3D pancreatic islet formation from endocrine precursor cells

Abstract

The present invention relates to a method for generating cells of the pancreatic lineage, such as pancreatic islet-like cell aggregates comprising pancreatic β cells, comprising providing a single cell suspension of a population of endocrine precursor cells (EP), allowing the EP cells in the single cell suspension to form 3D structures, and culturing the cells under conditions permissive for differentiation into pancreatic monohormonal β cells. The present invention also relates to the pancreatic islet-like cell aggregates obtained by the method and medical uses thereof.

Description

[Technical Field]

[0001] The present disclosure relates to a method for generating cells of the pancreatic lineage, such as pancreatic islet-like cell aggregates comprising pancreatic β cells, comprising providing a single-cell suspension of a population of endocrine precursor cells (EP), allowing the EP cells in the single-cell suspension to form 3D structures, and culturing the cells under conditions permissive for differentiation into pancreatic monohormonal β cells. The disclosure also relates to the pancreatic islet-like cell aggregates obtained by the method and medical uses thereof. [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 β cells. These β cells may be transplanted as pancreatic islets or islet-like structures. 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 the β-cell toxin streptozotocin were cured by infusion of allogeneic pancreatic islets (reviewed in Murtaugh 2007). Transplantation of pancreatic progenitor cells derived from human pluripotent stem cells 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 iPCs), 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 types. 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] Therefore, as is evident from various parts of this Background Description, it would be desirable to provide a differentiation strategy that overcomes the above-mentioned drawbacks. Therefore, it would be desirable to provide a differentiation strategy for efficiently obtaining high-quality islet-like cell aggregates (or islet-like structures) in vitro in a reproducible manner. Summary of the Invention

[0009] It is an object of the present disclosure to provide a differentiation strategy for generating pancreatic islet-like cell aggregates in vitro, which overcomes and / or mitigates the above 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 generating pancreatic islet-like cell aggregates that exhibit the desired ability to produce insulin in response to glucose stimulation.

[0011] An object of the present disclosure is to provide an efficient and reproducible in vitro differentiation protocol that allows for the generation of large quantities of pancreatic islet-like cell aggregates.

[0012] A further object of the present disclosure is to provide an in vitro differentiation protocol that allows for obtaining cultures of pancreatic islet-like cell aggregates with a high percentage of pancreatic monohormonal β cells and a low percentage of contaminating cell types, in which monohormonal α cells are also present.

[0013] It is also an object of the present disclosure to provide pancreatic islet-like cell aggregates having a high percentage of cells that exhibit functional characteristics of cells of the pancreatic beta cell lineage.

[0014] Another object of the present disclosure is to provide pancreatic islet-like cell aggregates that have a high percentage of pancreatic monohormonal beta cells and a low percentage of contaminating cell types. Monohormonal alpha cells are also present in the pancreatic islet-like cell aggregates. It is particularly important that the cells of interest are viable and healthy. Such islet-like cell aggregates, or cells obtained therefrom, may be useful for many applications, including therapeutic and scientific / biotechnological applications, such as in vitro drug development and screening. For example, such islet-like cell aggregates or cells may be used for cell transplantation (i.e., cell replacement therapy) into patients in need thereof.

[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 generating islet-like cell aggregates from pluripotent cells and / or cells derived from said aggregates 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.

[0017] The present disclosure is based on the surprising realization that by providing stage 5 endocrine precursor cells (EP) (also referred to herein as endocrine precursor cells) in a single-cell suspension, allowing the population of EP cells to form 3D structures, and continuing to culture the population of EP cells in the form of 3D structures in 3D culture conditions that allow differentiation into pancreatic monohormonal β cells, to provide islet-like cell aggregates, unexpectedly high-quality islet-like cell aggregates can be obtained. The islet-like cell aggregates contain an unexpectedly high proportion of monohormonal β cells, a low proportion of polyhormonal cells, a low proportion of monohormonal α cells (α cells) and / or δ cells, and a low proportion of proliferating cells. The inventors have found that culturing cells of the pancreatic lineage on a 2D substrate, such as by adherent culture on a 2D substrate, until they reach the endocrine precursor cell (EP) developmental stage, dissociating the cells, e.g., into a single-cell suspension, and then allowing the cells to form 3D structures, produces high-quality islet-like cell aggregates. As shown in the accompanying examples, the timing of the transition from 2D to 3D culture conditions is crucial. Without being bound by theory, the timing of cell transplantation appears to influence the development / differentiation of EP cells into high-quality pancreatic islet cells. Importantly, EP cells in single-cell suspension that are allowed to form 3D structures according to the methods disclosed herein have the potential to develop into mature, functional pancreatic beta cells.

[0018] Accordingly, in a first aspect of the present invention there is provided a method for producing pancreatic islet-like cell aggregates in vitro, comprising the steps of: i) providing a population of endocrine precursor cells (EP), e.g., EP cells characterized by expression of NEUROD1, e.g., EP cells characterized by expression of NKX6.1 and NEUROD1; ii) providing a single cell suspension of said population of EP cells; iii) allowing the population of EP cells in single cell suspension to form 3D structures; iv) culturing the population of EP cells in the form of a 3D structure under 3D culture conditions permissive for the differentiation of pancreatic monohormonal β cells to provide pancreatic islet-like cell aggregates; and v) thereby producing islet-like cell aggregates comprising a single hormonal beta cell; Methods are provided wherein the islet-like cell aggregates comprise at least about 25% monohormonal beta cells.

[0019] As used herein, the term "islet-like cell aggregate" or "islet-like aggregate" refers to a cellular aggregate of pancreatic cells that exhibits 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 that the islet-like cell aggregates obtained in vitro mimic the characteristics of a pancreatic islet in vivo, both in terms of the distribution of cell types present and their functional properties.

[0020] Those skilled in the art will understand that the percentages described herein relate to the average percentages exhibited by islet-like cell aggregates. Thus, for example, 100 aggregates are analyzed, and the average number of the cell types is as described herein. Therefore, step v) can be rephrased as "thereby generating a population of islet-like cell aggregates containing monohormonal β cells, wherein the population comprises islet-like cell aggregates containing at least 25% monohormonal β cells." In this context, the following listed characteristics refer to the average percentages in a population of islet-like cell aggregates according to the present disclosure.

[0021] Thus, in one embodiment, said islet-like cell aggregates comprise at least about 25%, such as at least about 30%, for example at least about 35%, such as at least about 40%, for example at least about 45%, such as at least about 50%, for example at least about 55%, such as at least about 60%, for example at least about 65%, such as at least about 70% monohormonal beta cells, or said population comprises islet-like cell aggregates comprising said monohormonal beta cells. In one embodiment, said islet-like cell aggregates comprise about 25-70%, such as 30-70%, for example 30-70% monohormonal beta cells, such as 35-70%, 35-70%, for example 40-70%, such as 45-70%, for example 45-65%, such as 45-60%, for example 45-55%, such as about 50% monohormonal beta cells, or said population comprises islet-like cell aggregates comprising said monohormonal beta cells. In one embodiment, the islet-like cell aggregates comprise about 35-65%, such as 40-65%, for example 40-60%, monohormonal beta cells, or the population comprises islet-like cell aggregates comprising the monohormonal beta cells.

[0022] In one embodiment, the islet-like cell aggregates comprise about 7-25%, such as 7-20%, 10-20%, such as 15-20%, such as about 20% monohormonal alpha cells, or the population comprises islet-like cell aggregates comprising the monohormonal alpha cells.

[0023] In one embodiment, said 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%, such as up to about 0.5%, for example up to about 0.3%, for example up to about 0.1% multihormonal alpha cells, or said population comprises islet-like cell aggregates comprising said multihormonal alpha cells.

[0024] 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%, such as up to about 0.5%, for example up to about 0.3%, for example up to about 0.1% multihormonal beta cells, or the population comprises islet-like cell aggregates comprising the multihormonal beta cells.

[0025] In one embodiment, the islet-like cell aggregates comprise less than 5%, such as less than 4%, such as less than 3%, such as less than 1%, such as less than 1% δ cells, or the population comprises islet-like cell aggregates comprising the δ cells.

[0026] 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%, for example 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, such as proliferating cells expressing Ki-67, or the population comprises islet-like cell aggregates comprising said proliferating cells.

[0027] 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, or the population comprises islet-like cell aggregates comprising the proliferating cells.

[0028] In one embodiment, the composition of the islet-like cell aggregates is scored, or in other words, examined, after 38 to 42 days of culture, e.g., on day 38, 39, 40, 41, or 42. That is, the composition of the islet-like cell aggregates is scored, or in other words, examined, at the end of stage 6.

[0029] Pancreatic islets, also known as the islets of Langerhans, are regions of the pancreas that contain endocrine (hormone-producing) cells. Approximately one million islets are distributed throughout the pancreas of a healthy adult, each measuring approximately 0.2 mm in diameter. Each islet is separated from the surrounding pancreatic tissue by a thin fibrous connective tissue capsule that is continuous with the fibrous connective tissue woven throughout the rest of the pancreas. Hormones produced in the islets are secreted directly into the bloodstream by at least five types of cells. The endocrine cell types in pancreatic islets include α cells, which produce glucagon; β cells, which produce insulin and amylin; PP cells (γ cells or F cells), which produce pancreatic polypeptide; δ cells, which produce somatostatin; and ε cells, which produce ghrelin. In humans, β cells account for approximately 40–50% of the cells. In addition to endocrine cells, there are stromal cells (fibroblasts), vascular cells (endothelial cells, pericytes), immune cells (granulocytes, lymphocytes, macrophages, dendritic cells), and neural cells. Those skilled in the art are familiar with the composition and cellular structure of pancreatic islets.

[0030] Endocrine precursor cells (EP) are assessed by the expression of a specific marker, NEUROD1, which is upregulated as cells transition to the developmental EP cell stage (also referred to as stage 5, see Figure 1A) during development along the pancreatic endocrine lineage. Cells at early developmental stage 4 (pancreatic precursor cells) do not express NEUROD1. EP cells can also be scored as double positive for NKX6.1 and at least one marker not expressed by pancreatic precursor cells (e.g., double positive for NKX6.1 and NEUROD1, or NKX6.1 and NGN3). Other markers and combinations thereof can also be used to identify EP cells, as described below. Pancreatic endocrine precursor cells express at least one, two, three, or all four of the following markers: PDX1, NKX6.1, NGN3, and NEUROD1.

[0031] Thus, in one embodiment of the method disclosed herein, the cell population of EP cells provided in step i) is characterized by expression of NEUROD1 and NKX6.1. In another embodiment, the cell population of EP cells provided in step i) is characterized by expression of NKX6.1 and NGN3, or expression of NEUROD1 and NGN3. Alternatively, the EP cells can be identified by expression of PDX1, NKX6.1, and NGN3, expression of PDX1, NKX6.1, and NEUROD1, or expression of PDX1, NKX6.1, NGN3, and NEUROD1.

[0032] Those skilled in the art will understand that step ii) of providing a single-cell suspension of EP cell populations involves dissociating EP cells from adherent culture on a 2D substrate 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.

[0033] In embodiments, step ii) of providing a single cell suspension of the EP cell population comprises dissociating EP cells from an adherent culture on a 2D substrate into single cells. In one embodiment, step ii) of providing a single cell suspension of the EP cell population involves dissociating the EP cells by enzymatic means, such as using a solution containing an enzyme, e.g., a proteolytic enzyme and / or a collagenolytic enzyme. For example, such a solution includes Accutase®. Those skilled in the art will be aware of suitable methods for dissociating EP cells and providing a single cell suspension of the EP cell population.

[0034] In one embodiment, steps ii) and iii) are 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.

[0035] 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.

[0036] Thus, in one embodiment, steps i), ii), and iii) are performed under conditions that allow differentiation into endocrine precursor cells, and step iv) is performed under conditions that allow differentiation into pancreatic monohormonal beta cells.

[0037] In step iii), 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.

[0038] In one embodiment, step iii) 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 0-10 μM. As shown in this example, without being bound by theory, the inventors believe that H1152 may promote survival of S5 EP cells as single cells in suspension.

[0039] Thus, in one embodiment of the methods disclosed herein, said ROCK inhibitor is present in the culture medium for about 24 hours during step iii).

[0040] Without being bound by theory, it is believed that self-aggregation can selectively enrich endocrine precursor cells. As shown in the accompanying examples, the formation of 3D structures, also referred to as aggregates, leads to selective enrichment, resulting in increased production of pancreatic monohormonal beta cells in the culture. As explained above, the aggregates exhibit desirable properties, including 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), and a low number of proliferating cells. In one embodiment, the pancreatic monohormonal beta cells are generated as part of a cell aggregate. In one embodiment, the aggregates comprise monohormonal beta cells. In one embodiment, the aggregates further comprise pancreatic monohormonal alpha cells. In one embodiment, the aggregates further comprise pancreatic monohormonal delta cells, e.g., less than 3%, e.g., less than 2%, e.g., less than 1% delta cells. Those skilled in the art will understand that the percentages recited herein should be interpreted as relating to the average percentages exhibited by pancreatic islet-like cell aggregates. The percentage can be assessed for the islet-like cell aggregates themselves, or for the population comprising the islet-like cell aggregates, which for clarity are referred to herein as islet-like cell aggregates and are obtained according to the methods defined herein.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Those skilled in the art are familiar with the different developmental stages of the pancreatic endocrine lineage, including the pancreatic β cell lineage.

[0046] 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.

[0047] 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.

[0048] Differentiation of cells along the pancreatic beta cell lineage involves differentiation from low-commitment to high-commitment cell types. Briefly, the development of insulin-producing pancreatic beta cells is the culmination of a complex developmental program, involving in vivo stages in which posterior foregut cells acquire pancreatic identity, the pancreatic primordium expands and commits to an endocrine fate, and a subset of these progenitor cells becomes capable of generating beta cells. Factors, e.g., transcription factors, that have been shown to be important for the development of pancreatic beta cell lineage cells 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.

[0049] 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.

[0050] 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.

[0051] 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).

[0052] 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.

[0053] In this stepwise progression, stage 0 refers to undifferentiated pluripotent cells such as hES cells or iPS 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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. As used herein, the term "pancreatic islet-like cell aggregates" or "pancreatic islet-like aggregates" refers to cellular aggregates of pancreatic cells that exhibit characteristics of pancreatic islets in vivo. In particular, the aggregates exhibit the properties described above.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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).

[0073] 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.

[0074] 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 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.

[0075] In one embodiment of the method disclosed herein, the population of EP cells in step i) is an adherent culture of EP cells on a 2D substrate.

[0076] In one embodiment of the methods disclosed herein, the cells are cultured on a 2D substrate, hi 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.

[0077] 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.

[0078] 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).

[0079] 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.

[0080] 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.

[0081] 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.

[0082] Laminins are macromolecular proteins of the extracellular matrix. They are the main components of basement membranes, the foundation of the 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 to use as cell culture substrates is important for providing cells with an appropriate chemo- and mechanosensitive microenvironment and optimal culture conditions.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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).

[0088] 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.

[0089] It will be appreciated that the EP cells provided in step i) of the methods of the present invention can be generated using a variety of differentiation protocols, such as those described above. It will be appreciated that it may be beneficial to provide a large number of EP cells in step i). Accordingly, it may be beneficial to generate said EP cells using the methods disclosed herein. Clearly, the utility of the methods described in steps i) through v) herein is in no way limited to EP cells generated by the methods disclosed herein.

[0090] In one embodiment disclosed herein, a method is provided in which, in step i), more than about 30%, for example, more than about 40%, 40%, for example, more than about 45%, for example, more than about 50% of the total cell population are EP cells characterized by the expression of NEUROD1. For example, the EP cells provided in step i) may be obtained in step c+1) disclosed herein. Thus, the population of EP cells in step i) may be a portion of the total population of cells, including a fraction of cells that do not express markers characteristic of EP cells. In one embodiment disclosed herein, a method is provided in which a population of EP cells is provided in step i), and more than about 30%, for example, more than about 40%, for example, more than about 45%, for example, more than about 50% of the total cell population are EP cells, for example, EP cells characterized by the expression of NKX6.1 and at least NEUROD1, for example, endocrine precursor cells characterized by the expression of NKX6.1 and at least NEUROD1. In one embodiment disclosed herein, in step i), the population comprising EP cells comprises more than about 30%, such as more than about 40%, such as more than about 45%, for example more than about 50% EP cells, e.g., EP cells characterized by expression of NKX6.1 and at least NEUROD1, e.g., endocrine precursor cells characterized by expression of NKX6.1 and at least NEUROD1. As discussed and exemplified above, other combinations of two or more of the markers PDX1, NKX6.1, NEUROD1, and NGN3 can be used to characterize EP cells.

[0091] In one embodiment of the method disclosed herein, step ii) of providing a single cell suspension of the EP cell population is performed when more than about 15%, such as more than about 20%, such as more than about 25%, such as more than about 30%, such as more than about 35%, such as more than about 40%, such as more than about 45%, such as more than about 50% of the total cell population are EP cells characterized by expression of NEUROD1 or NGN3. In one embodiment, the EP cells are characterized by expression of NEUROD1 and NGN3. In one embodiment, step ii) is performed when more than about 15%, such as more than about 20%, such as more than about 25%, such as more than about 30%, such as more than about 35%, such as more than about 40%, such as more than about 45%, such as more than about 50% of the total cell population are EP cells characterized by expression of NKX6.1 and NEUROD1. In one embodiment, step ii) is performed when the cells do not express hormones, for example, when they do not express hormones. In one embodiment, step ii) is performed when the cells do not exhibit hormone expression, e.g., do not exhibit insulin and / or glucagon expression. In this context, do not exhibit expression is understood to mean do not exhibit any detectable expression, such as detectable by the methods or means disclosed in the Examples section. In one embodiment, step ii) is performed before the cells exhibit hormone expression, e.g., before they exhibit insulin and / or glucagon expression.

[0092] In one embodiment, there is provided a method for producing pancreatic islet-like cell aggregates in vitro as defined herein, wherein step iv) culturing said population of EP cells in the form of a 3D structure under 3D culture conditions permissive for differentiation of pancreatic monohormonal beta cells to provide pancreatic islet-like cell aggregates comprises: The method comprises culturing in a culture medium suitable for culturing endocrine precursor cells under conditions that allow differentiation into monohormonal β cells. Non-limiting examples of the medium 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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. 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.

[0098] 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. 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.

[0099] In one embodiment, the culturing in step iv) is carried out on a shaker, such as an orbital shaker.

[0100] As described in connection with the methods of the present invention, the inventors have found that the timing of transferring EP cells from culture on a 2D substrate to culture on a 3D substrate is important for obtaining enriched islet-like cell aggregates in vitro that are enriched for β cells, have a low proportion of other monohormonal cells, a low proportion of polyhormonal cells, and a low proportion of proliferating cells.

[0101] 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 the expression of markers characteristic of endocrine precursor cells. As described above, endocrine precursor cells are characterized by the expression of PDX1, NKX6.1, and at least one of NEUROD1 and NGN3. As described above, EP cells do not express pancreatic hormones. In one embodiment, step ii) is performed about 24 hours after the EP cells begin to express NEUROD1 and / or NGN3, for example, 24 hours after the EP cells begin to express NEUROD1 and / or NGN3 in more than 15%, for example, more than about 20%, for example, more than about 25%, for example, more than about 30%, for example, more than about 35%, for example, more than about 40%, for example, more than about 45%, for example, more than about 50% of the total cells in the culture. In one embodiment, step ii) is performed within 6 days, for example, within 5 days, for example, within 1 to 4 days, for example, within 1 to 3 days, for example, within 1 to 2 days or 2 to 3 days, after the EP cells begin to express NEUROD1 or NGN3. Step ii) is performed before the expression of insulin and / or glucagon.

[0102] As described above, in one embodiment, the formation of the 3D structure in step iii) is spontaneous formation of the 3D structure. For example, such spontaneous formation of the 3D structure may occur by self-aggregation. Alternatively, the formation of the 3D structure in step iii) is forced (also referred to as assisted) formation of a 3D structure. Non-limiting examples of forced (or assisted) formation of a 3D structure include culture conditions in which cells are forced into close proximity due to the shape of a cell culture vial or flask. In one embodiment, the 3D culture conditions enable self-aggregation of cells. Without being bound by theory, this example demonstrates that self-aggregation can selectively enrich endocrine precursor cells that develop into stage 6 cells. Furthermore, the selective enrichment increases the production of pancreatic monohormonal β cells in the culture. In one embodiment, the pancreatic monohormonal β cells are generated as part of cell aggregates. In one embodiment, the aggregates comprise monohormonal β cells. In one embodiment, the pancreatic monohormonal β cells are generated in vitro as part of pancreatic islet-like cell aggregates. In one embodiment, said in vitro pancreatic islet-like cell aggregates further comprise pancreatic monohormonal cells alpha cells and / or delta cells.

[0103] In one embodiment, the in vitro islet-like cell aggregates are generated from human ES cells and comprise at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, for example at least 65% monohormonal β cells. In one embodiment, the in vitro islet-like cell aggregates are generated from human iPS cells and comprise at least 40% β cells, such as at least 45%, for example at least 50%, for example at least 55% β cells, such as at least 60% β cells, for example at least 65% monohormonal β cells. In a specific embodiment, the iPS cells are C7 cells, and the islet-like cell aggregates comprise at least 60% β cells, such as at least 65% monohormonal β cells. In one embodiment, the monohormonal β cells are characterized by insulin expression.

[0104] Thus, the present inventors have demonstrated that the present invention is equally applicable to the differentiation of ES cells and iPS cells, and that its beneficial effects are not limited to a particular cell line.

[0105] In one embodiment, the in vitro pancreatic 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% of proliferating cells, e.g., proliferating cells expressing Ki-67. In one embodiment, the in vitro pancreatic 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% of proliferating cells, e.g., proliferating cells expressing Ki-67. In one embodiment, the in vitro pancreatic 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% of proliferating cells, e.g., proliferating cells expressing Ki-67. In one embodiment, the Ki-67 is scored on day 1 after aggregate formation. In one embodiment, the Ki-67 is scored on day 15 of culture. Thus, the evaluation can be performed after a duration of about 4 weeks of step iv).

[0106] In one embodiment, the in vitro islet-like cell aggregates comprise at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75% of cells expressing NEUROD1 and NKX6.1. In one embodiment, the in vitro islet-like cell aggregates comprise about 60-90%, such as about 60-80%, such as about 65-80% of cells expressing NEUROD1 and NKX6.1. In one embodiment, the in vitro islet-like cell aggregates comprise at least 50%, such as at least 80%, such as at least 85%, such as at least 87%, such as at least 90% of cells expressing NEUROD1. In a specific embodiment, the islet-like cell aggregates are generated in vitro from human ES cells. In one embodiment, the expression of NEUROD1 and / or NKX6.1 is scored on day 15 of culture. Thus, the assessment may be performed in step iv) after the formation of the aggregates. In one embodiment, step iv) comprises culturing the cell population for about 2 weeks or more, such as about 3 weeks or more, for example, about 3 to 5 weeks, for example, about 4 weeks.

[0107] 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.

[0108] In vivo β cells are single hormonal cells, and it is beneficial that the population obtained by the methods of the present invention exhibits characteristics of natural in vivo β cells, i.e., endogenous in vivo β cells, such as healthy natural in vivo β cells or healthy endogenous in vivo β cells.

[0109] Thus, in one embodiment of the method of the present invention disclosed herein, the islet-like cell aggregates comprising beta cells produced in step v) comprise monohormonal beta cells. In one embodiment of the method of the present invention, the monohormonal beta cells in step v) express insulin. In one embodiment, the monohormonal beta cells in step v) express C-peptide upon glucose stimulation.

[0110] In particular, said monohormonal beta cells in step v) do not express glucagon or somatostatin. In particular, said monohormonal beta cells in step v) do not express glucagon and somatostatin.

[0111] In one embodiment, the monohormonal beta cells in step v) are characterized by the expression of insulin. The monohormonal beta cells may further express at least one of NKX6.1, PDX1, and NEUROD1.

[0112] In one embodiment, the monohormonal beta cells are characterized by expression of insulin and PDX1. In one embodiment, the monohormonal beta cells are characterized by expression of insulin and NKX6.1. In one embodiment, the monohormonal beta cells are characterized by expression of insulin and NEUROD1. In one embodiment, the monohormonal beta cells are 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 cells are characterized by expression of insulin, PDX1, NKX.1, and NEUROD1.

[0113] In one embodiment there is provided a method of producing in vitro pancreatic islet-like cell aggregates as defined herein, wherein said pancreatic islet-like cell aggregates in v) comprise: In one embodiment, the in vitro islet-like cell aggregates in v) comprise about 25-70%, such as about 30-70%, for example about 40-70%, such as about 40-60%, for example about 40-60% monohormonal beta cells.

[0114] In one embodiment, said in vitro pancreatic islet-like cell aggregates in v) 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.

[0115] In one embodiment, said in vitro pancreatic islet-like cell aggregates in v) 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, in one embodiment said monohormonal alpha cells are characterized by glucagon expression.

[0116] In one embodiment, there is provided a method of producing islet-like cell aggregates in vitro as defined herein, wherein the islet-like cell aggregates in v) comprise monohormonal beta cells and alpha cells, and up to 5% of any one or more cells selected from the group consisting of delta cells, acinar cells, ductal cells, and activated stellate cells. In one embodiment of the method, the islet-like cell aggregates in v) comprise up to about 5%, e.g., up to about 4, 3, 2, or 1%, of polyhormonal cells. In one embodiment of the method, the islet-like cell aggregates in v) comprise up to about 5%, e.g., up to about 4, 3, 2, or 1%, of non-endocrine cells.

[0117] In one embodiment, the in vitro islet-like cell aggregates in v) are scored at the end of S6, e.g., between 38 and 42 days of culture, e.g., day 38, 39, 40, 41, 42, or later.

[0118] Furthermore, the present inventors have demonstrated that short-term culture of posterior foregut (PF) cells under conditions permissive for differentiation into pancreatic progenitor (PP) cells results in 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 progenitor (EP) cells" and "endocrine precursor (EP) 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 after a short culture time, 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, such as monohormonal β cells capable of responding to glucose stimulation via the expression of C-peptide. As used herein, the term "corresponding method" refers to a method in which all steps are the same, except for those specifically indicated. Corresponding methods should therefore be construed as being the same method but with different steps indicated, e.g. the steps may relate to the incubation time, e.g. in step b-1), or b), or in both b-1) and b).

[0119] Thus, in one embodiment, there is provided a method for producing pancreatic islet-like cell aggregates in vitro, the method comprising, prior to step i), the following steps a) to c): 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] As mentioned above, the term "conditions permissive for differentiation" refers to conditions that allow cells to develop (i.e., 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. 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, the 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.

[0125] 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).

[0126] 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.

[0127] 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.

[0128] In one embodiment of the method, in step b), 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.

[0129] In one embodiment of the method, in step b), 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.

[0130] 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.

[0131] 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.

[0132] 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 described herein. Non-limiting examples of such agonists include other factors that bind to FGFR2b and signal through the receptor, such as FGF10.

[0133] In one embodiment, the KGF or derivative or agonist thereof is selected from the group consisting of KGF and FGF10, for example FGF10.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] Activin / Nodal has been reported to be involved in maintaining stem cell pluripotency, but Activin / Nodal signaling is also required for endoderm differentiation.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] In one embodiment, the medium in step b) contains about 50 to 150 nM RA, for example about 100 nM RA.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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. PDBu 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.

[0150] 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.

[0151] 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.

[0152] As described above, the present invention demonstrates that short-term culture of posterior foregut (PF) cells under conditions permissive for differentiation into pancreatic progenitor cells (PP) results in fewer PP cells than corresponding methods involving longer culture times, but produces higher numbers of endocrine precursor cells (EP) compared to corresponding methods involving longer culture times.

[0153] 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).

[0154] 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.

[0155] In one embodiment, said posterior foregut cells are characterized in a) by expression of PDX1 and lack expression of NKX6.1.

[0156] In one embodiment of the method for producing pancreatic islet-like cell aggregates in vitro disclosed herein, prior to step i), the method comprises 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; it will be appreciated that the posterior foregut cells may also be characterized by expression of HNF6, or co-expression of PDX1 and HNF6.

[0157] In one embodiment of the method, steps a-1) to c-1) are performed before steps a) to c).

[0158] In one embodiment of the method for producing pancreatic islet-like cell aggregates in vitro disclosed herein, prior to step i), the method comprises the following steps a-1) to c-1) and steps a) and c): 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 under conditions that allow differentiation into posterior foregut cells for about 54 hours or less; c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; a) providing a cell population of posterior foregut cells, such as posterior foregut cells, characterized by the expression of PDX1 produced in step c-1); 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.

[0159] 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.

[0160] [Table 1]

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] As mentioned above, "conditions permissive for differentiation" refers to conditions that allow cells to develop / differentiate to exhibit characteristics of said cell type, and may include the combination of cell culture medium, the presence or absence, and timing of exogenous factors. 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.

[0167] 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.

[0168] Thus, in one embodiment, there is provided a method 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 comprising 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, for example, a culture medium comprising 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.

[0169] 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.

[0170] 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.

[0171] In one embodiment, the medium in step b-1) contains about 250 to 750 nM PDBu, for example, about 500 nM PDBu.

[0172] 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.

[0173] 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.

[0174] 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 generated in step c; 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.

[0175] In one embodiment there is provided a method as disclosed herein, wherein prior to step i), said method comprises the steps of: 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 under conditions that allow differentiation into posterior foregut cells for about 54 hours or less; c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; a) providing a population of posterior foregut cells, such as posterior foregut cells characterized by expression of PDX1, produced in step c-1); 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; 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 the pancreatic progenitor cells generated in step c; 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.

[0176] 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.

[0177] 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.

[0178] 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 a population of endocrine precursor cells for step c+1).

[0179] Similar to the description of steps a)-c), steps a+1) to c+1) are performed in 2D culture on a 2D substrate. Note that the description of the 2D substrate related to steps a) to c) is similarly relevant to steps a+1) to c+1) and will not be repeated here for brevity. Similar to the description of steps a)-c), steps a-1) to c-1) are performed in 2D culture on a 2D substrate. Note that the description of the 2D substrate related to steps a) to c) is similarly relevant to steps a-1) to c-1) and will not be repeated here for brevity.

[0180] Thus, in one embodiment disclosed herein, the cells of step b+1) are cultured on a 2D substrate. Thus, in one embodiment disclosed herein, the cells of step b-1) are cultured on a 2D substrate.

[0181] In one embodiment, the cells are attached to the 2D substrate, which 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™.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] As mentioned above, "conditions permissive for differentiation" refers to conditions that allow cells to exhibit characteristics of said 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 relation to steps b) and b-1) 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 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 in which step b+1) comprises culturing the cell population in a culture medium comprising BTC and / or its derivatives and / or agonists, Alk5 inhibitors (e.g., Alk5iII) and / or their derivatives and / or agonists, γ-secretase inhibitors (e.g., GSI-XX) and / or their derivatives and / or agonists, GC-1 and / or its derivatives and / or agonists, LDN and / or its derivatives and / or agonists, retinoic acid and / or its derivatives and / or agonists, and SANT-1 and / or its derivatives and / or agonists. It will be understood that the culture medium may comprise a mixture of factors and their derivatives and / or agonists. In one embodiment of the method 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 said 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] In one embodiment, the medium in step b+1) contains about 5 to 15 μM Alk5iII, for example about 10 μM Alk5iII.

[0194] In one embodiment, the medium in step b+1) contains about 50 to 150 nM GSI-XX, for example about 100 nM GSI-XX.

[0195] 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.

[0196] 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.

[0197] In one embodiment, the medium in step b+1) contains about 50 to 150 nM RA, for example about 100 nM RA.

[0198] 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.

[0199] 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.

[0200] As described above, in one embodiment, a method for generating pancreatic islet-like cell aggregates in vitro as defined herein is provided, wherein the endocrine precursor cell population, e.g., endocrine precursor cells characterized by expression of NEUROD1, is generated. In one embodiment, the EP cells are characterized by expression of NGN3. In one embodiment, the EP cells are characterized by expression of NGN3 and NEUROD1. In another embodiment, the EP cells are characterized by expression of NKX6.1 and NEUROD1. In one embodiment, the EP cells are further characterized by expression of at least one of PDX1 and NGN3.

[0201] In one embodiment, in step b+1), the 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. In one embodiment, the pancreatic progenitor cells are cultured on a 2D substrate at least until the generation of endocrine precursor cells in step c+1).

[0202] During the differentiation protocol, cells may be cultured on a 2D substrate, such as by adhering to the 2D substrate. Note that the discussion above regarding the identity of the 2D substrate is equally relevant in the context of steps a) through c+1) and will not be repeated here for brevity.

[0203] It will be appreciated that the endocrine precursor cells obtained in step c+1) can be further differentiated into monohormonal pancreatic β cells. The inventors have found that it is beneficial to transfer the cells from 2D to 3D culture conditions at this step.

[0204] 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.

[0205] In one embodiment, there is provided a method as disclosed herein, the method further comprising steps i) to v) as defined herein after steps a+1) to c+1), comprising transferring the endocrine precursor cell population (e.g., endocrine precursor cells characterized by expression of NKX6.1 and NEUROD1) from culture on a 2D substrate to 3D culture conditions, culturing the endocrine precursor cell population under conditions permissive for differentiation into pancreatic monohormonal beta cells, and generating a pancreatic monohormonal beta cell population, such as pancreatic monohormonal beta cells characterized by expression of insulin. The monohormonal beta cells may further express at least one of NKX6.1, PDX1, and NEUROD1.

[0206] As detailed above, 3D culture conditions allow cells to self-aggregate.

[0207] In one embodiment, steps i)-v) follow steps a+1)-c+1), such as immediately after steps a+1)-c+1). It will be appreciated that the endocrine precursor cells can be cryopreserved for a desired period of time before steps i)-v). In this case, steps i)-v) follow the cryopreservation step and subsequent recovery of the cryopreserved cells. Those skilled in the art are familiar with the process of recovering cryopreserved cells, 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 i)-v) follow the cryopreservation and subsequent recovery of the EP cells obtained in c+1).

[0208] In one embodiment, step iv) comprises culturing the population of endocrine precursor cells for about 3 weeks or more, for example, about 3 to 5 weeks, for example, about 4 weeks.

[0209] 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.

[0210] 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.

[0211] Thus, in one embodiment of the method of the invention disclosed herein, the population of monohormonal beta cells generated in step v) is monohormonal. In particular, the population of monohormonal beta cells generated in step v) does not express glucagon or somatostatin. In particular, the population of monohormonal beta cells generated in step v) does not express glucagon or somatostatin.

[0212] It will be appreciated that the increased percentage and / or number of EP cells obtained by the method according to steps a-1) through c+1) compared to prior art methods including step b) culturing a population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for about 96 hours or more may result in improved quality of the islet-like cell aggregates, such as a higher percentage of monohormonal β cells as defined herein, and the islet-like cell aggregates may also contain a lower percentage of polyhormonal cells, a lower percentage of monohormonal cells that are not β cells, and / or a lower percentage of proliferating cells.

[0213] In one embodiment disclosed herein, the method is provided wherein in step c+1), more than about 30%, for example more than about 40%, 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 the 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. In one embodiment, in step c+1), more than about 30%, for example 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 the expression of NKX6.1 and NEUROD1.

[0214] 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. Thus, the culture time in step b) according to the present invention leads to an increased number of EP cells. As shown in the accompanying examples, the number of EP cells obtained by the method disclosed herein is significantly higher than methods using longer or shorter culture times in step b). This is particularly surprising and unexpected, since the number of PP cells is lower than the number of PP cells obtained when step b) is performed for about 96 hours or more.

[0215] 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.

[0216] In one particular embodiment of the method disclosed herein, when step b-1) of culturing the cell population of primitive intestinal cells under conditions permissive for differentiation into posterior foregut cells to obtain a posterior foregut cell population does not exceed 54 hours, the number of endocrine precursor cells in step c+1) is greater than the number of endocrine cells obtained using a corresponding method in which step b-1) of culturing the cell population of primitive intestinal cells under conditions permissive for differentiation into posterior foregut cells is about 56 hours or longer. In particular, the combination of the culture time in step b-1) and the culture time in step b) is considered to be particularly beneficial in terms of the number of EP cells obtained in step c+1).

[0217] This effect is demonstrated in the accompanying examples, particularly in Figure 4, where the number of endocrine precursor cells is scored by co-expression of NKX6.1 and NEUROD1. Importantly, this effect is shown in cell cultures of various human stem cell lines, including human ESC and iPSC lines.

[0218] As discussed above, it will be appreciated that an increase in the proportion and / or number of EP cells obtained by the methods of the present invention, compared to prior art methods including step b) culturing a population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for about 96 hours or more, may result in an increase in the proportion of monohormonal β cells as defined herein. It will be appreciated that an increase in the proportion and / or number of EP cells obtained by the methods of the present invention, compared to prior art methods in which step b-1) is for 56 hours or more and step b) is for 96 hours or more, may result in an increase in the proportion of monohormonal β cells as defined herein.

[0219] In one embodiment of the methods disclosed herein, in step v), more than about 40%, e.g., about 40-50%, e.g., about 40-60%, e.g., about 40-70%, of the total cells in the pancreatic islets 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 comprising step b) culturing the population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for about 96 hours. 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 comprising step b-1) for 56 hours or more and step b) for 96 hours or more. The monohormonal beta cells may be characterized by expression of insulin and NKX6.1, such as by expression of insulin and at least one of insulin and NKX6.1, PDX1, and NEUROD1.

[0220] In one embodiment, the monohormonal beta cells express insulin and are further characterized by expression of NKX6.1, PDX1 and / or NEUROD1. In one embodiment, the monohormonal beta cells are characterized by expression of insulin and NKX6.1, insulin and PDX1, or insulin and NEUROD1.

[0221] In one embodiment, the monohormonal beta cells are 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 cells are characterized by expression of insulin, PDX1, NKX6.1, and NEUROD1.

[0222] In one particular embodiment, the method results in at least two times more monohormonal pancreatic 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. Thus, the pancreatic islet-like cell aggregates obtained by the methods of the invention comprise at least two times more pancreatic monohormonal β cells than α cells.

[0223] 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.

[0224] Thus, in one embodiment, Provided are methods described herein wherein the monohormonal beta cells, e.g., the monohormonal beta cells of the pancreatic islet-like cell aggregates, are functional pancreatic beta cells, such as functional pancreatic beta cells, as assessed by expression of C-peptide upon glucose stimulation.

[0225] It is an object of the present invention to provide pancreatic islet-like cell aggregates comprising monohormonal β cells, for example human pancreatic islet-like cell aggregates comprising monohormonal β cells, by in vitro differentiation.

[0226] The source cells may be pluripotent cells, such as embryonic stem cells or induced pluripotent stem cell cell lines. Thus, the cells may be derived from an established cell line, or may be primary cells obtained directly from a patient, such as patient-specific cells. Thus, in one embodiment of the method disclosed herein, the cell population in step a) or a-1) 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) or a+1) is a population of primary cells derived directly from a patient. Alternatively, the cell population in step a) or a-1) or a+1) may be derived from a culture of pluripotent cells restricted to the endodermal lineage, such as a cell line. The cells may be human cells.

[0227] In one embodiment, the cell population in step a) or a-1) is a mammalian cell population, such as a human cell population. The same therefore applies to the cell population in step i). The cell population in step i) may therefore be 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. The cell population in step i) may be a population of primary cells derived directly from a patient, or may be derived from a culture of pluripotent cells restricted to the endoderm lineage, such as a cell line. The cells may be human cells.

[0228] As used herein, the term "derived" refers to the origin of the cells, and the cells in step i) may be derived from one or more differentiation steps (e.g., one of several of a-1-c-1), ac), and a+1-c+1) as defined herein) prior to step i).

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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 i) is derived from a human embryonic stem cell population. In one embodiment, the cell population in step i) is derived from a human embryonic stem cell population obtained without destroying a human embryo. In a specific embodiment, the cell population in step a) or step a-1) 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) or step a+1) is derived from a human embryonic stem cell population obtained without destroying a human embryo.

[0233] In a specific embodiment, the cell population in step a), a-1), a+1), or i) 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, e.g., 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 embryos. Solely for purposes of compliance with European patent practice, the above embodiments relating to HS980 cells, H1 cells, and / or H9 cells are to be regarded as reference examples in European jurisdictions.

[0234] In one particular embodiment, said cell population in step a), a-1), a+1), or i) is derived from iPS cells, such as human iPS cells.

[0235] In one specific embodiment, the iPS cells are selected from the group consisting of patient-derived iPS cells and iPS cell lines. In one specific embodiment, such an iPS cell line is CTRL-7-II (C7). C7 is described in Kele M et al. 2016.

[0236] As mentioned above, the method may further comprise a step of cryopreserving cells, and may be particularly suitable for cryopreserving endocrine precursor cells. Accordingly, in one embodiment, a method is provided comprising a step of cryopreserving endocrine precursor cells. In one embodiment, the cryopreservation is cryopreservation of the endocrine precursor cells generated in step c+1). In one embodiment, the method disclosed herein comprises cryopreserving EP cells prior to step i). In one embodiment, the EP cell population provided in step i) is a cryopreserved EP cell population or a previously cryopreserved EP cell population. As shown in the accompanying examples, cryopreservation does not adversely affect the generation of the islet-like cell aggregates or monohormonal β cells.

[0237] In a second aspect of the present disclosure, there is provided an isolated islet-like cell aggregate or a population of isolated islet-like cell aggregates obtained by the methods described herein. Also provided are cells obtained from said islet-like cell aggregates, as described in the third aspect below. In particular, said islet-like cell aggregates contain a single hormonal beta cell. As explained in the context of the first aspect, it is highly desirable that the islet-like cell aggregates obtained in vitro mimic the characteristics of in vivo pancreatic islets, both in terms of the distribution of cell types present and their functional properties. Those skilled in the art will appreciate these characteristics.

[0238] In particular, the isolated islet-like cell aggregates or isolated populations of islet-like cell aggregates are characterized by 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.

[0239] Thus, in one embodiment, the isolated islet-like cell aggregates comprise at least about 25%, such as at least about 30%, for example at least about 35%, such as 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 70% monohormonal beta cells, hi one embodiment, the islet-like cell aggregates comprise about 25-70%, such as about 30-70%, for example about 40-70%, for example about 40-60% monohormonal beta cells.

[0240] In one embodiment, said isolated islet-like cell aggregates 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, hi one embodiment, said monohormonal beta cells are characterized by insulin expression.

[0241] In one embodiment, said islet-like cell aggregates 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.

[0242] In one embodiment, the isolated islet-like cell aggregates 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 in v) 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 in v) comprise at most about 5%, e.g., at most about 4, 3, 2, or 1%, of non-endocrine cells. Thus, in one embodiment, the isolated islet-like cell aggregates comprise at least about 25%, such as at least about 30%, for example at least about 35%, such as at least about 40%, for example at least about 45%, such as at least about 50%, for example at least about 55%, such as at least about 60%, for example at least about 65%, such as at least about 70% monohormonal beta cells, or the isolated population comprises islet-like cell aggregates comprising said monohormonal beta cells. In one embodiment, the isolated islet-like cell aggregates comprise about 25-70%, such as 30-70%, for example 30-70% monohormonal beta cells, such as 35-70%, 35-70%, for example 40-70%, such as 45-70%, for example 45-65%, such as 45-60%, for example 45-55%, such as about 50% monohormonal beta cells, or the isolated population comprises islet-like cell aggregates comprising said monohormonal beta cells. In one embodiment, the islet-like cell aggregates comprise about 35-65%, such as 40-65%, for example 40-60%, monohormonal beta cells, or the population comprises islet-like cell aggregates comprising the monohormonal beta cells.

[0243] In one embodiment, the islet-like cell aggregates comprise about 7-25%, such as 7-20%, 10-20%, such as 15-20%, such as about 20% monohormonal alpha cells, or the population comprises islet-like cell aggregates comprising the monohormonal alpha cells.

[0244] In one embodiment, said 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%, such as up to about 0.5%, for example up to about 0.3%, for example up to about 0.1% multihormonal alpha cells, or said population comprises islet-like cell aggregates comprising said multihormonal alpha cells.

[0245] 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%, such as up to about 0.5%, for example up to about 0.3%, for example up to about 0.1% multihormonal beta cells, or the population comprises islet-like cell aggregates comprising the multihormonal beta cells.

[0246] In one embodiment, the islet-like cell aggregates comprise less than 5%, such as less than 4%, such as less than 3%, such as less than 1% δ cells, or the population comprises islet-like cell aggregates comprising the δ cells.

[0247] 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%, for example 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, such as proliferating cells expressing Ki-67, or the population comprises islet-like cell aggregates comprising said proliferating cells.

[0248] In one embodiment, the isolated in vitro islet-like cell aggregates 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.

[0249] Isolated islet-like cell aggregates, populations thereof, or cells therefrom are believed to be useful for treatments such as cell replacement therapy, as well as for drug development or other research applications. 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, particularly without the need for additional selection or sorting of the cells. For clarity, the term "isolated" with respect to isolated islet-like cell aggregates, populations thereof, or cells from islet-like cell aggregates refers to cells that are removed from their natural environment, such as the in vivo environment (i.e., isolated). It will be understood that the isolated islet-like cell aggregate populations, populations thereof, or cells from islet-like cell aggregates disclosed herein may be part or all of a cell aggregate formed during cell culture. The aggregates (also referred to as islet-like cell aggregates) may, for example, contain pancreatic alpha cells and / or delta cells, or cells of the pancreatic alpha cell and / or delta cell lineage, in addition to pancreatic beta cells.

[0250] Thus, in one embodiment, the isolated islet-like cell aggregates or population of islet-like cell aggregates are provided, wherein the cells comprising the islet-like cell aggregates have not been enriched for a desired phenotype, e.g., have not been enriched by manual intervention or machine (in other words, automated sorting). In one embodiment, the cells have not been enriched prior to forming the 3D structure in step iii). Thus, in one embodiment of the isolated islet-like cell aggregates or population of isolated islet-like cell aggregates, the cells comprising the islet-like cell aggregates have not been enriched for a desired phenotype, e.g., have not been sorted for a desired phenotype, such as sorting based on desired marker expression or FACS-based sorting for a desired phenotype. Those skilled in the art will appreciate that selection / sorting can be performed based on the presence of a desired phenotype (e.g., based on marker expression) or the absence of an undesired phenotype, in which case undesired cells are removed from the population, thus enriching the population for cells exhibiting the desired phenotype. In another embodiment, the cells have not been sorted prior to forming the 3D structure in step iii). For clarity, the term "enrichment," as used herein, refers to the enrichment of cells by intervention (manual or mechanical), e.g., sorting based on cell characteristics, and not to a process that occurs naturally in cell culture. Thus, in one embodiment, the cells comprising the islet-like cell aggregates (i.e., the cells of the islet-like cell aggregates) have not been subjected to selection for a desired phenotype, such as selection based on expression of a desired marker. In one embodiment, the cells comprising the islets have not been subjected to selection for a desired phenotype based on FACS. In one embodiment, the cells comprising the islets have not been enriched by removing undesired phenotypes, e.g., based on marker expression, e.g., by FACS.

[0251] The islet-like cell aggregates of the present 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 present invention, isolated islet-like cell aggregates are provided, wherein greater than about 40%, e.g., about 40-60%, e.g., about 40-50%, of the total cell population of said islets are monohormonal β cells, such as monohormonal β cells characterized by insulin expression. In one embodiment, said population comprises at least two-fold, e.g., three, four, or five-fold more monohormonal pancreatic β cells characterized by insulin expression than monohormonal pancreatic α cells characterized by glucagon expression.

[0252] 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. In one embodiment, the monohormonal beta cells may comprise part of a cell aggregate, such as a cell aggregate further comprising pancreatic monohormonal alpha cells and / or delta cells.

[0253] In a related third aspect, provided is a cell obtained from the isolated islet-like cell aggregate obtained by isolating pancreatic islets as defined herein.Therefore, provided is a population of such cells.For the sake of brevity, such cells are referred to herein as "cells from them".

[0254] As noted above, it is contemplated that the isolated pancreatic islets or isolated pancreatic islet-like cell aggregates disclosed herein or cells obtained therefrom are useful for the treatment and / or prevention of diabetes.

[0255] Thus, in a third aspect of the present disclosure, there is provided an isolated islet-like cell aggregate, a population of isolated islet-like cell aggregates, or cells obtained therefrom, as disclosed herein, for use in therapy (in other words, for use as a medicament).

[0256] It will be appreciated that the islet-like cell aggregates or cells therefrom can be transplanted into patients in need thereof for cell replacement therapy, which may be for the purpose of providing pancreatic beta cells to patients who have no endogenous beta cells or only non-functional beta cells, or to patients who require more pancreatic beta cells because their endogenous beta cell population is reduced or is less functional than required for a healthy patient condition.

[0257] The islet-like cell aggregates or cells may be donor-derived and therefore allogeneic. For example, the islet-like cell aggregates or 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 for endocrine development (e.g., pluripotent, oligopotent, or unipotent cell lines), iPS cell lines, and iPS cell-derived cell lines capable of developing along the pancreatic beta 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 that have the potential to develop along the pancreatic beta cell lineage.

[0258] The islet-like cell aggregates or cells 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. Therefore, the islet-like cell aggregates or cells obtained by the disclosed methods need not be subjected to cell sorting or similar stressful, potentially damaging techniques to obtain a homogeneous population containing a high percentage of the desired cell type, at least to a lesser extent, or even at all, compared to islet-like cell aggregates or cells produced by methods known in the prior art. Therefore, treatments, such as transplants (e.g., forms of cell replacement therapy), according to the present disclosure are expected to be performed using healthier and more viable islets or cell populations than those produced by methods known in the art, and the islet-like cell aggregates or cell populations of the present invention contain a lower percentage of damaged and / or unhealthy cells, which is believed to be beneficial to patients in terms of fewer potential side effects and better clinical outcomes.

[0259] In a fourth aspect, there is provided an isolated islet-like cell aggregate, an isolated population of islet-like cell aggregates, or cells therefrom, as disclosed herein, for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 diabetes 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.

[0260] In one embodiment, isolated pancreatic islets, isolated islet-like cell aggregates, or cells therefrom as disclosed herein are provided for use in therapeutic treatment, and the isolated pancreatic islets, isolated islet-like cell aggregates, or cells therefrom as disclosed herein are produced by the methods defined herein.

[0261] In one embodiment there is provided an isolated islet-like cell aggregate, an isolated population of islet-like cell aggregates or cells therefrom for use in therapy, i.e. as a medicament, said use comprising: Producing cells of the islet-like cell aggregates isolated according to the methods defined herein, an isolated population of islet-like cell aggregates, or cells obtained therefrom; and administering a therapeutically effective amount of said islet-like cell aggregates or cells to the patient.

[0262] In one embodiment, the use further comprises isolating cells from the patient, e.g., cells or stem cells for the generation of iPS cells from the patient, and using the cells to generate pancreatic islet-like cell aggregates or cells according to the methods defined herein.

[0263] In one embodiment there is provided isolated pancreatic islet-like cell aggregates or cells obtained therefrom for use in the treatment, prevention and / or amelioration of diabetes, such as type 1 diabetes or type 2 diabetes, wherein said pancreatic islet-like cell aggregates or cells have been produced by a method defined herein.

[0264] In another embodiment, there is provided isolated pancreatic islet-like cell aggregates or cells therefrom for use in the treatment, prevention and / or amelioration of diabetes, such as type 1 diabetes or type 2 diabetes, the use comprising: generating said islet-like cell aggregates or cells according to the methods defined herein; and The method includes administering a therapeutically effective amount of the cells to a patient. In one embodiment, the islet-like cell aggregates may be administered in the form of islet-like cell aggregates, i.e., in the form of cell aggregates as defined above. Alternatively, the islet-like cell aggregates may be dissociated and the cells administered in the form of a cell suspension, such as a single cell suspension. In one embodiment, the use further comprises isolating cells from the patient, e.g., cells for the generation of iPS cells or stem cells from the patient, and using the cells to generate cells of the islet-like cell aggregates or cells therefrom, according to the methods defined herein. As described with respect to the third aspect, other cell types, such as allogeneic and endogenous cells, are also contemplated as useful in this regard.

[0265] Thus, in one embodiment, the use comprises: generating isolated islet-like cell aggregates according to the methods defined herein; and administering a therapeutically effective amount of said islet-like cell aggregates to the patient.

[0266] Thus, in one embodiment, the use comprises: generating isolated pancreatic islet-like cell aggregates according to the methods defined herein; isolating the islet-like cell aggregates; and administering a therapeutically effective amount of the isolated pancreatic islet cells to the patient.

[0267] It will be understood that cell replacement therapy includes the transplantation of pancreatic islet-like cell aggregates (cell aggregates) or cells derived therefrom, which results in the production of insulin in the patient's body and the ability of the patient to respond to glucose stimulation.

[0268] Thus, in one embodiment, the use comprises transplanting the islet-like cell aggregates into a patient in need thereof, hi one embodiment, the use comprises transplanting all cells or a subset thereof, e.g., monohormonal pancreatic beta cells, from the dissociated islet-like cell aggregates into a patient in need thereof.

[0269] The isolated islet-like cell aggregates or cells therefrom are believed to be useful as pharmaceutical compositions. As used herein, the term "pharmaceutical composition" includes compositions derived from cell therapy. Accordingly, in a related fifth aspect of the present disclosure, there is provided a pharmaceutical composition comprising the isolated islet-like cell aggregates disclosed herein or cells therefrom and at least one pharmaceutically acceptable excipient or carrier.

[0270] It is also contemplated that it may be beneficial to administer the isolated islet-like cell aggregates or cells therefrom, 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 fourth to sixth aspects of the present disclosure, kits of parts are provided that include the isolated islet-like cell aggregates or cells therefrom, or the pharmaceutical compositions disclosed herein, and a suitable carrier substrate. The suitable carrier substrate may be a 3D scaffold.

[0271] In one embodiment, a kit of parts is provided, wherein the carrier substrate is a 3D substrate and the cells are single hormonal beta cells. In another embodiment, the kit of parts comprises a 2D substrate. Non-limiting examples of such 2D substrates include those described herein.

[0272] It will be appreciated that the isolated pancreatic islets or cells therefrom disclosed herein may have many applications in biological research, such as drug screening, for example, in vitro drug screening. It will be appreciated that the isolated pancreatic islets or cells therefrom disclosed herein are advantageous because they contain a higher percentage of total cells of a desired cell type than the islet-like cell aggregates of cell populations obtained by the aforementioned culture methods. Thus, the islet-like cell aggregates of the present invention or cells therefrom need not 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, compared to islet-like cell aggregates or cells produced according to conventional techniques. A homogenous population with 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 islet-like cell aggregates disclosed herein or cells therefrom in drug screening, such as in vitro drug screening.

[0273] In this regard, there is provided an in vitro drug screening method comprising generating an isolated islet-like cell aggregate or cells therefrom according to the methods defined herein, and exposing said islet-like cell aggregate or cells therefrom to at least one candidate drug compound, said method optionally further comprising assessing the response of said isolated islet-like cell aggregate or cells therefrom to said candidate drug compound.

[0274] In one embodiment, an in vitro drug screening method is provided, comprising generating islet-like cell aggregates isolated according to the methods defined herein, and exposing the islet-like cell aggregates to at least one candidate drug compound.

[0275] In one embodiment, an in vitro drug screening method is provided, comprising the steps of generating islet-like cell aggregates isolated according to the methods defined herein, separating the islet-like cell aggregates, and exposing at least a portion of the separated islet cells to at least one candidate drug compound.

[0276] In an eighth aspect, there is provided a method of treating a patient in need of treatment, comprising administering to said patient a therapeutically effective amount of isolated islet-like cell aggregates or cells therefrom as disclosed herein, further comprising producing the isolated islet-like cell aggregates or cells therefrom according to the methods defined herein, and A method for treating diabetes in a patient in need thereof is provided, comprising administering to said patient a therapeutically effective amount of said isolated islet-like cell aggregates or cells therefrom, hi one embodiment, said isolated islet-like cell aggregates or cells therefrom are allogeneic, and in another embodiment, said isolated islet-like cell aggregates or cells therefrom are endogenous to the patient.

[0277] In one embodiment, the method is for the treatment of diabetes, such as type 1 or type 2 diabetes. Thus, the patient may have type 1 or type 2 diabetes.

[0278] Accordingly, there is provided a method for treating diabetes in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of an isolated islet-like cell aggregate or cells therefrom as disclosed herein, further comprising producing the isolated islet-like cell aggregate or cells therefrom according to the methods defined herein; Methods for treating a patient in need thereof are provided, comprising administering to said patient a therapeutically effective amount of said isolated islet-like cell aggregates or cells therefrom. The methods may further comprise isolating cells, such as cells for generating iPS cells or stem cells, from said patient and using said cells to generate cells of the isolated islet-like cell aggregates or cells therefrom according to the methods defined herein. The islet-like cell aggregates or cells may be administered by transplantation.

[0279] Thus, in one embodiment, said administering comprises transplanting said islet-like cell aggregates or cells into said patient.

[0280] In a related ninth aspect, there is provided a use of an isolated islet-like cell aggregate, or cells therefrom, as described herein, for the manufacture of a medicament for the treatment of diabetes in a patient in need thereof. In one embodiment, the manufacture of the medicament comprises producing the isolated islet-like cell aggregate, or cells therefrom, by a method defined herein. In one embodiment, the isolated islet-like cell aggregate, or cells therefrom, is endogenous to the patient, i.e., islet-like cell aggregate or cells therefrom, specific to the patient. In one embodiment, the isolated islet-like cell aggregate, or cells therefrom, is an allogeneic islet-like cell aggregate or cells.

[0281] 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.

[0282] In yet another related aspect, 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 under conditions that allow differentiation into posterior foregut cells for no more than about 54 hours; c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; a) providing a population of posterior foregut cells, such as posterior foregut cells characterized by expression of PDX1, produced in step c-1); 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; 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 the pancreatic progenitor cells produced in step c; b+1) culturing the cell population of pancreatic precursor cells under conditions that allow differentiation into endocrine precursor cells; c+1) thereby generating a population of endocrine precursor cells characterized by expression of NEUROD1, such as expression of NKX6.1 and NEUROD1; i) providing a population of endocrine precursor cells (EP) generated in step c+1), such as EP cells characterized by expression of NEUROD1, such as EP cells characterized by expression of NKX6.1 and NEUROD1; ii) providing a single cell suspension of said population of EP cells; iii) allowing the population of EP cells in single cell suspension to form 3D structures; iv) culturing the population of EP cells in the form of a 3D structure under 3D culture conditions that allow differentiation of pancreatic monohormonal β cells to provide pancreatic islet-like cell aggregates; and v) thereby producing islet-like cell aggregates comprising a single hormonal beta cell; A method is provided for producing islet-like cell aggregates in vitro, wherein said islet-like cell aggregates comprise at least 25% monohormonal beta cells.

[0283] As with the first embodiment, in this context of the present invention, step v) can be rephrased as "thereby producing a population of islet-like cell aggregates comprising monohormonal β cells, said population comprising islet-like cell aggregates comprising at least 25% monohormonal β cells." It will be understood that the characterizations disclosed in the context of the first embodiment, in particular those relating to the proportions of different cells, are equally relevant here and will not be repeated here for the sake of brevity.

[0284] 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 "a population of EP cells" 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.

[0285] 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.

[0286] 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.

[0287] 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]

[0288] [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]

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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 ).

[0293] 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. Differentiation was divided into six stages, S1 to S6, and the media used at each stage were as follows:

[0294] 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).

[0295] 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).

[0296] 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).

[0297] 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).

[0298] 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).

[0299] 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.

[0300] 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.

[0301] 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).

[0302] 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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] The medium was changed daily from stage 1 to stage 5, and every 2–3 days in stage 6.

[0308] 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.

[0309] 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.

[0310] 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. Aggregate formation in microwells

[0311] 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.

[0312] 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.

[0313] 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).

[0314] 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 and 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).

[0315] 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).

[0316] 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.

[0317] 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.

[0318] 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.

[0319] 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).

[0320] 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.

[0321] 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).

[0322] 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.

[0323] Example 2 In this example, the effect of the duration of stage 4 on endocrine differentiation was examined.

[0324] 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.

[0325] 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).

[0326] 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 stage 4 duration on differentiation into endocrine precursor cells

[0327] 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 ).

[0328] 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 significant increase in the number of NKX6.1 cells in the subsequent S5 culture. + / 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).

[0329] 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.

[0330] 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.

[0331] 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 ).

[0332] 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).

[0333] Example 3 In this example, various cell culture coated substrates were evaluated for pancreatic differentiation.

[0334] 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.

[0335] [Table 2]

[0336] 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). 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).

[0337] 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).

[0338] Taken together, these results demonstrate that the short-term protocol is highly efficient for defined substrates that do not contain multiple heterogeneous materials.

[0339] Example 4 In this example, it was investigated whether S4 cells or S5 cells are more suitable for in vitro pancreatic islet formation.

[0340] 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.

[0341] 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).

[0342] 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).

[0343] 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 duration under S5 was sufficient for aggregate formation in suspension.

[0344] Example 5 In this example, the effect of culturing cells in 3D single-cell suspension was investigated.

[0345] 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.

[0346] 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).

[0347] 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.

[0348] 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).

[0349] 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 ).

[0350] 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.

[0351] 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; days 8, 13, and 17 of culture for the long-term protocol).

[0352] 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.

[0353] 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).

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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).

[0359] Taken together, these results suggest that S5 EP cells can separate from other non-endocrine cell types and then spontaneously self-aggregate.

[0360] Example 8 In this example, we evaluated cell line variability among multiple hPSC lines differentiated using a short-term pancreatic differentiation protocol.

[0361] 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.

[0362] Results: The results were consistent with 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).

[0363] 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.

[0364] 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).

[0365] 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.

[0366] 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).

[0367] 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.

[0368] Example 10 In this example, the ability of frozen and non-frozen S5 EP cells to generate 3D islets was compared.

[0369] 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.

[0370] 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).

[0371] 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.

[0372] 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.

[0373] Itemized List of Embodiments 1. A method for generating pancreatic islet-like cell aggregates in vitro, comprising: i) providing a population comprising endocrine precursor cells (EP), e.g., EP cells, e.g., EP cells characterized by expression of NEUROD1, e.g., a population of EP cells characterized by expression of NKX6.1 and NEUROD1; ii) providing a single cell suspension of said population of EP cells; iii) allowing the population of EP cells in single cell suspension to form 3D structures; iv) culturing the population of EP cells in the form of a 3D structure under 3D culture conditions permissive for the differentiation of pancreatic monohormonal β cells to provide pancreatic islet-like cell aggregates; and v) thereby producing islet-like cell aggregates comprising a single hormonal beta cell; The method, wherein said pancreatic islet-like cell aggregates comprise at least about 25%, such as at least about 30%, such as at least about 35%, such as at least about 40%, such as at least about 45% monohormonal beta cells.

[0374] 2. The method for generating pancreatic islet-like cell aggregates in vitro according to item 1, wherein the population of EP cells in step i) is an adherent culture of EP cells on a 2D substrate.

[0375] 3. The method for generating pancreatic islet-like cell aggregates in vitro according to item 2, 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™.

[0376] 4. The method for generating pancreatic islet-like cell aggregates in vitro according to item 3, wherein 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 group consisting of LN-521 and fragments thereof and LN-511 and fragments thereof, for example, the group consisting of LN-521 and fragments thereof, or the group consisting of LN-511 and fragments thereof.

[0377] 5. The method for generating pancreatic islet-like cell aggregates in vitro according to item 3 or 4, 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 are LN-521, or for example, the laminin and fragments thereof are LN-511.

[0378] 6. The laminin (LN) and fragments thereof are selected from the group consisting of an E8 fragment of laminin, 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, 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. 5. The method for generating pancreatic islet-like cell aggregates in vitro according to item 3 or 4, comprising an E8 fragment of laminin selected from the group consisting of E8 fragments of LN-511, LN-521, and 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.

[0379] 7. In step i), more than about 15% of the total cell population, such as more than about 20%, for example more than about 25%, for example more than about 30%, for example more than about 35%, for example more than about 40%, for example more than about 45%, for example more than about 50% are EP cells characterized by expression of NEUROD1; or 7. A method for producing pancreatic islet-like cell aggregates in vitro according to any one of items 1 to 6, wherein in step i), more than about 15%, for example more than about 20%, for example more than about 25%, for example more than about 30%, for example more than about 35%, for example more than about 40%, for example more than about 45%, for example more than about 50% of the total cell population are EP cells characterized by expression of NKX6.1 and NEUROD1.

[0380] 8. Step ii) of providing a single cell suspension of the population of EP cells is performed when more than about 15%, for example more than about 20%, for example more than about 25%, for example more than about 30%, for example more than about 35%, for example more than about 40%, for example more than about 45%, for example more than about 50% of the total cell population are EP cells characterized by expression of NEUROD1; or A method for producing pancreatic islet-like cell aggregates in vitro described in any one of items 1 to 7, wherein step ii) of providing a single cell suspension of the population of EP cells is performed when more than about 15%, for example more than about 20%, for example more than about 25%, for example more than about 30%, for example more than about 35%, for example more than about 40%, for example more than about 45%, for example more than about 50% of the total cell population are EP cells characterized by expression of NKX6.1 and NEUROD1.

[0381] 9. The method for generating islet-like cell aggregates in vitro according to any one of items 1 to 8, wherein the islet-like cell aggregates generated in step v) comprise about 7-25%, for example 7-20%, 10-20%, for example 15-20%, for example about 20% monohormonal alpha cells.

[0382] 10. A method for producing islet-like cell aggregates in vitro according to any one of items 1 to 9, wherein the islet-like cell aggregates produced in step v) comprise up to about 5%, for example up to about 4%, for example up to about 3%, for example 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, for example proliferating cells expressing Ki-67.

[0383] 11. A method for generating islet-like cell aggregates in vitro according to any one of items 1 to 10, wherein the islet-like cell aggregates generated in step v) are scored after 38 to 42 days of culture.

[0384] 12. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 1 to 11, wherein step ii) is performed before culturing the cells in a medium permissive for differentiation into pancreatic monohormonal beta cells.

[0385] 13. A method for generating islet-like cell aggregates in vitro according to any one of items 1 to 12, wherein step ii) is performed within about 6 days after the EP cells start to express NEUROD1.

[0386] 14. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 1 to 13, wherein step ii) is performed within 1 to 5 days after the EP cells start to express NEUROD1.

[0387] 15. A method for generating islet-like cell aggregates in vitro according to any one of items 1 to 14, wherein the formation of a 3D structure in step iii) is spontaneous formation of a 3D structure.

[0388] 16. The method for generating islet-like cell aggregates in vitro according to any one of items 1 to 14, wherein the formation of the 3D structure in step iii) is forced or assisted formation of the 3D structure.

[0389] 17. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 1 to 16, wherein step iv) comprises culturing the EPs for at least about 2 weeks, for example at least about 3 weeks, for example about 3 to 5 weeks, for example about 4 weeks.

[0390] 18. The method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 1 to 17, wherein the monohormonal beta cells generated in step v) have the ability to express insulin, e.g., the monohormonal beta cells generated in step v) express insulin.

[0391] 19. The method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 1 to 18, wherein the monohormonal beta cells generated in step v) have the ability to express C-peptide upon glucose stimulation, e.g., the monohormonal beta cells generated in step v) express C-peptide upon glucose stimulation.

[0392] 20. A method for generating islet-like cell aggregates in vitro according to any one of items 1 to 19, wherein the monohormonal beta cells generated in step v) do not express glucagon and / or somatostatin.

[0393] 21. The method for generating islet-like cell aggregates in vitro according to any one of items 1 to 20, wherein the islet-like cell aggregates generated in step v) comprise at least 40% monohormonal beta cells, 7-25% monohormonal alpha cells, and less than 2% proliferating cells.

[0394] 22. A method for generating islet-like cell aggregates in vitro according to any one of items 1 to 21, comprising, before step i), the following steps a) to c): 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.

[0395] 23. A method for generating islet-like cell aggregates in vitro according to any one of items 1 to 22, comprising, before step i), 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;

[0396] 24. A method for generating islet-like cell aggregates in vitro according to any one of items 22 to 23, wherein steps a-1) to c-1) are performed before steps a) to c).

[0397] 25. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 24, comprising, before step i), the following steps a-1) to c-1) and steps a) and c): 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 under conditions that allow differentiation into posterior foregut cells for no more than about 54 hours; c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; a) providing a population of posterior foregut cells, such as posterior foregut cells characterized by expression of PDX1, produced in step c-1); 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.

[0398] 26. The method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 25, further comprising the following steps a+1) to c+1) after steps a) to c): a+1) providing a cell population of pancreatic progenitor cells generated in step c; 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.

[0399] 27. A method for generating islet-like cell aggregates in vitro according to any one of items 22 to 26, comprising the following step before step i): 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 under conditions that allow differentiation into posterior foregut cells for no more than about 54 hours; c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; a) providing a population of posterior foregut cells, such as posterior foregut cells characterized by expression of PDX1, produced in step c-1); 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; 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 the pancreatic progenitor cells generated in step c; 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.

[0400] 28. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 27, 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, for example about 46 to 74 hours, for example about 48 to 72 hours.

[0401] 29. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 28, 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, for example about 70 to 74 hours, for example about 72 hours.

[0402] 30. A method for generating islet-like cell aggregates in vitro according to any one of items 22 to 29, 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, for example about 46 to 50 hours, for example about 48 hours.

[0403] 31. The method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 30, 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, e.g., a marker selected from the group consisting of PTF1A and SOX9.

[0404] 32. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 31, 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.

[0405] 33. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 32, 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.

[0406] 34. The method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 32 to 33, 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.

[0407] 35. A method for producing pancreatic islet-like cell aggregates in vitro according to any one of items 32 to 34, wherein the effective amount of the NIC or its derivative or agonist is about 5 to 20 mM, for example, about 5 to 15 mM, for example, about 8 to 12 mM, for example, about 10 mM.

[0408] 36. The method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 35, wherein step b) comprises culturing the cell population in a culture medium further comprising KGF, activin A, retinoic acid, SANT-1, PDBu, and LDN.

[0409] 37. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 36, wherein the cells are cultured on a 2D substrate, for example, the cells are cultured as adherent cells on the 2D substrate.

[0410] 38. The method for generating pancreatic islet-like cell aggregates in vitro according to item 37, 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™.

[0411] 39. 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; 39. The method for generating pancreatic islet-like cell aggregates in vitro according to item 38, wherein the aggregates are selected from the group consisting of LN-521 and fragments thereof, or LN-511 and fragments thereof.

[0412] 40. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 38 to 39, 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.

[0413] 41. The laminin (LN) and fragments thereof are selected from the group consisting of laminin E8 fragments, 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 E8 fragments of LN-121. 40. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 38 to 39, comprising an E8 fragment of laminin selected from the group consisting of fragments thereof, 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.

[0414] 42. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 41, 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, e.g., pancreatic progenitor cells, in c).

[0415] 43. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 42, wherein in step c) at least about 75%, for example at least about 80%, for example about 80-85%, for example about 80-90% of the total cell population expresses PDX1.

[0416] 44. A method for generating islet-like cell aggregates in vitro according to any one of items 22 to 43, wherein in step c), a maximum of about 10% of the total cell population expresses NEUROD1.

[0417] 45. A method for generating islet-like cell aggregates in vitro according to any one of items 22 to 44, wherein in step c), about 40 to 70%, for example about 30 to 50%, of the total cell population expresses NKX6.1.

[0418] 46. ​​A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 23 to 45, wherein in step b-1), the cell population is cultured for not more than about 52 hours, for example not more than about 50 hours, for example not more than about 48 hours.

[0419] 47. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 23 to 46, wherein in step b-1), the cell population 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.

[0420] 48. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 23 to 47, wherein in step b-1), 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.

[0421] 49. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 23 to 47, wherein in step b-1), the cell population 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.

[0422] 50. The method according to any one of items 23 to 49, wherein step b-1) comprises culturing the cell population in a culture medium containing KGF, retinoic acid, SANT-1, PDBu, and LDN.

[0423] 51. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 50, wherein the endocrine precursor cell population, e.g., endocrine precursor cells characterized by expression of NEUROD1 or expression of NKX6.1 and NEUROD1, is further characterized by expression of at least one of PDX1 and NGN3.

[0424] 52. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 51, 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 about 5 days.

[0425] 53. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 52, wherein step b+1) comprises culturing the cell population in a culture medium containing BTC, Alk5i II, GSI-XX, GC-1, LDN, retinoic acid, and SANT-1.

[0426] 54. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 53, wherein the cells are cultured on a 2D substrate at least until endocrine precursor cells are generated in step c+1).

[0427] 55. The method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 54, 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™. 56. The method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 54, wherein 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 group consisting of LN-521 and fragments thereof and LN-511 and fragments thereof; 56. A method for generating islet-like cell aggregates in vitro according to any one of items 26 to 55, for example selected from the group consisting of LN-521 and fragments thereof, or LN-511 and fragments thereof.

[0428] 57. The method for generating islet-like cell aggregates in vitro according to any one of items 26 to 56, 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 are LN-521, or for example, the laminin and fragments thereof are LN-511.

[0429] 58. 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 E8 fragments of LN-121. 57. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 56, comprising an E8 fragment of laminin selected from the group consisting of fragments thereof, 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.

[0430] 59. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 58, wherein in step c+1), more than about 30%, for example 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.

[0431] 60. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 58, 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.

[0432] 61. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 60, 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.

[0433] 62. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 61, further comprising culturing the endocrine precursor cells under conditions that allow differentiation into monohormonal pancreatic β cells.

[0434] 63. A method for generating islet-like cell aggregates in vitro according to any one of items 26 to 62, wherein the cells are cultured on a 2D substrate, such as in an adherent state on the 2D substrate, during steps a-1) to c+1).

[0435] 64. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 26 to 63, 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.

[0436] 65. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 1 to 64, wherein the population of EP cells in step i) 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.

[0437] 66. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 22 to 65, wherein the population of cells provided in step a-1), 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.

[0438] 67. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 65 to 66, wherein the provided cell population is derived from a culture of human embryonic stem cells.

[0439] 68. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 65 to 67, wherein the cell population is a mammalian cell population, such as a human cell population.

[0440] 69. A method for producing pancreatic islet-like cell aggregates in vitro according to any one of claims 65 to 68, wherein the cell population is derived from a human embryonic stem cell line 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.

[0441] 70. A method for generating pancreatic islet-like cell aggregates in vitro according to any one of items 65 to 68, wherein the cell population is derived from a human induced pluripotent stem cell population.

[0442] 71. A method for generating islet-like cell aggregates in vitro according to any one of items 1 to 70, further comprising cryopreserving the EP cells prior to step i).

[0443] 72. An isolated pancreatic islet-like cell aggregate obtained by the method according to any one of items 1 to 71.

[0444] 73. An isolated population of pancreatic islet-like cell aggregates obtained by the method according to any one of items 1 to 72.

[0445] 74. The isolated islet-like cell aggregate of item 72 or the isolated population of islet-like cell aggregates of item 73, wherein the islet-like cell aggregates have more than about 40% of the total cell population, such as about 40% to 70%, for example about 40% to 60%, for example about 40% to 50%, which are monohormonal beta cells, such as monohormonal beta cells characterized by the expression of insulin.

[0446] 75. An isolated islet-like cell aggregate according to any one of items 72 to 74 or an isolated population of islet-like cell aggregates according to any one of items 73 to 74, wherein the cells constituting the islet-like cell aggregates have not been enriched for a desired phenotype, for example have not been enriched by manual or automated intervention, for example have not been enriched before the cells form the 3D structure in step iii).

[0447] 76. An isolated islet-like cell aggregate according to any one of items 72 to 75 or an isolated population of islet-like cell aggregates according to any one of items 73 to 75, wherein the cells comprising the islet-like aggregates have not been subjected to selection for a desired phenotype based on marker expression, e.g., the cells have not been subjected to selection prior to forming the 3D structure in step iii).

[0448] 77. An isolated islet-like cell aggregate according to any one of items 72 to 76 or an isolated population of islet-like cell aggregates according to any one of items 73 to 76, wherein the cells prior to forming the islet-like cell aggregates have not been subjected to FACS-based sorting for a desired phenotype, e.g., the cells have not been subjected to sorting prior to forming the 3D structure in step iii).

[0449] 78. The isolated islet-like cell aggregate according to any one of items 72 to 77 or the isolated population of islet-like cell aggregates according to any one of items 73 to 77, wherein the islet-like cell aggregates comprise 7% to 25%, such as 7% to 20%, 10% to 20%, such as 15% to 20%, such as about 20% monohormonal alpha cells.

[0450] 79. The isolated islet-like cell aggregate according to any one of items 72 to 78 or the isolated population of islet-like cell aggregates according to any one of items 73 to 78, wherein the islet-like cell aggregates comprise at least 40%, such as at least 50%, monohormonal beta cells, about 15-20%, such as about 20%, monohormonal alpha cells, and less than about 2%, such as less than about 1%, proliferating cells.

[0451] 80. Cells obtained from at least one isolated pancreatic islet as defined in any one of items 72 to 79, for example cells obtained by isolation of said islet-like cell aggregates.

[0452] 81. An isolated islet-like cell aggregate according to any one of items 72 to 79, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79, or a cell according to item 80, for use in therapy.

[0453] 82. An isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81, for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes.

[0454] 83. An isolated islet-like cell aggregate according to any one of items 72 to 79, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79, for use in treatment in a therapy, wherein the islet-like cell aggregates or cells have been produced by a method according to any one of items 1 to 71.

[0455] 84. An isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81, for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes, wherein the islet-like cell aggregates or cells have been produced by a method according to any one of items 1 to 71.

[0456] 85. An isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81, for use in therapy, The use, Producing isolated islet-like cell aggregates according to the method defined in any one of items 1 to 71; and administering a therapeutically effective amount of the islet-like cell aggregates to a patient; or The use, generating isolated islet-like cell aggregates according to the method defined in any one of items 1 to 71; isolating the islet-like cell aggregates; and 2. A method for treating a patient with an isolated islet-like cell aggregate, or an isolated population of islet-like cell aggregates, or cells, comprising administering a therapeutically effective amount of said isolated islet cells to a patient.

[0457] 86. An isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81, for use in the treatment, prevention, and / or amelioration of diabetes, such as type 1 or type 2 diabetes, said use comprising: Producing isolated islet-like cell aggregates according to the method defined in any one of items 1 to 71; and administering a therapeutically effective amount of said cells to a patient. or The use, generating isolated islet-like cell aggregates according to the method defined in any one of items 1 to 71; isolating the islet-like cell aggregates; and 2. A method for treating a patient with an isolated islet-like cell aggregate, or an isolated population of islet-like cell aggregates, or cells, comprising administering a therapeutically effective amount of said isolated islet cells to a patient.

[0458] 87. An isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81 to 87, wherein said use comprises transplanting said islet-like cell aggregates or cells into a patient in need thereof.

[0459] 88. A pharmaceutical composition comprising an isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81, and at least one pharmaceutically acceptable excipient or carrier.

[0460] 89. A kit of parts comprising an isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81, or a pharmaceutical composition according to item 88, and a suitable carrier substrate.

[0461] 90. The kit of parts according to item 89, wherein the suitable carrier substrate is a 3D substrate.

[0462] 91. Use of an isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81, in drug screening, for example in vitro drug screening.

[0463] 92. An in vitro drug screening method comprising: Producing isolated islet-like cell aggregates according to the method defined in any one of items 1 to 71; and The method comprises exposing said islet-like cell aggregates to at least one candidate drug compound.

[0464] 93. Producing isolated islet-like cell aggregates according to the method defined in any one of items 1 to 71; isolating the islet-like cell aggregates; and 93. The in vitro drug screening method of claim 92, comprising exposing at least a portion of the isolated pancreatic islet cells to at least one candidate drug compound.

[0465] 94. A method of treating a patient in need thereof, comprising administering to said patient a therapeutically effective amount of an isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81.

[0466] 95. A method of treating a patient in need of treatment, e.g., a method of treating diabetes in a patient in need of treatment, comprising: Producing isolated islet-like cell aggregates according to the method defined in any one of items 1 to 71; and administering a therapeutically effective amount of the islet-like cell aggregates to the patient. or generating isolated islet-like cell aggregates according to the method defined in any one of items 1 to 71; isolating the islet-like cell aggregates; and administering to said patient a therapeutically effective amount of said isolated pancreatic islet cells.

[0467] 96. A method for treating diabetes in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of an isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81.

[0468] 97. The method for treating diabetes in a patient in need thereof according to item 95 or 96, wherein said patient suffers from type 1 or type 2 diabetes.

[0469] 98. A method for treating diabetes in a patient in need thereof according to any one of items 94 to 97, wherein said administering comprises transplanting said islet-like cell aggregates or cells into said patient.

[0470] 99. Use of an isolated islet-like cell aggregate according to any one of items 72 to 79 and 81, or an isolated population of islet-like cell aggregates according to any one of items 73 to 79 and 81, or a cell according to item 80 or 81, for the manufacture of a medicament for the treatment of diabetes in a patient in need thereof.

[0471] 100. Use according to item 99, wherein the preparation of the medicament comprises producing islet-like aggregates by a method as defined in any one of items 1 to 71, and optionally isolating them.

[0472] 101. A method for producing pancreatic islet-like cell aggregates in vitro, comprising: 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 under conditions that allow differentiation into posterior foregut cells for no more than about 54 hours; c-1) thereby generating a population of posterior foregut cells, such as posterior foregut cells, characterized by expression of PDX1; a) providing a population of posterior foregut cells, such as posterior foregut cells characterized by expression of PDX1, produced in step c-1); 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; 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 the pancreatic progenitor cells produced in step c; b+1) culturing the cell population of pancreatic precursor cells under conditions that allow differentiation into endocrine precursor cells; c+1) thereby generating a population of endocrine precursor cells characterized by expression of NEUROD1, such as expression of NKX6.1 and NEUROD1; i) providing a population of endocrine precursor cells (EP) generated in step c+1), such as EP cells characterized by expression of NEUROD1, such as EP cells characterized by expression of NKX6.1 and NEUROD1; ii) providing a single cell suspension of said population of EP cells; iii) allowing the population of EP cells in single cell suspension to form 3D structures; iv) culturing the population of EP cells in the form of a 3D structure under 3D culture conditions that allow differentiation of pancreatic monohormonal β cells to provide pancreatic islet-like cell aggregates; and v) thereby producing pancreatic islet-like cell aggregates, wherein the islet-like cell aggregates comprise at least about 25% monohormonal beta cells; or v) thereby generating a population of islet-like cell aggregates comprising single hormonal β cells, The method includes a step in which the population comprises pancreatic islet-like cell aggregates comprising at least about 25% monohormonal beta cells.

Claims

1. A method for generating pancreatic islet-like cell aggregates in vitro, i) A step of providing a population comprising endocrine progenitor cells (EPs) characterized by the expression of NKX6.1 and Neurod1, for example, a population comprising EP cells characterized by the expression of NKX6.1, Neurod1 and PDX1, wherein the population is an adherent culture of EP cells on a 2D substrate; ii) A step of providing a single-cell suspension of the population of EP cells; iii) A step of causing a population of EP cells in a single-cell suspension to form a 3D structure; iv) A step of providing islet-like cell aggregates by culturing a population of EP cells in a 3D structural form under 3D culture conditions that allow differentiation into monohormonal β cells of the pancreas; and v) The process of generating pancreatic islet-like cell aggregates containing single hormone-producing β-cells. Includes, A method wherein the islet-like cell aggregate contains up to approximately 2% proliferating cells, and the islet-like cell aggregate contains at least approximately 40% monohormonal β-cells.

2. The method according to claim 1, wherein the 2D substrate comprises one or more components selected from the group consisting of laminin (LN) and its fragments, vitronectin and its fragments, fibronectin and its fragments, collagen and its fragments, gelatin and its fragments, functionalized silk (FN silk), and Matrigel®, for example, one or more components selected from the group consisting of laminin (LN) and its fragments, vitronectin and its fragments, fibronectin and its fragments, and Matrigel®.

3. The method according to claim 2, wherein the laminin (LN) and its fragments are selected from the group consisting of LN-521 and its fragments, LN-511 and its fragments, LN-332 and its fragments, LN-421 and its fragments, LN-121 and its fragments, and LN-111 and its fragments, for example, the group consisting of LN-521 and its fragments, LN-511 and its fragments, LN-332 and its fragments, LN-421 and its fragments, and LN-121 and its fragments, for example, the group consisting of LN-521 and its fragments, LN-511 and its fragments, and LN-332 and its fragments, for example, the group consisting of LN-521 and its fragments, for example, the group consisting of LN-511 and its fragments, or the group consisting of LN-511 and its fragments.

4. The method according to claim 2, wherein the laminin (LN) and its fragments are selected from the group consisting of LN-521, LN-511, LN-332, LN-421, LN-121, and LN-111, for example the group consisting of LN-521, LN-511, LN-332, LN-421, and LN-121, for example the group consisting of LN-521, LN-511, and LN-332, for example the group consisting of LN-521 and LN-511, for example the laminin and its fragments are LN-521, or for example the laminin and its fragments are LN-511.

5. The method according to claim 1, wherein step ii) providing a single-cell suspension of the population of EP cells is performed when more than about 15%, for example more than about 20%, for example more than about 25%, for example more than about 30%, for example more than about 35%, for example more than about 40%, for example more than about 45%, for example more than about 50% of the total cell population are EP cells characterized by the expression of NEUROD1.

6. The method according to claim 1, wherein the islet-like cell aggregates generated in step v) contain about 7-25%, for example 7-20%, 10-20%, for example 15-20%, for example about 20% single hormone-mediated α cells.

7. The method according to claim 1, wherein the islet-like cell aggregates generated in step v) contain up to about 1%, for example, up to about 0.5%, for example, up to about 0.1%, of proliferating cells, for example, proliferating cells expressing Ki-67.

8. The method according to claim 1, wherein the islet-like cell aggregates generated in step v) are scored after being cultured for 38 to 42 days.

9. The method according to claim 1, wherein step ii) is performed before culturing the cells in a medium that allows differentiation into pancreatic monohormonal β-cells.

10. The method according to claim 1, wherein the formation of the 3D structure in step iii) is the spontaneous formation of the 3D structure.

11. The method according to claim 1, wherein the formation of the 3D structure in step iii) is forced formation or assisted formation of the 3D structure.

12. The method according to claim 1, wherein step iv) includes culturing the EP for about two weeks or more, for example, about three weeks or more, for example, about three to five weeks, for example, about four weeks.

13. The method according to claim 1, wherein the monohormone β-cells produced in step v) have the ability to express insulin.

14. The method according to claim 1, wherein the monohormone β-cells produced in step v) have the ability to express C-peptide upon glucose stimulation.

15. The method according to claim 1, wherein the monohormone-mediated β-cells generated in step v) do not express glucagon and / or somatostatin.

16. The method according to claim 1, wherein the islet-like cell aggregate produced in step v) comprises at least 40% monohormonal β cells, 7-25% monohormonal α cells, and less than 2% proliferating cells.

17. The method according to claim 1, comprising the following steps a-1) to c-1) and steps a) and c) prior to step i): a-1) A step of providing a cell population of primitive intestinal cells characterized by the expression of HNF1β and / or HNF4α; b-1) A step of culturing the cell population of primitive intestinal cells for approximately 54 hours or less under conditions that allow differentiation into posterior foregut cells; c-1) A step of generating a population of posterior foregut cells characterized by PDX1 expression; a) A step of providing a cell population of posterior foregut cells characterized by the expression of PDX1 produced in step c-1); b) A step of culturing the posterior foregut cell population under conditions that allow differentiation into pancreatic progenitor cells for about 78 hours or less, for example, about 72 hours or less; and c) A step of generating a cell population of pancreatic progenitor cells characterized by the expression of both PDX1 and NKX6.

1.

18. The method according to claim 17, further comprising the following steps a) to c): a+1) A step of providing a cell population of pancreatic progenitor cells generated in step c); b+1) A step of culturing the cell population of pancreatic progenitor cells under conditions that allow differentiation into endocrine progenitor cells; and c+1) A step of generating a population of endocrine progenitor cells, for example, endocrine progenitor cells characterized by the expression of NKX6.1 and NEUROD1.

19. The method according to claim 17, 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.

20. The method according to claim 17, wherein the cells are cultured on a 2D substrate, for example, the cells are adherently cultured on a 2D substrate.

21. The method according to claim 20, wherein the 2D substrate is as defined in any one of claims 2 to 4.

22. The method according to claim 17, wherein in step b-1), the cell population 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.

23. The method according to claim 18, 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 about 5 days.

24. The method according to claim 18, wherein the cells are cultured on a 2D substrate, for example, in an adherent state on the 2D substrate, from step a-1) to c+1).

25. The method according to claim 1, wherein the cells are not transferred from culture on a 2D substrate, such as adherent culture on a 2D substrate, to culture on a 3D substrate before they express markers characteristic of endocrine progenitor cells.

26. The method according to claim 1, wherein the population of EP cells in step i) is derived from a culture of pluripotent stem cells, for example, a culture of induced pluripotent stem cells or a culture of embryonic stem cells, for example, a culture of human induced pluripotent stem cells or a culture of human embryonic stem cells.

27. The method according to claim 26, wherein the cell population is a mammalian cell population such as a human cell population.

28. The method according to claim 26, wherein the cell population is derived from a human embryonic stem cell line selected from the group of embryonic stem cell lines consisting of HS980 cells, H1 cells, and H9 cells.

29. The method according to claim 26, wherein the cell population is derived from a human induced pluripotent stem cell population.

30. Isolated islet-like cell aggregates obtained by the method of claim 1, wherein the islet-like cell aggregates comprise at least 40% monohormonal β cells, about 15-20% monohormonal α cells, and less than 2% proliferating cells.

31. An isolated population of islet-like cell aggregates obtained by the method of claim 1, wherein the islet-like cell aggregates comprise at least 40% monohormonal β-cells, about 15-20% monohormonal α-cells, and less than 2% proliferating cells.

32. The islet-like cell aggregate described in claim 30, or the isolated population of islet-like cell aggregates described in claim 31, wherein approximately 40-70%, for example, approximately 40-60%, or for example, approximately 40-50%, of the islet-like cell aggregates are monohormonal β-cells, such as monohormonal β-cells characterized by insulin expression.

33. The islet-like cell aggregate described in claim 30, or the islet-like cell aggregate described in claim 31, wherein the islet-like cell aggregate contains 7-20%, 10-20%, for example 15-20%, for example about 20% monohormonal α cells.

34. The islet-like cell aggregate described in claim 30, or the islet-like cell aggregate described in claim 31, wherein the islet-like cell aggregate comprises at least 50% monohormonal β cells, about 15-20%, for example, about 20% monohormonal α cells, and less than about 2%, for example, less than 1% proliferating cells.

35. An isolated islet-like cell aggregate according to claim 30 or an isolated population of islet-like cell aggregates according to claim 31, for use in therapeutic purposes.

36. Isolated islet-like cell aggregates according to claim 30 or isolated populations of islet-like cell aggregates according to claim 31 for use in the treatment, prevention, and / or improvement of diabetes such as type 1 or type 2 diabetes.

37. For use of claim 35, an isolated islet-like cell aggregate according to claim 30 or an isolated population of islet-like cell aggregates according to claim 31, wherein the use is the following steps: A step of generating isolated islet-like cell aggregates according to the method defined in claim 1; and The process of administering a therapeutically effective amount of the islet-like cell aggregates to a patient. Isolated islet-like cell aggregates or isolated populations, including [specific cells].

38. The islet-like cell aggregates according to claim 30 or an isolated population of islet-like cell aggregates according to claim 31 for use according to claim 35, wherein the use comprises transplanting the islet-like cell aggregates into a patient who needs them.

39. A pharmaceutical composition comprising an isolated islet-like cell aggregate according to claim 30 or an isolated population of islet-like cell aggregates according to claim 31, and at least one pharmaceutically acceptable excipient or carrier.

40. A component kit comprising an isolated islet-like cell aggregate according to claim 30, an isolated population of islet-like cell aggregates according to claim 31, or a pharmaceutical composition comprising the islet-like cell aggregate or the isolated population of islet-like cell aggregates, and a suitable carrier substrate.

41. The component kit according to claim 40, wherein the appropriate carrier substrate is a 3D substrate.

42. Use of the isolated islet-like cell aggregates described in claim 30 or the isolated population of islet-like cell aggregates described in claim 31 in drug screening, for example, in vitro drug screening.

43. An in vitro drug screening method, A step of generating isolated islet-like cell aggregates according to the method defined in claim 1; and The process of exposing the pancreatic islet-like cell aggregates to at least one candidate drug compound. Methods that include...

44. Use of the isolated islet-like cell aggregates described in claim 30 or the isolated population of islet-like cell aggregates described in claim 31 for the manufacture of a pharmaceutical product for the treatment of diabetes in patients requiring treatment for diabetes.

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