markers was examined by flow cytometry at day 4 after the start of S5 and at the end of S6. In vitro glucose-stimulated insulin C-peptide secretion was examined at the end of S6. Results were calculated from multiple independent experiments. Unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. Representative dot plots of INS and GCG expression at S6 and NKX6.1 and NEUROD1 expression at S5 are shown in (A) and (B). Bar graphs showing quantification of INS+ / GCG-, INS- / GCG+, and INS+ / GCG+ cell populations (left) and the results of in vitro glucose-stimulated insulin secretion (right) are shown in (A). (A) Differentiation of hESC lines. (B) Differentiation of human iPSC line C7. [Figure 12B] Figure 12 shows pancreatic differentiation of human ESC and iPSC lines. Cells were differentiated using a short-term protocol. Expression of key markers was examined by flow cytometry at day 4 after the start of S5 and at the end of S6. In vitro glucose-stimulated insulin C-peptide secretion was examined at the end of S6. Results were calculated from multiple independent experiments. Unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. Representative dot plots of INS and GCG expression at S6 and NKX6.1 and NEUROD1 expression at S5 are shown in (A) and (B). Bar graphs showing quantification of INS+ / GCG-, INS- / GCG+, and INS+ / GCG+ cell populations (left) and the results of in vitro glucose-stimulated insulin secretion (right) are shown in (A). (A) Differentiation of hESC lines. (B) Differentiation of human iPSC line C7. [Figure 13A]Figure 13 shows the results of a comparison of the short-term protocol and two published protocols using scRNA sequencing. H1 cells were differentiated using the short-term protocol and scRNA sequencing was performed at the end of S6. (A) Schematic of the short-term protocol and the two published protocols. The duration, factors, and 2D / 3D culture systems of each stage are shown. (B) UMAP with cell type prediction (top) and expression levels of the marker genes INS and GCG in the predicted cell types (bottom). [Figure 13B] Figure 13 shows the results of a comparison of the short-term protocol and two published protocols using scRNA sequencing. H1 cells were differentiated using the short-term protocol and scRNA sequencing was performed at the end of S6. (A) Schematic of the short-term protocol and the two published protocols. The duration, factors, and 2D / 3D culture systems of each stage are shown. (B) UMAP with cell type prediction (top) and expression levels of the marker genes INS and GCG in the predicted cell types (bottom). [Figure 14] Figure 14 shows that frozen S5 EP cells were able to generate S6 islet-like aggregates. HS980 and H1 cells were differentiated to S5 on a 2D surface coated with LN-521. Cells were dissociated into single cells on day 4 of S5 and then maintained in 3D suspension to generate S6 aggregates. Alternatively, the single cells were frozen and maintained in liquid nitrogen. To generate 3D aggregates, frozen S5 cells were thawed and cultured in 3D suspension. At the end of S6, expression of the INS and GCG markers and in vitro glucose-stimulated insulin C-peptide release were examined. Bar graphs (left) show quantification of the INS+ / GCG-, INS- / GCG+, and INS+ / GCG+ cell populations, and bar graphs (right) show the results of in vitro glucose-stimulated insulin secretion. Results were calculated from multiple independent experiments. Unpaired two-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01. [Example]

[0246] SUMMARY: This example demonstrates that the methods of the invention disclosed herein, which include culturing a population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for up to about 78 hours, e.g., 72 hours or 48 hours, result in an expansion of the number of endocrine progenitor cells that have the potential to develop into mature pancreatic β cells.

[0247] Materials and Methods hESC culture The hESC line HS980 was derived under defined xeno-free conditions as previously described (Rodin, S., et al., 2014). The hESC lines WA01 / H1 and WA09 / H9 were obtained from WiCell Research Institute (Madison, WI). The human iPSC line CTRL-7-II (C7) was described in Kele M et al., 2016. The hESC lines were maintained in NutriStem hPSC XF medium (Biological Industries, Israel, 05-100-1A) on Sarstedt multiwell cell culture plates coated with 10 μg / mL human recombinant laminin (LN)-521 (BioLamina, Sweden, LN-521) in a 37°C incubator with 5% CO2, 5% O2, and 100% humidity. hESCs were cultured every 3–5 days at 1 cm 2 The cells were enzymatically passaged at 12,000–24,000 cells per well.

[0248] In routine subculture, hESC cultures on LN-521 are incubated with Ca 2+ and Mg 2+The cells were briefly washed with HCl-free D-PBS (Thermo Fisher Scientific, 14190169) and incubated with Gibco TrypLE Select (Thermo Fisher Scientific, A1285901) for 5 minutes at 37°C. The cells were harvested into fresh NutriStem hPSC XF Medium by gently pipetting 5–10 times using a P1000 pipette, centrifuged at 300 g for 4–5 minutes, resuspended in fresh NutriStem hPSC XF Medium, and plated onto newly coated cell culture plates.

[0249] In vitro pancreatic differentiation of hESCs The stepwise pancreatic differentiation protocol described herein was modified from previously published protocols ( Pagliuca et al., 2014 ; Rezania et al., 2014 ; Millman et al., 2016 ; Vegas et al., 2016 ).

[0250] hESC lines H1, H9, and HS980 were cultured at 1 cm in NutriStem hPSC XF Medium. 2 Cells were seeded onto LN-521-coated cell culture plates at a density of 24,000 cells per plate. Pancreatic differentiation was initiated after 4 days, when a confluency of 95%-100% was achieved. Differentiated cell cultures were maintained in a 37°C incubator with 5% CO2, 20% O2, and 100% humidity.

[0251] Differentiation was divided into six stages, S1 to S6, and the media used at each stage were as follows: S1 medium: MCDB131 (Thermo Fisher Scientific, 10372019) + 25 mM sodium bicarbonate (NaHCO3) (Sigma, S6297) + 1x GlutaMAX (Thermo Fisher Scientific, 35050038) + 50 U / mL penicillin-streptomycin (Thermo Fisher Scientific, 15140122) + 2.5 mM D-glucose (final concentration 8 mM, Sigma, G8769) + 0.2% or 0.5% fatty acid-free bovine serum albumin (FAF-BSA, Sigma, A8806).

[0252] S2 medium: MCDB131 + 25 mM sodium bicarbonate (NaHCO3) + 1x GlutaMAX + 50 U / mL penicillin-streptomycin + 2.5 mM D-glucose (final concentration 8 mM) + 0.2% or 0.5% FAF-BSA + 0.25 mM vitamin C (Sigma, A4544).

[0253] S3 medium: MCDB131 + 25 mM sodium bicarbonate (NaHCO3) + 1x GlutaMAX + 50 U / mL penicillin-streptomycin + 2.5 mM D-glucose (final concentration 8 mM) + 0.5% FAF-BSA + 0.25 mM vitamin C + 1:200 ITS-X (Thermo Fisher Scientific, 51500056).

[0254] S5 medium: MCDB131 + 25 mM sodium bicarbonate (NaHCO3) + 1x GlutaMAX + 50 U / mL penicillin-streptomycin + 14.5 mM D-glucose (final concentration 20 mM) + 0.5% FAF-BSA + 1:200 ITS-X + 10 μM zinc sulfate (ZnSO4) (Sigma, Z0251) + 10 μg / mL heparin (Sigma, H3149).

[0255] S6 medium: CMRL (Thermo Fisher Scientific, 11530037) + 14 mM sodium bicarbonate (NaHCO3) + 1x GlutaMAX + 50 U / mL penicillin-streptomycin + 14.5 mM D-glucose (final concentration 20 mM) + 1% FAF-BSA + 1:200 ITS-X (3 weeks) + 10 μM zinc sulfate (ZnSO4) + 10 μg / mL heparin + 1x NEAA (Thermo Fisher Scientific, 11140035).

[0256] Short-term differentiation protocol Stage 1 Definitive Endoderm (Day 3): Undifferentiated H1, H9, and HS980 cells were cultured in Ca 2+ and Mg 2+ The cells were washed once with D-PBS containing HS980 cells (Thermo Fisher Scientific, 14040091) and then induced for 24 hours with S1 medium containing 5 μM CHIR99021 (Tocris, 4423) and 100 ng / mL activin A (R&D, 338-AC). For the next two days, the cells were fed daily with S1 medium containing 100 ng / mL activin A alone. The concentration of FAF-BSA in S1 medium was 0.2% for HS980 cells and 0.5% for H1 and H9 cells.

[0257] Stage 2 primitive gut (3 days): The cells were induced for 3 days in S2 medium containing 50 ng / mL KGF (R&D, 251-KG). The concentration of FAF-BSA was 0.2% for HS980 cells and 0.5% for H1 and H9 cells.

[0258] Stage 3 Posterior Foregut (Day 1): The cells were induced for 24 hours in S3 medium containing 50 ng / mL KGF, 2 μM retinoic acid (Sigma, R2625), 0.25 μM SANT-1 (Sigma, S4572), 0.5 μM PDBu (Tocris, 4153), and 200 nM LDN193189 (Tocris, 6053).

[0259] Stage 4 Pancreatic Progenitor Cells (2 or 3 days): Cells were induced for 3 days in S3 medium containing 50 ng / mL KGF, 100 ng / mL EGF (R&D 236-EG), 5 ng / mL activin A, 10 mM nicotinamide (Sigma N0636), 100 nM retinoic acid, 0.25 μM SANT-1, 0.5 μM PDBu, and 200 nM LDN193189. To analyze the effect of the duration of Stage 4, cells were differentiated for 1 to 5 days in this step.

[0260] Stage 5 Endocrine Progenitor Cells (Day 5): Cells were induced in S5 medium containing 20 ng / mL Betacellulin (R&D; 261-CE), 100 nM Retinoic Acid, 0.25 μM SANT-1, 100 nM GSI-XX (Sigma, 565789), 10 μM ALK5 Inhibitor II (Cayman Chemical, 14794), 1 μM GC-1 (Tocris, 4554), and 100 nM LDN193189.

[0261] On day 4 of stage 5, cells were incubated with Ca 2+ and Mg 2+ After washing once with DPBS without ATP, they were treated with Accutase at 37°C for 10 minutes and then dissociated into single cells in S5 medium by multiple pipetting using a P1000 pipette. The single cells were pelleted by centrifugation at 300g for 5 minutes and then cultured in S5 medium supplemented with 10 μM H1152 (Tocris, 2414) and other factors at a concentration of 1.0–1.5 × 10 cells / mL. 6 To generate islet-like aggregates, cells were transferred to an ultra-low attachment 6-well plate (Corning, 3471) at a total of 4–6 × 10 cells / mL in 4 mL per well. 4 The cells were placed in an incubator and incubated overnight on an orbital shaker (Infors HT Celltron) at 95 rpm.

[0262] To examine the effect of the ROCK inhibitor H1152 on cell survival and aggregate formation, different concentrations of H1152, ranging from 0 to 10 μM, were added to single cell suspensions for 1 day.

[0263] Stage 6 (4 weeks): Cell aggregates were maintained in S6 medium supplemented with 10 μM H1152, 1 μM GC-1, 10 μM Trolox (Merck Millipore, 648471), and 1 mM N-acetyl-L-cysteine (Sigma, A9165). ITS-X and H1152 were removed from the medium after 3 weeks. The aggregates were maintained in an incubator on an orbital shaker at 95 rpm.

[0264] The medium was changed daily from stage 1 to stage 5, and every 2–3 days in stage 6.

[0265] Long-term differentiation protocol: hESCs were differentiated into endocrine precursor cells using similar factors and media as in the short-term protocol above, but the duration of stages 3 and 4 was 2 and 5 days, respectively.

[0266] Stage 5 freezing of endocrine precursor cells On day 4 of S5, cells were dissociated into single cells as described above. Single cells were pelleted by centrifugation at 300 g for 5 min and then transferred to cold STEM-CELLBANKER GMP-grade solution (AMSBIO, 11924) at 1 × 10 7 The cells were resuspended at 1000 cells / mL. The cell suspension was dispensed into Nunc cryogenic tubes (Thermo Fisher Scientific, 377267) at 1-1.5 mL aliquots and cooled to -80°C using a Mr. Frosty freezing container (Thermo Fisher Scientific, 5100-0001). For controlled cooling, a programmable cooling unit was used to cool the cells at 1°C per minute. For long-term cryopreservation, the cryogenic tubes were transferred to liquid nitrogen storage.

[0267] Thawing of endocrine precursor cells at stage 5 Frozen S5 cells in cryogenic tubes were removed from liquid nitrogen storage and rapidly thawed at 37°C. Each 1 mL cell suspension was diluted and gently mixed with 5 mL of prewarmed complete S5 medium. Cells were pelleted by centrifugation at 300 g for 5 min and then diluted to 1.0–1.5 × 10 cells in complete S5 medium. 6 The cells were resuspended at 1000 cells / mL. Further differentiation procedures were performed as described above.

[0268] Aggregate formation in microwells To generate islet-like aggregates within the microwells, S5 cells were dissociated into single cells as described above. Single cells were collected at 1.5 × 10 6 Resuspend in 100 cells / mL and seed into AggreWell400 6-well plates (Stem Cell Technologies, 34421) for a total of 6 × 10 cells / mL in 4 mL per well. 4 Alternatively, 10 cells in 50 μL 4 Single cells were seeded into each well of a 96-well Microtest plate (Sarstedt, 82.1583.001), and then cultured overnight in an incubator.

[0269] Differentiation into S6 islet-like cell aggregates was performed as described above, and the aggregates were maintained without shaking in AggreWell 400 6-well plates.

[0270] Aggregate and cell counting The S6 islet-like aggregates were counted under a bright-field microscope. 2+ and Mg 2+ The cells were rinsed once with Ca-free D-PBS and incubated with Accutase for 15 min at 37°C. The aggregates were dissociated into single cells by multiple pipetting with a P1000 pipette and centrifuged at 300g for 4–5 min to remove Ca. 2+ and Mg 2+ The cells were resuspended in D-PBS without HCl. The cell number was counted using an ORFLO MOXI Z Mini Automated Cell Counter (ORFLO, MXZ001).

[0271] Flow cytometry The cells were treated with Accutase for 15 min at 37°C to dissociate them into single cells. Then, the cells were washed twice and diluted with Mg 2+ and Ca 2+ 10 in D-PBS containing 6 The cells were resuspended at 2000 cells / mL. The cells were incubated with LIVE / DEAD Fixable Dead Cell Stain Kit (Thermo Fisher Scientific, L34963 and L34965) on ice for 30 minutes. 2+ and Ca 2+ After washing twice with D-PBS without Ca, the cells were fixed with BD Bioscience Cytofix / Cytoperm buffer (554722) for 20 minutes on ice. The cells were then washed twice with 1x BD Bioscience Perm / Wash buffer (554723) and incubated with conjugated antibodies diluted in 1x BD Bioscience Perm / Wash buffer for 30 minutes on ice. After washing twice with 1x BD Bioscience Perm / Wash buffer, the cells were fixed with FACS buffer (Ca). 2+ or Mg 2+The cells were resuspended in D-PBS containing 2% fetal bovine serum (Thermo Fisher Scientific, 10082147) and 1 mM EDTA (Thermo Fisher Scientific, 15575020) without ATP and analyzed using a Beckman Coulter CytoFLEX S flow cytometer. Flow cytometry data were analyzed using BD Bioscience FlowJo v10.8 software. All conjugated antibodies were purchased from BD Bioscience: Alexa Fluor 647 mouse anti-insulin (1 / 20, 565689), PE mouse anti-glucagon (1 / 20, 565860), Alexa Fluor 488 mouse anti-human somatostatin (1 / 20, 566032), PE mouse anti-NEUROD1 (1 / 20, 563001), Alexa Fluor 647 mouse anti-NKX6.1 (1 / 20, 563338), Alexa Fluor 488 mouse anti-PDX-1 (1 / 20, 562274), and V450 mouse anti-Ki-67 (1 / 20, 561281).

[0272] Immunofluorescence Cells were fixed with 4% (wt / vol) paraformaldehyde for 20 min at room temperature (RT), followed by Mg 2+ and Ca 2+ For immunostaining, the cells were washed three times with D-PBS containing 0.3% (vol / vol) Triton X-100 (Sigma, T9284) and Mg. 2+ and Ca 2+ Block with 5% normal donkey serum (Merck Millipore, S30-100mL) in D-PBS without Mg for 1 hour at RT, followed by 0.1% (vol / vol) Triton X-100 or Tween 20 (Sigma, P9416) and 5% normal donkey serum. 2+ and Ca 2+ The cells were incubated overnight at 4°C with primary antibodies diluted in D-PBS without Mg. The next day, the cells were incubated with secondary antibodies for 1 h at RT. After each incubation step, the cells were incubated with Mg. 2+ and Ca 2+The sections were washed three times with D-PBS without HCl. The primary antibodies used were: goat anti-human PDX-1 (1 / 300, R&D, AF2419), mouse anti-Nkx6.1 (1 / 100, DSHB, F55A12-s), sheep anti-human neurogenin-3 (NGN3) (1 / 100, R&D, AF3444), goat anti-human / mouse NeuroD1 (1 / 100, R&D, AF2746), guinea pig anti-C-peptide (1 / 100, Abcam, ab30477), rat anti-C-peptide (1 / 50, DSHB, GN-ID4-s), mouse anti-glucagon (1 / 1000, Sigma, G2654), and rabbit anti-somatostatin (1 / 500, Sigma, 332A-1).

[0273] In vitro glucose stimulation Twenty to thirty hESC-derived aggregates at the end of stage 6 were incubated overnight in S6 medium without ITS-X and supplemental glucose (final concentration: 5 mM). The next day, the aggregates were transferred to a 24-well ultra-low attachment plate (Corning, 3473) and washed twice with 2 mL of Krebs buffer (129 mM sodium chloride (NaCl), 4.8 mM potassium chloride (KCl), 2.5 mM calcium chloride (CaCl), 1.2 mM magnesium sulfate (MgSO), 1 mM disodium hydrogen phosphate (NaHPO), 1.2 mM potassium dihydrogen phosphate (KHPO), 5 mM sodium bicarbonate (NaHCO), 10 mM HEPES, and 0.1% FAF-BSA). To remove residual insulin, the aggregates were then preincubated for 2 hours in 2 mL of Krebs buffer containing 2 mM glucose. The aggregates were then washed twice with 2 mL of Krebs buffer and incubated for 30 minutes in 2 mL of Krebs buffer containing 2 mM glucose. After incubation, a 500 μL supernatant sample (low glucose sample) was collected. The aggregates were washed once with 2 mL of Krebs buffer and incubated for 30 minutes in 2 mL of Krebs buffer containing 2 mM glucose. After incubation, a 500 μL supernatant sample (high glucose sample) was collected. The aggregates were washed twice with 2 mL of Krebs buffer and then incubated again for 30 minutes in 2 mL of Krebs buffer containing 2 mM glucose. A 500 μL supernatant sample (low glucose sample) was collected. The aggregates were washed once with 2 mL of Krebs buffer and then incubated for 30 minutes in 2 mL of Krebs buffer containing 2 mM glucose and 30 mM potassium chloride (KCl) (polarization challenge). A 500 μL supernatant sample (KCl challenge sample) was collected. After potassium chloride loading, the aggregates were treated with Accutase for 15 minutes to disperse them into single cells, and the total cell number was counted using an ORFLO MOXI Z cell counter. The collected supernatant samples containing secreted insulin were processed using a human C-peptide ELISA kit (R&D, DICP00). The measured values of human insulin C-peptide were normalized by the total cell number and 10 3The results were expressed as pmol C-peptide released per cell. If the ELISA was not performed on the same day, the samples were stored at -80°C.

[0274] scRNA sequencing sample preparation hESC-derived islets were harvested at the end of S6 for scRNA sequencing. 2+ and Mg 2+ The cells were rinsed twice with D-PBS without Ca and incubated with TrypLE on an orbital shaker at 37°C for 15-20 minutes. The aggregates were dissociated into single cells by multiple pipetting with a P1000 pipette and centrifuged at 300g for 4-5 minutes. 2+ and Mg 2+ The cells were resuspended in D-PBS containing 0.04% FAF-BSA without HCl and filtered through a 40 μm cell strainer (VWR, 732-2760). To determine the total cell number and viability, the single-cell suspension was stained with 0.4% trypan blue solution (Thermo Fisher Scientific, 15250061) and counted using a hemocytometer.

[0275] Single-cell RNA-seq analysis 3000 cells were used for scRNA sequencing library construction. RNA sequencing was performed on an Illumina Nextseq 2000 machine using the 10x Genomics Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics, CG000315 or CG000388), optionally combined with the Cell Multiplexing Oligo Labeling protocol (10x Genomics, CG000391). Analysis steps were performed in Cell Ranger 3.1.0 to generate FastQ files and functional barcode matrices. Uniform manifold approximate projection (UMAP) was performed for dimensionality reduction, cell type identification, and differential gene expression analysis.

[0276] Example 1 In this example, various hESC lines were used to compare cell culture substrates for their ability to support pancreatic differentiation, which was assessed based on the expression of key markers for stage 4 (S4) pancreatic progenitor cells (PP) and stage 5 (S5) endocrine progenitor cells (EP).

[0277] Protocols for in vitro pancreatic differentiation of hPSCs cultured on 2D surfaces coated with feeder cells or Matrigel have been developed (Pagliuca et al. 2014; D'Amour et al., 2006; Kroon et al., 2008). However, for clinical application of hPSC-derived islets, xeno-free, chemically defined coated substrates are highly preferable. Therefore, we compared human recombinant LN-511 and LN-521 with Matrigel for their ability to support pancreatic differentiation. hESC lines HS980 and H1 were differentiated on these three substrates using a long-term differentiation protocol (see Figure 1B, top panel), and the expression of key progenitor markers PDX1, NKX6.1, and NEUROD1 was measured by flow cytometry at the end of S4 and on day 4 of S5.

[0278] Results: The results showed that S4 PDX1 + / NKX6.1 + We demonstrated that the proportion of pancreatic progenitor cells (PPs) was similar on all substrates (Figure 2A). However, PDX1 + / NKX6.1 + The level of S5 NKX6.1 cells was dramatically reduced on Matrigel and to a lesser extent on LN-511, but remained relatively stable on LN-521, even as S4 cells further differentiated to S5 (Figure 2B). + / NEUROD1 + The percentage of endocrine precursor cells (EP) was much higher in LN-521 than in Matrigel, but not significantly higher in LN-511 (Fig. 2C).

[0279] Taken together, the data indicate that Matrigel, LN-511, and LN-521 can support differentiation into S4 PP cells. LN-521, in particular, provided a suitable surface environment for the endocrine differentiation of S4 PP cells. Therefore, we decided to continue all experiments with LN-521 instead of Matrigel. Because the percentage of S5 EP cells was only 20-30% in LN-521, modifications were necessary to optimize the differentiation protocol.

[0280] Example 2 In this example, the effect of the duration of stage 4 on endocrine differentiation was examined.

[0281] Stage 3: Evaluation of different lengths To date, the short duration of S3 has resulted in S4 PDX1 + / NKX6.1 + It has been reported that S4 promotes the PP cell population and inhibits the immature polyhormonal cell population (Nostro et al., 2015). However, it was unclear whether the duration of S4 also affects subsequent differentiation stages. Therefore, we decided to analyze and optimize the duration of both S3 and S4. To identify the minimum duration of S3, HS980 and H1 cells were differentiated into S3 posterior foregut (PF) cells, and the expression of the key PF marker PDX1 was measured at the end of S2 and on days 1 or 2 of S3.

[0282] Results: PDX1 was already expressed in most cells one day after the start of S3. Immunocytochemical analysis and PDX1 expression on days 6, 7, and 8 of culture showed no significant differences. + There was only a slight increase in expression after 2 days, as shown in a representative dot blot showing cell number (Fig. 3).

[0283] Thus, the data suggest that a 1-day duration of S3 may contribute to the differentiation of S2 PGT cells into S3 PDX1 + This indicates that the 2-day duration of S3 PDX1 was sufficient to induce differentiation into PF cells. +The number of PF cells also increased. Therefore, we concluded that the duration of S3 should be up to 48 hours, e.g., 24 hours. Effect of the duration of stage 4 on differentiation into endocrine precursor cells

[0284] To analyze the effect of S4 duration on S5 EP differentiation, HS980, H1, and H9 cells were induced under S3 for 1 day and then under S4 for 1, 2, 3, 4, or 5 days (schematically shown in Figure 4A ).

[0285] Results: NKX6.1 and NEUROD1 expression was measured at the end of S4 and on day 4 of S5 (representative dot plots are shown in the upper and lower panels of Figure 4B, respectively). The results showed that increasing the S4 duration from 2 to 5 days in HS980 cells significantly increased S4 NKX6.1 expression. + The proportion of PP cells was significantly increased. In contrast, S5 NKX6.1 + / NEUROD1 + The percentage of EP cells decreased with increasing duration of S4 (Figure 4B). Similar inhibitory effects were observed in both H1 and H9 cells. When the duration exceeded 2–3 days, NKX6.1 + / NEUROD1 + The proportion of EP cells began to decrease (Figure 4C). The experiment was repeated several times, and the results showed that 2-3 days under S4 resulted in a decrease in the proportion of EP cells, followed by S5 NKX6.1 + / NEUROD1 + This suggests that the NKX6.1 expression at S5 was optimal for differentiation into EP cells (Figure 4D). In contrast, a longer duration at S4 resulted in a higher expression of NKX6.1 at S5. + / NEUROD1 - The proportion of NKX6.1 cells significantly increased (Figure 4D). + / NEUROD1 - The cells are actually S4 NKX6.1 + PP cells, and long-term inhibitory effects on S5 NKX6.1 + / NEUROD1 +This likely resulted in a failure of further differentiation into EP cells (Figure 4D). The inhibitory effect of a prolonged S4 duration was particularly pronounced in H9 cells, and shortening it from 5 days to 2 days significantly reduced the total NEUROD1 levels in HS980 and H1 cells. + The proportion of endocrine cells increased to a similar level (Figure 4E). The results also showed that one day under S4 was insufficient to induce NKX6.1 (Figure 4E).

[0286] Therefore, if the S4 duration is set to 2-3 days, NKX6.1 + / NEUROD1 + It was concluded that a higher percentage of EP cells was obtained and was beneficial for differentiation into S5 EP cells. An optimal S4 duration also minimized cell line variation during pancreatic differentiation.

[0287] Effect of the duration of stage 4 on islet cell maturation The effect of S4 duration on the maturation of S6 islet cells was also analyzed. To generate S6 islet-like cell aggregates, S5 EP cells at different S4 durations were dissociated into single cells and maintained in suspension as described above. Expression of key markers, insulin (INS) and glucagon (GCG), was analyzed at the end of S6.

[0288] Results: As shown in the representative dot plot (Figure 4E) and bar graph (Figure 4F), S4 durations longer than 3 days resulted in INS + The proportion of monohormonal β cells decreased, and GCG + The percentage of monohormonal α-cells was significantly increased. Continuing S4 for 2 days increased the percentage of EP cells in H1 and H9 cell cultures, and continuing S4 for 3 days increased the percentage of EP cells in H980 cultures (Figure 4D). However, the 3-day duration did not increase the percentage of EP cells in S6 INS. + The proportion of β cells was the highest ( Fig. 4F ).

[0289] Summary: Taken together, these results suggest that long-term S4 duration is associated with S4 NKX6.1 + S5 NKX6.1 promoted differentiation into PP cells+ / NEUROD1 + On the other hand, a shorter S4 duration inhibited the further differentiation of S4 NKX6.1 cells. + PP cells were reduced, but S5 NKX6.1 + / NEUROD1 + The EP cells were increased. The results also showed that the S4 duration had opposite effects on the α-cell and β-cell populations. A 3-day duration under S4 significantly increased the INS + β cells are promoted, and GCG is + Alpha cells were strongly promoted. Importantly, the results were confirmed in three different hESC lines (H1, H9, and HS980).

[0290] Example 3 In this example, various cell culture coated substrates were evaluated for pancreatic differentiation.

[0291] Chemically defined, xeno-free cell culture substrates such as LN-521 can provide more consistent and reliable conditions for the growth and differentiation of pluripotent stem cells. To confirm whether hESCs can differentiate into pancreatic endocrine cells on other matrix proteins, HS980, H1, and H9 cells were passaged on cell culture plates coated with Matrigel (1 / 100, Corning, 354277) or human recombinant LN-111, LN-121, LN-211, LN-221, LN-332, LN-411, LN-421, LN-511, or LN-521 (all from BioLamina). HS980, H1, and H9 cells adhered to LN-111, LN-121, LN-332, LN-421, LN-511, LN-521, and Matrigel (Table 2).

[0292] [Table 2]

[0293] Cells were differentiated into S5 EP cells using a short-term protocol and analyzed for NKX6.1 and NEUROD1 expression on day 4 of stage 5 (Figure 5A). The results showed that LN-332, LN-521, and LN-511 differentiated S5 NKX6.1 more efficiently than Matrigel. + / NEUROD1 + We demonstrated that LN-511 promoted S5 EP differentiation in both HS980 and H1 cells as efficiently as LN-521 when a short-term protocol was used instead of a long-term protocol (Figures 5A and 5B). H1 cells were also differentiated on plates coated with 10 μg / mL fibronectin (FN, Sigma, F0895) or vitronectin (VTN, Sigma, 5051). The results indicated that FN and VTN could support S5 EP differentiation (Figure 5B).

[0294] S5 EP cells generated on LN-332, LN-511, LN-521, and Matrigel were selected for further differentiation into S6 islet cells as described above. Expression of INS and GCG was measured at the end of S6. Results showed that S5 EP cells from these substrates differentiated into S6 monohormonal INS cells. + β cells and GCG + We demonstrated that α-cells could be generated (Fig. 5C).

[0295] Taken together, these results demonstrate that the short-term protocol is highly efficient for defined substrates that do not contain multiple heterogeneous materials.

[0296] Example 4 In this example, it was investigated whether S4 cells or S5 cells are more suitable for in vitro pancreatic islet formation.

[0297] Previous reports have used purified S4 PP cells for islet generation (Cogger et al., 2017; Ameri et al., 2017; Kelly et al., 2011). To determine which cell populations were suitable for islet formation, HS980 cells were dissociated into single cells at the end of S4 or on day 4 of S5. The cells were then maintained in suspension to generate islet-like aggregates (Figure 6A). Expression of key endocrine markers INS and GCG, number of islet-like aggregates, and number of islet cells were analyzed at the end of S6.

[0298] Results: The results showed that aggregates generated from S5 EP cells were more INS than aggregates from S4 PP cells. + β cells and GCG + The proportion of α-cells was much higher in S5 EP cells (Fig. 6B). S5 EP cells generated more than 10 times more islet-like aggregates and islet cells than S4 PP cells (Fig. 6B).

[0299] To determine whether a longer S5 duration would improve islet formation, S5 EP cells were dissociated into single cells on day 6 after the initiation of S5 and maintained in 3D suspension. However, the proportion of α- and β-cells remained unchanged, and the number of aggregates / cells actually decreased at S6 (Figure 6B).

[0300] Taken together, these data indicate that S5 EP cells, but not S4 PP cells, efficiently formed islet-like aggregates in 3D suspension, and that a 4-day period under S5 was sufficient for aggregate formation in suspension.

[0301] Example 5 In this example, the effect of culturing cells in 3D single-cell suspension was investigated.

[0302] To investigate whether 3D single-cell suspension selectively promotes aggregate formation from S5 EP cells, HS980 and H1 cells were differentiated to S5 EP cells on LN-521 and then dissociated into single cells in 3D suspension. Expression of NKX6.1, NEUROD1, and the cell proliferation marker Ki-67 was analyzed before and after aggregate formation in 3D suspension.

[0303] Results: Results showed that in 3D aggregates at day 15 compared to 2D cells at day 14, S5 NKX6.1 in newly formed aggregates was significantly higher. + / NEUROD1 + Cellular and total NEUROD1 + The proportion of cells clearly increased, and NEUROD1 - The results also show that the proportion of non-endocrine cells was significantly reduced (Figure 7A). + EP cells are non-proliferative Ki-67 - cells and proliferative Ki-67 + / NEUROD1 - Cellular and non-proliferative Ki-67 - / NEUROD1 - We showed that most of the cells were removed during aggregate formation in the 3D suspension (Figure 7B).

[0304] To determine whether the ROCK inhibitor was required for S5 EP cell survival and aggregate formation, we added various concentrations of H1152 (0-10 μM) to 3D single-cell suspensions for 1 day. One day after aggregate formation in the 3D suspension, we measured the expression of NKX6.1, NEUROD1, and Ki-67, and the number of aggregated cells.

[0305] Results: The results showed that the concentration of H1152 did not affect the proportion of S5 EP cells in the newly formed aggregates (Figure 7C). However, the number of islet cells significantly increased in the presence of low concentrations of H1152, indicating that H1152 promoted the survival of S5 EP cells as single cells in suspension (Figure 7D).

[0306] Taken together, these results demonstrate the selective enrichment of S5 EP cells by 3D aggregate formation from single cells, which is consistent with previous studies on fetal pancreatic development ( Gouzi et al., 2017 ).

[0307] Example 6 In this example, short-term and long-term pancreatic differentiation protocols were compared by comparing the percentages of pancreatic progenitor cells (PP) and endocrine progenitor cells (EP) obtained.

[0308] Next, we compared short-term and long-term protocols. HS980 cells on LN-521 were differentiated into S5 EP cells using short-term and long-term differentiation protocols as described above (schematically shown in Figure 1B). Expression of key progenitor markers was analyzed at the end of S3, at the end of S4, and on day 4 from the start of S5 (days 7, 10, and 14 of differentiation for the short-term protocol, and days 8, 13, and 17 of culture for the long-term protocol).

[0309] Results: The results showed that the short-term and long-term protocols had opposite effects. The short-term protocol significantly increased S4 NKX6.1 activity compared to the long-term protocol. + Although the proportion of PP cells was lower, S5 NKX6.1 + / NEUROD1 + The proportion of EP cells increased (Figures 8A and 8B). + / NEUROD1 - The percentage of non-endocrine cells was lower in the short-term protocol than in the long-term protocol (Figures 8A and 8B). These results indicate that the short duration of S3+S4 has a positive effect on S5 EP differentiation.

[0310] To investigate whether the duration of S3+S4 also affects islet maturation during S6, S5 EP cells were dissociated into single cells and maintained in suspension as described above. Expression of key endocrine markers INS and GCG, number of islet-like aggregates and islet cells, and in vitro glucose-stimulated insulin secretion were analyzed at the end of S6 (Figures 8C and 8D).

[0311] Results: The short-term protocol resulted in significantly more INS than the long-term protocol. + The proportion of monohormonal β cells was increased, but GCG + The results showed a lower percentage of monohormonal α-cells (Figure 8C). A similar effect of the S3+S4 duration was observed in H1 cells (Figure 8C). Furthermore, the short-term protocol generated significantly more islet-like aggregates and islet cells than the long-term protocol (Figure 8D). In vitro glucose stimulation experiments confirmed that aggregates at the end of S6 were fully functional and capable of increasing insulin secretion 10-fold in response to high glucose concentrations, while aggregates generated using the short-term protocol secreted more insulin at high glucose levels (Figure 8D). Taken together, these results indicated that a short-term S3+S4 duration has a strong positive impact on islet maturation during S6.

[0312] The expression of stage-specific markers was also analyzed using immunocytochemistry (ICC) (Figure 9). The long-term protocol resulted in the expression of mainly PDX1 at S4. + / NKX6.1 + A highly dense layer containing PP cells was induced (Figure 9A). In contrast, the short-term protocol did not induce the formation of S4 PDX1 cells. + / NKX6.1 + PP cells were less induced, and some cells were NKX6.1 - The NKX6.1 gene expression remained unchanged (Fig. 9A). - Early induction of NEUROD1 in S4 cells was also observed in both protocols (Figure 9A). Further differentiation into S5 EP cells showed the opposite effect: the short-term protocol produced more S5 NKX6.1 than the long-term protocol. + / NEUROD + EP cells were generated (Figure 9B). The results also showed that the ratio of β cells to α cells differed between aggregates generated using the short-term and long-term protocols (Figure 9C). The short-term protocol induced more insulin C-peptide (CPEP)-positive β cells, while the long-term protocol induced more GCG-positive β cells. +Alpha cells were induced (Figure 9C). A small number of somatostatin (SST)-positive delta cells were also observed (Figure 9C). Taken together, the ICC results support the conclusions from the flow cytometry analysis above.

[0313] Example 7 In this example, we investigate the impact of spontaneous aggregation of S5 cells into 3D culture on the generation of monohormonal pancreatic β-cells compared to forced aggregation using AggreWell 400 plates and 96-well plates.

[0314] For spontaneous aggregation, H1 cells were dissociated into single cells at S5 and maintained in 3D suspension as described above. For forced aggregation within microwells, S5 cells were instead transferred to AggreWell or 96-well plates. The identity of the generated cells was examined by flow cytometry 1 day after 3D aggregation and at the end of S6.

[0315] Results: The results showed that the aggregates generated in the microwells were primarily NKX6.1, whereas the aggregates generated spontaneously in the 3D suspension cultures were primarily NKX6.1. + / NEUROD1 + / Ki-67 - The results showed that EP cells were included (Fig. 10A, Fig. 10B). + Cells were removed from the aggregates in the microwells in the same manner as in the 3D suspension (Figure 10A, Figure 10B). At the end of S6, the aggregates formed in the microwells had INS comparable to those formed spontaneously. + β cells and GCG + These aggregates contained α-cells (Fig. 10C). + Only proliferating cells remained (Fig. 10C).

[0316] Taken together, these results suggest that S5 EP cells can separate from other non-endocrine cell types and then spontaneously self-aggregate.

[0317] Example 8 In this example, we evaluated cell line variability among multiple hPSC lines differentiated using a short-term pancreatic differentiation protocol.

[0318] hESC lines HS980, H1, and H9 were differentiated into S6 islet cells as described above. Expression of key markers INS and GCG, as well as in vitro glucose-stimulated insulin secretion, were analyzed at the end of S6. To determine whether the short-term differentiation protocol also works with human iPSC lines, C7 cells were differentiated in LN-521 and key marker expression was examined at the end of S5 and S6.

[0319] Results: The results showed that the single hormone INS + β cells and GCG + The proportion of α-cells was similar in all three hESC lines, indicating that the aggregates were functional and capable of increasing insulin secretion in response to high glucose concentrations in a similar manner (Figure 12A). The results also showed that the human iPSC lines S5 NKX6.1 + / NEUROD1 + Differentiation into EP cells followed by S6 monohormonal INS + β cells and GCG + We demonstrated that they could differentiate into α cells (Fig. 12B).

[0320] Taken together, these results demonstrate relatively low cell line variability among multiple human ES and iPSC cell lines differentiated using short-term pancreatic differentiation protocols.

[0321] Example 9 In this example, we compared our short-term pancreatic differentiation protocol with two previously published protocols (Augsornworawat et al., 2020, and Balboa et al., 2022) using datasets from single-cell RNA sequencing (scRNAseq) experiments (Figure 13A).

[0322] H1 cells were differentiated into S6 islet cells using the short-term pancreatic differentiation protocol described above. The gene expression profile of islet cells was examined by single-cell RNA sequencing at the end of S6. The resulting dataset was processed and visualized by homogeneous manifold projection (Figure 13B). The cell type identity and expression of key marker genes in each cell population were analyzed (Figure 13B). Two published datasets (Augsornworawat et al., 2020, and Balboa et al., 2022) were also included in the analysis.

[0323] Results: The results showed that S6 islets generated using the short-term differentiation protocol contained two mature, monohormonal cell populations: INS-expressing β cells and GCG-expressing α cells (Figure 13B, left). The islets generated by Augsornworawat et al. also contained two major endocrine cell populations (Figure 13B, center). However, most cells coexpressed both INS and GCG, representing immature, polyhormonal β and α cells. In contrast, the islets generated by Balboa et al. contained both endocrine and non-endocrine cell types, although the expected β and α cells were monohormonal (Figure 13B, right).

[0324] Taken together, these results indicate that islets generated by short-term pancreatic differentiation protocols contain primarily mature monohormonal β- and α-cells. In contrast, two recently published protocols generated islets containing either immature polyhormonal or non-endocrine cells.

[0325] Example 10 In this example, the ability of frozen and non-frozen S5 EP cells to generate 3D islets was compared.

[0326] HS980 and H1 cells were differentiated to S5 as described above. The cells were dissociated into single cells in 3D suspension and then further differentiated to S6. Alternatively, S5 single cells were frozen and maintained in liquid nitrogen. The frozen cells were then thawed and differentiated to S6 as described above. At the end of S6, islet cell identity and in vitro glucose-stimulated insulin release were examined.

[0327] Results: The results showed that the freeze / thaw procedure at S5 increased INS in HS980 and H1 cells. + The proportion of β cells did not change (Fig. 14, left). After freeze / thaw treatment, GCG was not observed in HS980 cells. + The proportion of α-cells, but not H1 cells, was significantly reduced (Figure 14, left). The results also showed that islets differentiated from both frozen and non-frozen S5 EP cells were functional and capable of increasing insulin release in response to high blood glucose levels (Figure 14, right).

[0328] Taken together, these results demonstrate that S5 EP cells can be cryopreserved and that frozen cells are an ideal source for generating functional islets in vitro.

[0329] References 1. Rodin, S., et al., Clonal culturing of human embryonic stem cells on laminin-521 / E-cadherin matrix in defined and xeno-free environment. Nat Commun, 2014. 5: p. 3195. 2. Pagliuca, FW, et al., Generation of functional human pancreatic β-cells in vitro. Cell, 2014. 159(2): p. 428-39. 3. Rezania, A., et al., Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol, 2014. 32(11): p. 1121-33. 4. Millman, J.R., et al., Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nat Commun, 2016. 7: p. 11463. 5. Vegas, A.J., et al., Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nat Med, 2016. 22(3): p. 306-11. 6. D’Amour, K.A., et al., Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol, 2006. 24(11): p. 1392-401. 7. Kroon, E., et al., Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol, 2008. 26(4): p. 443-52. 8. Nostro, M.C., et al., Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines. Stem Cell Reports, 2015. 4(4): p. 591-604. 9. Cogger, K.F., et al., Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors. Nat Commun, 2017. 8(1): p. 331. 10. Ameri, J., et al., Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2. Cell Rep, 2017. 19(1): p. 36-49. 11. Gouzi, M., et al., Neurogenin3 initiates stepwise delamination of differentiating endocrine cells during pancreas development. Dev Dyn, 2011. 240(3): p. 589-604. 12. Augsornworawat, P., et al., Single-Cell Transcriptome Profiling Reveals β Cell Maturation in Stem Cell-Derived Islets after Transplantation. Cell Rep, 2020. 32(8): p. 1-13. 13. Balboa, D., et al., Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nat Biotechnol, 2022. 40(7): p. 1042-55. 14. Kele, M., et al., Generation of human iPS cell line CTL07-II from human fibroblasts, under defined and xeno-free conditions. Stem Cell Res. 2016 Nov;17(3): p. 474-478.

[0330] Itemized List of Embodiments 1. A method for generating cells of the pancreatic endocrine lineage, such as the pancreatic beta cell lineage, comprising the steps of: a) generating a cell of the pancreatic endocrine lineage; a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells for about 78 hours or less, e.g., about 72 hours or less, under conditions permissive for differentiation into pancreatic progenitor cells; and c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1.

[0331] 2. The method according to item 1, wherein in step b), the cell population is cultured for a period of about 42 to 78 hours, for example, about 44 to 76 hours, for example, about 46 to 74 hours, for example, about 48 to 72 hours.

[0332] 3. The method according to item 1 or 2, wherein in step b) the cell population is cultured for a period of about 66 to 78 hours, such as about 68 to 76 hours, such as about 70 to 74 hours, for example about 72 hours.

[0333] 4. The method according to item 1 or 2, wherein in step b) the cell population is cultured for a period of about 42 to 54 hours, such as about 44 to 52 hours, such as about 46 to 50 hours, for example about 48 hours.

[0334] 5. The method of any one of items 1 to 4, wherein in step c), the cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1, is further characterized by expression of at least one marker selected from the group consisting of PTF1A, SOX9, HNF6, and CPA, for example, a marker selected from the group consisting of PTF1A and SOX9.

[0335] 6. The method according to any one of items 1 to 5, wherein step b) comprises culturing the cell population in a culture medium in the presence of an effective amount of epidermal growth factor (EGF), such as human EGF, or a derivative or agonist thereof, and an effective amount of nicotinamide (NIC) or a derivative or agonist thereof.

[0336] 7. The method according to any one of items 1 to 6, wherein step b) comprises culturing the cell population in a culture medium in the presence of an effective amount of EGF, such as human EGF, and an effective amount of NIC.

[0337] 8. The method according to any one of items 6 to 7, wherein the effective amount of EGF or a derivative or agonist thereof is about 50 to 200 ng / mL, for example, about 50 to 150 ng / mL, for example, about 75 to 125 ng / mL, for example, about 100 ng / mL.

[0338] 9. The method according to any one of items 6 to 8, wherein the effective amount of the NIC or a derivative or agonist thereof is about 5 to 20 nM, for example, about 5 to 15 mM, for example, about 8 to 12 mM, for example, about 10 mM.

[0339] 10. The method according to any one of items 1 to 9, wherein step b) comprises culturing the cell population in a culture medium further comprising KGF, activin A, retinoic acid, SANT-1, PDBu, and LDN.

[0340] 11. The method according to any one of items 1 to 10, wherein the cells are cultured on a 2D substrate, for example, the cells are cultured as adhesion on the 2D substrate.

[0341] 12. The method according to any one of items 1 to 11, wherein the 2D substrate comprises one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and Matrigel™, such as one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and Matrigel™.

[0342] 13. The laminin (LN) and its fragments are the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, LN-121 and fragments thereof, and LN-111 and fragments thereof; For example, the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof; For example, the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, and LN-332 and fragments thereof; 13. The method according to item 12, wherein the antibody is selected from the group consisting of LN-521 and fragments thereof, or LN-511 and fragments thereof.

[0343] 14. The method of any one of items 12 to 13, wherein the laminin (LN) and fragments thereof are selected from the group consisting of LN-521, LN-511, LN-332, LN-421, LN-121, and LN-111, such as the group consisting of LN-521, LN-511, LN-332, LN-421, and LN-121, such as the group consisting of LN-521, LN-511, and LN-332, such as the group consisting of LN-521 and LN-511, for example, the laminin and fragments thereof is LN-521 or the laminin and fragments thereof is LN-511.

[0344] 15. The laminin (LN) and fragments thereof are selected from the group consisting of E8 fragments of laminins, such as E8 fragments of LN-511, E8 fragments of LN-521, E8 fragments of LN-332, E8 fragments of LN-421, E8 fragments of LN-121, and E8 fragments of LN-111, such as E8 fragments of LN-511, E8 fragments of LN-521, E8 fragments of LN-332, E8 fragments of LN-421, and 14. The method according to any one of items 12 to 13, comprising an E8 fragment of laminin selected from the group consisting of the E8 fragment of LN-121, for example the E8 fragment of LN-511, the E8 fragment of LN-521, and the E8 fragment of LN-332, for example the group consisting of the E8 fragment of LN-511 and the E8 fragment of LN-521, for example the E8 fragment of LN-511 or the E8 fragment of LN-521.

[0345] 16. The method of any one of items 1 to 15, wherein at least about 60%, such as at least about 55%, such as at least about 70%, such as at least about 75%, such as at least about 80% of the posterior foregut cells in a), e.g., posterior foregut cells characterized by expression of PDX1, are differentiated into pancreatic progenitor cells in c), e.g., pancreatic progenitor cells characterized by expression of PDX1 and NKX6.1.

[0346] 17. The method according to any one of items 1 to 16, wherein in step c) at least about 75%, such as at least about 80%, such as about 80-85%, such as about 80-90% of the total cell population expresses PDX1.

[0347] 18. The method according to any one of items 1 to 17, wherein in step c), at most about 10% of the total cell population expresses NEUROD1.

[0348] 19. The method according to any one of items 1 to 18, wherein in step c), approximately 40 to 70% of the total cell population expresses NKX6.1.

[0349] 20. Before steps a) to c), carry out the following steps a-1) to c-1): a-1) providing a cell population of primitive intestinal cells, such as primitive intestinal cells, characterized by the expression of HNF1β and / or HNF4α; b-1) culturing the population of primitive intestinal cells for about 54 hours or less under conditions that allow differentiation into posterior foregut cells; and c-1) The method according to any one of items 1 to 19, thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by the expression of PDX1.

[0350] 21. The method according to item 20, wherein in step b-1), the cell population is cultured for about 52 hours or less, for example, about 50 hours or less, for example, about 48 hours or less.

[0351] 22. The method according to item 20 or 21, wherein in step b-1), the cell population is cultured for a period of about 18 to 54 hours, such as about 20 to 52 hours, such as about 22 to 50 hours, such as about 24 to 48 hours.

[0352] 23. The method according to any one of items 20 to 22, wherein in step b-1), the cell population is cultured for about 42 to 54 hours, for example, about 44 to 52 hours, for example, about 46 to 50 hours, for example, about 48 hours.

[0353] 24. The method according to any one of items 20 to 22, wherein in step b-1), the cell population is cultured for about 18 to 30 hours, for example, about 20 to 28 hours, for example, about 22 to 26 hours, for example, about 24 hours.

[0354] 25. The method according to any one of items 20 to 24, wherein step b-1) comprises culturing the cell population in a culture medium containing KGF, retinoic acid, SANT-1, PDBu, and LDN.

[0355] 26. After steps a) to c), perform the following steps a+1) to c+1): a+1) providing a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of PDX1 and NKX6.1; b+1) culturing the population of pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells; and c+1) thereby generating a population of endocrine precursor cells, such as endocrine precursor cells, characterized by expression of NKX6.1 and NEUROD1; 26. The method according to any one of items 1 to 25, comprising:

[0356] 27. The method of item 26, wherein the endocrine precursor cell population, e.g., endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1, is further characterized by expression of at least one of PDX1 and NGN3.

[0357] 28. The method according to item 26 or 27, wherein in step b+1), the cell population of pancreatic progenitor cells is cultured for about 3 to 5 days, for example, about 3 to 4 days, or about 4 to 5 days, for example, about 4 days, or for example, about 5 days.

[0358] 28. The method according to any one of items 26 to 28, wherein the cells are cultured on a 2D substrate at least until the generation of endocrine precursor cells in step c+1).

[0359] 29. The method according to any one of items 25 to 28, wherein the 2D substrate comprises 25 or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and Matrigel™, such as one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and Matrigel™.

[0360] 30. The laminin (LN) and fragments thereof are selected from the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, LN-121 and fragments thereof, and LN-111 and fragments thereof; For example, the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof; For example, the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, and LN-332 and fragments thereof; 30. The method according to item 29, wherein the antibody is selected from the group consisting of LN-521 and fragments thereof, or LN-511 and fragments thereof.

[0361] 31. The method of any one of items 29 to 30, wherein the laminin (LN) and fragments thereof are selected from the group consisting of LN-521, LN-511, LN-332, LN-421, LN-121, and LN-111, such as the group consisting of LN-521, LN-511, LN-332, LN-421, and LN-121, such as the group consisting of LN-521, LN-511, and LN-332, such as the group consisting of LN-521 and LN-511, for example, the laminin and fragments thereof is LN-521 or the laminin and fragments thereof is LN-511.

[0362] 32. The laminin (LN) and fragments thereof are selected from the group consisting of E8 fragments of laminins, such as E8 fragments of LN-511, E8 fragments of LN-521, E8 fragments of LN-332, E8 fragments of LN-421, E8 fragments of LN-121, and E8 fragments of LN-111, such as E8 fragments of LN-511, E8 fragments of LN-521, E8 fragments of LN-332, E8 fragments of LN-421, and 31. The method according to any one of items 29 to 30, comprising an E8 fragment of laminin selected from the group consisting of the E8 fragment of LN-121, for example the E8 fragment of LN-511, the E8 fragment of LN-521, and the E8 fragment of LN-332, for example the E8 fragment of LN-511 and the E8 fragment of LN-521, for example the E8 fragment of LN-511 or the E8 fragment of LN-521.

[0363] 33. The method according to any one of items 26 to 32, wherein in step c+1), more than about 30%, such as more than about 40%, for example more than about 45%, for example more than about 50% of the total cell population are endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1.

[0364] 34. The method according to any one of items 1 to 33, wherein the number of endocrine precursor cells in step c+1) is higher compared to the number of endocrine cells obtained using a corresponding method, in which step b) of culturing the cell population of posterior foregut cells under conditions permissive for differentiation into pancreatic precursor cells is performed for about 24 hours or less and / or for about 96 hours or more.

[0365] 35. The method according to any one of items 1 to 34, wherein the method results in at least about 10%, such as at least about 15%, for example at least about 20%, such as at least about 30%, for example at least about 40%, for example at least 50%, for example at least 60% more endocrine precursor cells than a corresponding method in which step b) of culturing the cell population of posterior foregut cells under conditions permissive for differentiation into pancreatic precursor cells is for less than about 24 hours and / or for more than about 96 hours.

[0366] 36. The method according to any one of items 26 to 35, wherein step b+1) comprises culturing the cell population in a culture medium containing BTC, Alk5iII, GSI-XX, GC-1, LDN, retinoic acid, and SANT-1.

[0367] 37. The method of any one of items 1 to 36, further comprising culturing the endocrine precursor cells under conditions that allow differentiation into monohormonal pancreatic β cells.

[0368] 38. The method of any one of items 1 to 37, wherein the cells are not transferred from culture on a 2D substrate to culture on a 3D substrate before they exhibit expression of markers characteristic of endocrine precursor cells, e.g., the cells are not transferred from adherent culture on a 2D substrate to culture on a 3D substrate before they exhibit expression of markers characteristic of endocrine precursor cells.

[0369] 39. After steps a+1) to c+1), the following steps a+2) to c+2): a+2) transferring a population of endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1, from culture on a 2D substrate to 3D culture conditions; b+2) culturing the endocrine precursor cell population under conditions that allow differentiation into pancreatic monohormonal beta cells; and c+2) thereby generating a monohormonal β-cell population, such as monohormonal β-cells, characterized by the expression of insulin, e.g., said pancreatic monohormonal β-cell population being part of at least one pancreatic islet-like cell aggregate; 39. The method according to any one of items 1 to 38, comprising:

[0370] 40. The method according to item 39, wherein step b+2) comprises culturing the endocrine precursor cell population for about 2 weeks or more, for example, about 3 weeks or more, for example, about 3 to 5 weeks, for example, about 4 weeks.

[0371] 41. The method according to any one of items 38 to 40, wherein the monohormonal beta cell population generated in step c+2) does not express glucagon or somatostatin.

[0372] 42. The method according to any one of items 1 to 41, wherein in step c+2) more than about 40%, such as about 40-70%, such as about 40-60%, such as about 40-50% of the total cell population are monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin.

[0373] 43. The method according to any one of items 1 to 42, wherein the method results in at least about 30%, such as at least about 35%, such as at least about 40% more monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin, than a corresponding method comprising step b) culturing the cell population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for at least about 96 hours.

[0374] 44. The method of any one of items 1 to 43, wherein the method results in at least two times more monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin, than monohormonal pancreatic alpha cells characterized by the expression of glucagon.

[0375] 45. The method of any one of items 1 to 44, wherein the monohormonal beta cells are functional pancreatic beta cells, such as functional pancreatic beta cells scored by expression of C-peptide upon glucose stimulation.

[0376] 46. The method according to any one of items 1 to 45, wherein the cell population in step a) or a-1) is derived from a culture of pluripotent stem cells, such as a culture of induced pluripotent stem cells or a culture of embryonic stem cells, such as a culture of human induced pluripotent stem cells or a culture of human embryonic stem cells.

[0377] 47. The method according to any one of items 1 to 46, wherein the cell population in step a) or a-1) is a mammalian cell population, such as a human cell population.

[0378] 48. The method according to any one of items 46 to 47, wherein the cell population in step a) or a-1) is derived from a human embryonic stem cell population, such as a human embryonic stem cell population selected from the group of embryonic stem cell lines consisting of HS980 cells, H1 cells, and H9 cells, for example the group of embryonic stem cell lines consisting of HS980 cells and H1 cells, or the group of embryonic stem cell lines consisting of H1 cells and H9 cells, or the group of embryonic stem cell lines consisting of HS980 cells and H9 cells.

[0379] 49. The method according to any one of items 46 to 47, wherein the cell population in step a) or a-1) is derived from a human induced pluripotent stem cell population.

[0380] 50. The method of any one of items 1 to 25 and 46 to 49, wherein the cells of the pancreatic β cell lineage are pancreatic progenitor cells.

[0381] 51. The method of any one of items 1 to 38 and 46 to 49, wherein the cells of the pancreatic beta cell lineage are endocrine precursor cells.

[0382] 52. The method of any one of items 1 to 49, wherein the cells of the pancreatic β cell lineage are pancreatic β cells.

[0383] 53. The method according to any one of items 1 to 49 and 51 to 52, further comprising cryopreservation of endocrine precursor cells.

[0384] 54. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage, obtained by the method according to any one of items 1 to 53.

[0385] 55. The isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to item 54, wherein the cell population has not been subjected to enrichment for a desired phenotype, e.g., sorting based on expression of a desired marker, or sorting for a desired phenotype based on FACS.

[0386] 56. The isolated population of pancreatic beta cells according to any one of items 54 to 55, wherein more than about 40%, such as about 40-70%, such as about 40-60%, such as about 40-50% of the total cell population are monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin.

[0387] 57. The isolated population of pancreatic beta cells according to any one of items 54 to 56, wherein the population comprises at least two times more monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin, than monohormonal alpha cells characterized by the expression of glucagon.

[0388] 58. The isolated cell population of pancreatic beta cell lineage according to item 54 or 55, wherein the population comprises pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of PDX1 and NKX6.1.

[0389] 59. The isolated cell population of pancreatic beta cell lineage according to item 54 or 55, wherein the population is a population comprising endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1.

[0390] 60. The isolated cell population of pancreatic beta cell lineage according to item 59, wherein at least 30%, such as at least 40%, such as at least 50%, such as at least 60% of the total cells are endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1.

[0391] 61. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage according to any one of items 54 to 60 for use in therapy.

[0392] 62. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage according to any one of items 54 to 61 for use in the treatment, prevention and / or amelioration of diabetes, such as type 1 or type 2 diabetes.

[0393] 63. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage for use in therapeutic treatment, wherein the cell population has been produced by a method according to any one of items 1 to 53.

[0394] 64. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage, produced by the method of any one of items 1 to 53, for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes.

[0395] 65. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage for use in treatment in therapy, said use comprising: generating cells of pancreatic β cells or cells of a pancreatic β cell lineage according to the method of any one of items 1 to 53; and 2. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage, comprising administering to a patient a therapeutically effective amount of said cells.

[0396] 66. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes, said use comprising: generating cells of pancreatic β cells or cells of a pancreatic β cell lineage according to the method of any one of items 1 to 53; and 2. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage, comprising administering to a patient a therapeutically effective amount of said cells.

[0397] 67. An isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage for use according to any one of items 61 to 66, wherein said use comprises transplanting said population into a patient in need thereof.

[0398] 68. The isolated population of pancreatic beta cells for use according to item 67, wherein the cells of the pancreatic beta cell lineage are monohormonal pancreatic beta cells, and the use comprises transplanting the monohormonal pancreatic beta cells into a patient in need thereof.

[0399] 69. The isolated cell population of pancreatic beta cell lineage for use according to item 67, wherein the cells of the pancreatic beta cell lineage are endocrine precursor cells, and the use comprises transplantation of endocrine precursor cells into a patient in need thereof.

[0400] 70. A pharmaceutical composition comprising an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of items 54 to 69 and at least one pharmaceutically acceptable excipient or carrier.

[0401] 71. A kit of parts comprising an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of items 54 to 60, an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage for use according to any one of items 61 to 69, or a pharmaceutical composition according to item 70, and a suitable carrier substrate.

[0402] 72. A kit of parts according to item 71, wherein the suitable carrier substrate is a 2D substrate according to any one of items 12 to 15 and the cells of the pancreatic beta cell lineage are endocrine precursor cells.

[0403] 73. The kit of parts according to item 71, wherein the suitable carrier substrate is a 3D substrate and the cells are monohormonal beta cells.

[0404] 74. Use of an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of items 53 to 59 in drug screening, such as in vitro drug screening.

[0405] 75. An in vitro drug screening method comprising: generating cells of the pancreatic endocrine lineage, such as the pancreatic β cell lineage, according to the method defined in any one of items 1 to 53; and The method comprising exposing said cells to at least one candidate drug compound.

[0406] 76. A method of treating a patient in need thereof, comprising administering to said patient a therapeutically effective amount of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of items 54 to 60.

[0407] 77. A method of treating a patient in need thereof, comprising: generating cells of the pancreatic endocrine lineage, such as the pancreatic β cell lineage, according to the method defined in any one of items 1 to 53; and administering to said patient a therapeutically effective amount of said pancreatic beta cells or cells of a pancreatic beta cell lineage.

[0408] 78. A method for treating diabetes in a patient in need thereof, the method comprising administering to said patient a therapeutically effective amount of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of items 54 to 60.

[0409] 79. A method of treating a patient in need thereof, comprising generating cells of the pancreatic endocrine lineage, such as the pancreatic beta cell lineage, according to a method defined in any one of items 1 to 53; and administering to said patient a therapeutically effective amount of said pancreatic beta cells or cells of a pancreatic beta cell lineage.

[0410] 80. A method for treating diabetes in a patient in need thereof according to item 78 or 79, wherein said patient suffers from type 1 or type 2 diabetes.

[0411] 81. A method for treating diabetes in a patient in need thereof according to any one of items 76 to 80, wherein said administering comprises transplanting said cells into said patient.

[0412] 82. Use of an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of items 54 to 62 in the manufacture of a medicament for the treatment of diabetes in a patient in need thereof.

[0413] 83. Use of an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to item 82, wherein said manufacturing of said medicament comprises producing pancreatic beta cells or cells of a pancreatic beta cell lineage by a method defined in any one of items 1 to 53.

[0414] 84. A method for generating pancreatic progenitor cells, comprising the steps of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for no more than about 78 hours; and c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1.

[0415] 85. A method for generating endocrine precursor cells, comprising the steps of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for no more than about 78 hours; c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1; a+1) providing a cell population of pancreatic progenitor cells; b+1) culturing the population of pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells; and c+1) thereby generating a population of endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1.

[0416] 86. A method for generating monohormonal beta cells, comprising the steps of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for no more than about 78 hours; c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1; a+1) providing a cell population of pancreatic progenitor cells; b+1) culturing the population of pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells; c+1) thereby generating a population of endocrine precursor cells, such as endocrine precursor cells, characterized by expression of NKX6.1 and NEUROD1; a+2) transferring the endocrine precursor cell population from culture on a 2D substrate to 3D culture conditions; b+2) culturing the endocrine precursor cell population under conditions that allow differentiation into pancreatic monohormonal beta cells; and c+2) thereby generating a population of monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin.

[0417] 87. The method for generating pancreatic progenitor cells according to Item 84, the method for generating endocrine progenitor cells according to Item 85, or the method for generating monohormonal beta cells according to Item 86, further comprising the following steps a-1) to c-1) before steps a) to c): a-1) providing a cell population of primitive intestinal cells, such as primitive intestinal cells, characterized by the expression of HNF1β and HNF4α; b-1) culturing the population of primitive intestinal cells for about 54 hours or less under conditions that allow differentiation into posterior foregut cells; and c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1.

[0418] 88. A method for generating pancreatic progenitor cells according to item 84 or 87, which is a method as defined in any one of items 1 to 25.

[0419] 89. A method for generating endocrine precursor cells according to item 85 or 87, which is a method as defined in any one of items 1 to 36.

[0420] 90. A method for generating monohormonal beta cells according to item 86 or 87, which is a method as defined in any one of items 1 to 53.

Claims

1. 1. A method for producing cells of the pancreatic endocrine lineage, such as the pancreatic β cell lineage, comprising the steps of: a) generating a cell of the pancreatic endocrine lineage, such as a pancreatic β cell lineage; a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for about 78 hours or less, e.g., about 72 hours or less; and c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.

1.

2. Before the steps a) to c), the following steps a-1) to c-1): a-1) providing a cell population of primitive intestinal cells, such as primitive intestinal cells, characterized by the expression of HNF1β and / or HNF4α; b-1) culturing the population of primitive intestinal cells for about 36 hours or less under conditions that allow differentiation into posterior foregut cells; and c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; further comprising 2. The method of claim 1, wherein the cells in steps b and b-1) are cultured as adherent cells on a 2D substrate, the 2D substrate comprising one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and Matrigel™.

3. 3. The method of claim 1 or 2, wherein the cells are cultured adherently on a 2D substrate selected from the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof.

4. 4. The method according to any one of claims 1 to 3, wherein in step b) the cell population is cultured for a period of about 42 to 78 hours, such as about 44 to 76 hours, such as about 46 to 74 hours, such as about 48 to 72 hours.

5. 5. The method according to any one of claims 1 to 4, wherein in step b) the cell population is cultured for a period of about 66 to 78 hours, such as about 68 to 76 hours, such as about 70 to 74 hours, such as about 72 hours.

6. 6. The method of claim 1, wherein in step b) the cell population is cultured for a period of not more than about 72 hours.

7. 7. The method according to any one of claims 1 to 4 and 6, wherein in step b) the cell population is cultured for a period of about 42 to 54 hours, such as about 44 to 52 hours, such as about 46 to 50 hours, such as about 48 hours.

8. 8. The method of claim 1, wherein in step c), the cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1, is further characterized by expression of at least one marker selected from the group consisting of PTF1A, SOX9, HNF6, and CPA, for example, a marker selected from the group consisting of PTF1A and SOX9.

9. 9. The method of claim 1, wherein step b) comprises culturing the cell population in a culture medium in the presence of an effective amount of epidermal growth factor (EGF), such as human EGF, or a derivative or agonist thereof, and an effective amount of nicotinamide (NIC), or a derivative or agonist thereof.

10. 10. The method of claim 1, wherein step b) comprises culturing the cell population in a culture medium in the presence of an effective amount of EGF, such as human EGF, and an effective amount of NIC.

11. 11. The method of any one of claims 9 to 10, wherein the effective amount of EGF or a derivative or agonist thereof is about 50 to 200 ng / mL, such as about 50 to 150 ng / mL, for example about 75 to 125 ng / mL, for example about 100 ng / mL.

12. 12. The method of any one of claims 9 to 11, wherein the effective amount of NIC or a derivative or agonist thereof is about 5 to 20 mM, such as about 5 to 15 mM, for example about 8 to 12 mM, such as about 10 mM.

13. 13. The method of any one of claims 1 to 12, wherein step b) comprises culturing the cell population in a culture medium further comprising KGF, activin A, retinoic acid, SANT-1, PDBu, and LDN.

14. 14. The method of any one of claims 2 and 4 to 13, wherein the 2D substrate comprises one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and Matrigel™.

15. The laminin (LN) and fragments thereof are the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof; For example, the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, and LN-332 and fragments thereof; The method according to any one of claims 2 to 14, wherein the antibody is selected from the group consisting of LN-521 and fragments thereof, or LN-511 and fragments thereof.

16. 16. The method of any one of claims 2 to 15, wherein the laminin (LN) and fragments thereof are selected from the group consisting of LN-521, LN-511, LN-332, LN-421, and LN-121, such as the group consisting of LN-521, LN-511, and LN-332, such as the group consisting of LN-521 and LN-511, for example, the laminin and fragments thereof is LN-521.

17. 17. The method of any one of claims 2 to 16, wherein the laminin and fragments comprise an E8 fragment of laminin, such as the group consisting of the E8 fragment of LN-511, the E8 fragment of LN-521, the E8 fragment of LN-332, the E8 fragment of LN-421, and the E8 fragment of LN-121, such as the group consisting of the E8 fragment of LN-511, the E8 fragment of LN-521, and the E8 fragment of LN-332, such as the group consisting of the E8 fragment of LN-511 and the E8 fragment of LN-521, such as the E8 fragment of LN-511 or the E8 fragment of LN-521.

18. 18. The method of any one of claims 1 to 17, wherein at least about 60%, such as at least about 55%, such as at least about 70%, such as at least about 75%, such as at least about 80% of the posterior foregut cells in a), e.g., posterior foregut cells characterized by expression of PDX1, are differentiated into pancreatic progenitor cells in c), e.g., pancreatic progenitor cells characterized by expression of PDX1 and NKX6.

1.

19. 19. The method of any one of claims 1 to 18, wherein in step c) at least about 75%, such as at least about 80%, such as about 80-85%, such as about 80-90% of the total cell population expresses PDX1.

20. 20. The method of claim 1, wherein in step c) at most about 10% of the total cell population expresses NEUROD1.

21. 21. The method according to any one of claims 1 to 20, wherein in step c) approximately 40-70% of the total cell population expresses NKX6.

1.

22. 22. The method according to any one of claims 2 to 21, wherein in step b-1) the cell population is cultured for about 18 to 30 hours, such as about 20 to 28 hours, such as about 22 to 26 hours, such as about 24 hours.

23. 23. The method of any one of claims 2 to 22, wherein step b-1) comprises culturing the cell population in a culture medium containing KGF, retinoic acid, SANT-1, PDBu, and LDN.

24. After steps a) to c), the following steps a+1) to c+1): a+1) providing a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells, characterized by expression of PDX1 and NKX6.1; b+1) culturing the population of pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells; and c+1) thereby generating a population of endocrine precursor cells, such as endocrine precursor cells, characterized by expression of NKX6.1 and NEUROD1; 24. The method of any one of claims 1 to 23, comprising:

25. 25. The method of claim 24, wherein the endocrine precursor cell population, e.g., endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1, is further characterized by expression of at least one of PDX1 and NGN3.

26. 26. The method of claim 24 or 25, wherein in step b+1) the cell population of pancreatic progenitor cells is cultured for about 3 to 5 days, such as for about 3 to 4 days, or for about 4 to 5 days, such as for about 4 days, or for example for about 5 days.

27. 27. The method of any one of claims 2 to 26, wherein the cells in steps b-1), b), and b+1) are cultured as adherent cells on a 2D substrate, the 2D substrate comprising one or more components selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and Matrigel™.

28. 28. The method of any one of claims 24 to 27, wherein the cells are cultured adherently on a 2D substrate at least until the generation of endocrine precursor cells in step c+1).

29. 29. The method of any one of claims 27 to 28, wherein the 2D substrate is as defined in any one of claims 3, 14 and 17.

30. 30. The method of any one of claims 24 to 29, wherein in step c+1) more than about 30%, such as more than about 40%, such as more than about 45%, such as more than about 50% of the total cell population are endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1.

31. 31. The method of any one of claims 24 to 30, wherein the number of endocrine precursor cells in step c+1) is higher compared to the number of endocrine cells obtained using a corresponding method, in which step b) of culturing the cell population of posterior foregut cells under conditions permissive for differentiation into pancreatic precursor cells is for about 24 hours or less and / or about 96 hours or more.

32. 32. The method of any one of claims 24 to 31, wherein the method results in at least about 10%, such as at least about 15%, for example at least about 20%, such as at least about 30%, for example at least about 40%, such as at least 50%, for example at least 60% more endocrine precursor cells than a corresponding method in which step b) of culturing the cell population of posterior foregut cells under conditions permissive for differentiation into pancreatic precursor cells is for about 24 hours or less and / or for about 96 hours or more.

33. 33. The method of any one of claims 24 to 32, wherein step b+1) comprises culturing the cell population in a culture medium comprising BTC, Alk5iII, GSI-XX, GC-1, LDN, retinoic acid, and SANT-1.

34. 34. The method of any one of claims 24 to 33, further comprising culturing the endocrine precursor cells under conditions that allow differentiation into monohormonal pancreatic beta cells.

35. 35. The method of any one of claims 2 to 34, wherein the cells are not transferred from adherent culture on a 2D substrate to culture on a 3D substrate before they exhibit expression of markers characteristic of endocrine precursor cells.

36. After steps a+1) to c+1), the following steps a+2) to c+2): a+2) transferring said population of endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1, from culture on a 2D substrate to 3D culture conditions; b+2) culturing the endocrine precursor cell population under conditions that allow differentiation into pancreatic monohormonal β cells; and c+2) thereby generating a monohormonal β-cell population, such as monohormonal β-cells, characterized by the expression of insulin, e.g., said pancreatic monohormonal β-cell population being part of at least one pancreatic islet-like cell aggregate; 36. The method of any one of claims 1 to 35, comprising:

37. 37. The method of claim 36, wherein step b+2) comprises culturing the population of endocrine precursor cells for about 2 weeks or more, such as about 3 weeks or more, such as about 3-5 weeks, such as about 4 weeks.

38. 38. The method of any one of claims 36 to 37, wherein the population of monohormonal beta cells generated in step c+2) does not express glucagon or somatostatin.

39. 39. The method according to any one of claims 36 to 38, wherein in step c+2) more than about 40%, such as about 40-70%, such as about 40-60%, such as about 40-50% of the total cell population are monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin.

40. 40. The method of any one of claims 1 to 39, wherein the method results in at least about 30%, such as at least about 35%, such as at least about 40% more monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin, than a corresponding method comprising step b) culturing the cell population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for about 96 hours or more.

41. 41. The method of any one of claims 1 to 40, wherein the method results in at least two times more monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin, than monohormonal pancreatic alpha cells characterized by the expression of glucagon.

42. 44. The method of any one of claims 36 to 43, wherein the monohormonal beta cells are functional pancreatic beta cells, such as functional pancreatic beta cells scored by expression of C-peptide upon glucose stimulation.

43. 43. The method of any one of claims 1 to 42, wherein the cell population in step a) or a-1) is derived from a culture of pluripotent stem cells, such as a culture of induced pluripotent stem cells or a culture of embryonic stem cells, such as a culture of human induced pluripotent stem cells or a culture of human embryonic stem cells.

44. 44. The method of any one of claims 1 to 43, wherein the cell population in step a) or a-1) is a mammalian cell population, such as a human cell population.

45. 45. The method of any one of claims 43 to 44, wherein the cell population in step a) or a-1) is derived from a human embryonic stem cell population, such as a human embryonic stem cell population selected from the group of embryonic stem cell lines consisting of HS980 cells, H1 cells, and H9 cells, for example the group of embryonic stem cell lines consisting of HS980 cells and H1 cells, or the group of embryonic stem cell lines consisting of H1 cells and H9 cells, or the group of embryonic stem cell lines consisting of HS980 cells and H9 cells.

46. 45. The method of any one of claims 43 to 44, wherein the cell population in step a) or a-1) is derived from a human induced pluripotent stem cell population.

47. 47. The method of any one of claims 1 to 23 and 43 to 46, wherein the cells of the pancreatic beta cell lineage are pancreatic progenitor cells.

48. 47. The method of any one of claims 1 to 35 and 43 to 46, wherein the cells of the pancreatic beta cell lineage are endocrine precursor cells.

49. 47. The method of any one of claims 1 to 46, wherein said cells of a pancreatic beta cell lineage are pancreatic beta cells.

50. 50. The method of any one of claims 1 to 46 and 48 to 49, further comprising cryopreservation of the endocrine precursor cells.

51. 51. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage, obtainable by the method of any one of claims 1 to 50.

52. 52. The isolated population of pancreatic beta cells of claim 51, wherein the isolated population of pancreatic beta cells is part of at least one pancreatic islet-like cell aggregate, e.g., the isolated population of pancreatic beta cells is in the form of a pancreatic islet-like aggregate.

53. 53. The isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage described in claim 51 or 52, wherein the cell population has not been enriched for a desired phenotype, e.g., has not been subjected to selection for a desired phenotype, such as selection based on expression of a desired marker or selection for a desired phenotype based on FACS.

54. 54. The isolated population of pancreatic beta cells of any one of claims 51 to 53, wherein more than about 40%, such as about 40-70%, such as about 40-60%, such as about 40-50% of the total cell population are monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin.

55. 55. The isolated population of pancreatic beta cells of any one of claims 51 to 54, wherein the population comprises at least two times more monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin, than monohormonal alpha cells characterized by the expression of glucagon.

56. 54. The isolated cell population of pancreatic beta cell lineage of claim 51 or 53, wherein the population is a population comprising pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of PDX1 and NKX6.

1.

57. 54. An isolated cell population of pancreatic beta cell lineage according to claim 51 or 53, wherein the population is a population comprising endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1.

58. 58. The isolated cell population of pancreatic beta cell lineage of claim 57, wherein at least 30%, such as at least 40%, such as at least 50%, such as at least 60% of the total cells are endocrine precursor cells, such as endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1.

59. 60. An isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of claims 51 to 58 for use in therapy.

60. 60. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage according to any one of claims 51 to 59 for use in the treatment, prevention and / or amelioration of diabetes, such as type 1 or type 2 diabetes.

61. 60. The isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage of any one of claims 51 to 59, wherein said population of cells is produced by a method of any one of claims 1 to 50.

62. 51. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes, wherein said population of cells has been produced by the method of any one of claims 1 to 50.

63. 1. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage for use in therapeutic treatment, said use comprising: generating cells of pancreatic β cells or cells of a pancreatic β cell lineage according to the method of any one of claims 1 to 50; and 2. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage, comprising administering to a patient a therapeutically effective amount of said cells.

64. 1. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes, said use comprising: generating cells of pancreatic β cells or cells of a pancreatic β cell lineage according to the method of any one of claims 1 to 50; and 2. An isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage, comprising administering to a patient a therapeutically effective amount of said cells.

65. 65. An isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage for use according to any one of claims 59 to 64, wherein said use comprises transplanting said population into a patient in need thereof.

66. 66. An isolated population of pancreatic beta cells for use according to claim 65, wherein the cells of the pancreatic beta cell lineage are monohormonal pancreatic beta cells, and the use comprises transplanting the monohormonal pancreatic beta cells into a patient in need thereof, for example, transplanting the monohormonal pancreatic beta cells in the form of islet-like aggregates.

67. 66. The isolated cell population of pancreatic beta cell lineage for use according to claim 65, wherein the cells of the pancreatic beta cell lineage are endocrine precursor cells, and the use comprises transplantation of endocrine precursor cells into a patient in need thereof.

68. 68. A pharmaceutical composition comprising an isolated population of pancreatic beta cells or cells of the pancreatic beta cell lineage described in any one of claims 51 to 67 and at least one pharmaceutically acceptable excipient or carrier.

69. 68. A kit of parts comprising an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of claims 51 to 58, an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage for use according to any one of claims 59 to 67, or a pharmaceutical composition according to claim 68, and a suitable carrier substrate.

70. 70. The kit of parts of claim 69, wherein said suitable carrier substrate is a 2D substrate according to any one of claims 2, 3 and 14 to 17, and said cells of a pancreatic beta cell lineage are endocrine precursor cells.

71. 70. The kit of parts according to claim 69, wherein said suitable carrier substrate is a 3D substrate and said cells are monohormonal beta cells.

72. 60. Use of an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of claims 51 to 58 in drug screening, such as in vitro drug screening.

73. 1. An in vitro drug screening method comprising: generating cells of the pancreatic endocrine lineage, such as the pancreatic β cell lineage, according to a method as defined in any one of claims 1 to 50; and The method comprising exposing said cells to at least one candidate drug compound.

74. 60. A method of treating a patient in need thereof, comprising administering to said patient a therapeutically effective amount of pancreatic beta cells or cells of a pancreatic beta cell lineage described in any one of claims 51 to 58.

75. 1. A method of treating a patient in need thereof, comprising: generating cells of the pancreatic endocrine lineage, such as the pancreatic β cell lineage, according to a method as defined in any one of claims 1 to 50; and administering to said patient a therapeutically effective amount of said pancreatic beta cells or cells of a pancreatic beta cell lineage.

76. 60. A method for treating diabetes in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of claims 51 to 58.

77. A method of treating a patient in need thereof, comprising generating cells of the pancreatic endocrine lineage, such as the pancreatic β-cell lineage, according to a method of any one of claims 1 to 50; administering to said patient a therapeutically effective amount of said pancreatic beta cells or cells of a pancreatic beta cell lineage.

78. 78. A method of treating diabetes in a patient in need thereof according to claim 76 or 77, wherein said patient has type 1 or type 2 diabetes.

79. 79. A method of treating diabetes in a patient in need thereof according to any one of claims 74 to 78, wherein the administration comprises transplanting the cells into the patient, for example comprising transplantation of monohormonal pancreatic beta cells in the form of islet-like aggregates.

80. 60. Use of an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage according to any one of claims 51 to 58 in the manufacture of a medicament for the treatment of diabetes in a patient in need thereof.

81. 83. The use of an isolated population of pancreatic beta cells or cells of a pancreatic beta cell lineage described in claim 82, wherein the manufacture of the pharmaceutical product comprises producing pancreatic beta cells or cells of a pancreatic beta cell lineage by a method defined in any one of claims 1 to 53.

82. 1. A method for generating pancreatic progenitor cells, comprising the steps of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for no more than about 78 hours; and c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.

1.

83. A method for generating endocrine precursor cells, comprising the steps of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for up to about 78 hours; c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells, characterized by expression of both PDX1 and NKX6.1; a+1) providing a cell population of pancreatic progenitor cells; b+1) culturing the population of pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells; and c+1) thereby generating a population of endocrine precursor cells, such as endocrine precursor cells, characterized by expression of NKX6.1 and NEUROD1.

84. A method for generating monohormonal beta cells, comprising the steps of: a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for up to about 78 hours; c) thereby generating a cell population of pancreatic progenitor cells, such as pancreatic progenitor cells, characterized by expression of both PDX1 and NKX6.1; a+1) providing a cell population of pancreatic progenitor cells; b+1) culturing the population of pancreatic progenitor cells under conditions that allow differentiation into endocrine precursor cells; c+1) thereby generating a population of endocrine precursor cells, such as endocrine precursor cells, characterized by expression of NKX6.1 and NEUROD1; a+2) transferring the endocrine precursor cell population from culture on a 2D substrate to 3D culture conditions; b+2) culturing the endocrine precursor cell population under conditions that allow differentiation into pancreatic monohormonal β cells; and c+2) thereby generating a population of monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin.

85. The method for generating pancreatic progenitor cells of claim 82, the method for generating endocrine precursor cells of claim 83, or the method for generating monohormonal beta cells of claim 84, further comprising the following steps a-1) to c-1) prior to steps a) to c): a-1) providing a cell population of primitive intestinal cells, such as primitive intestinal cells, characterized by the expression of HNF1β and HNF4α; b-1) culturing the population of primitive intestinal cells for about 36 hours or less under conditions that allow differentiation into posterior foregut cells; and c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1.

86. 86. A method of generating pancreatic progenitor cells as described in claim 82 or 85, which is a method as defined in any one of claims 1 to 55.

87. 86. A method for generating endocrine precursor cells according to claim 83 or 85, which is a method as defined in any one of claims 1 to 36.

88. 86. A method for generating monohormonal beta cells according to claim 84 or 85, which is a method as defined in any one of claims 1 to 53.

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