3d islet formation from endocrine progenitor cells

Abstract

The present disclosure relates to a method of generating pancreatic lineage cells, e.g. islet-like cell aggregates comprising pancreatic beta cells, comprising the steps of providing a single cell suspension of a population of endocrine progenitor cells (EP), allowing the EP cells in the single cell suspension to form 3D structures, and culturing the cells under conditions allowing differentiation into pancreatic mono-hormonal beta cells. The present disclosure also relates to islet-like cell aggregates obtainable by said method and their medical use.

Description

Technical Field

[0001] This disclosure relates to a method for generating pancreatic lineage cells, such as islet-like cell aggregates comprising pancreatic β cells, the method comprising the steps of: providing a single-cell suspension of a population of endocrine progenitor cells (EPs), allowing the EP cells in the single-cell suspension to form 3D structures, and culturing the cells under conditions that allow differentiation into pancreatic monohormonal β cells. This disclosure also relates to islet-like cell aggregates obtainable by said method and their medical uses. Background Technology

[0002] According to the International Diabetes Federation, diabetes is a major global health crisis affecting more than 200-300 million people worldwide. Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells that produce insulin, 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, adult-onset diabetes in adolescents, neonatal diabetes, and islet loss in pancreatitis. Patients with type 1 diabetes receive exogenous insulin injections, which can control blood sugar levels to some extent and significantly reduce the incidence of diabetes, but this is not a cure and is associated with both short-term and long-term complications. Therefore, while insulin therapy has saved countless people with diabetes from premature death, it represents a moderating treatment, not a cure.

[0003] The islets of Langerhans, also known as the pancreatic islets, are regions of the pancreas containing its endocrine cells. These islets are arranged in a density pathway throughout the human pancreas and are important in glucose metabolism. Hormones produced by the islets are secreted directly into the bloodstream by at least five types of cells: α cells that produce glucagon; β cells that produce insulin and amylin; δ cells that produce somatostatin; ε cells that produce ghrelin; and PP cells (γ cells or F cells) that produce pancreatic polypeptides. The cellular structure of the islets is crucial for cell-cell communication and coordinated hormone secretion.

[0004] Patients with diabetes, especially those with type 1 diabetes, can be cured by transplanting insulin-producing pancreatic beta cells. These pancreatic beta cells can be transplanted as islets or islet-like structures. An unlimited supply of human beta cells derived from pluripotent stem cells could provide therapy for millions of patients. Therefore, a cure for diabetes could be achieved by replenishing lost beta cells in patients in need. This approach was demonstrated decades ago in animal models: rats with diabetes caused by the beta-cytotoxin streptozotocin could be cured by injection of syngeneic islets (see Murtaugh's 2007 review). Transplantation of pancreatic progenitor cells derived from human pluripotent stem cells represents a promising therapeutic approach for diabetes. However, reliable and safe strategies are needed to obtain the desired cell masses, and efficient differentiation protocols are required to scale up the treatment to meet therapeutic needs.

[0005] Pluripotent cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (referred to herein as iPS cells or IPCs), have the ability to differentiate into any somatic cell type, and the potential to utilize the therapeutic potential of pluripotent cells has considerable scientific and public interest.

[0006] Numerous studies over the past decade have demonstrated that pancreatic cells, including those expressing the multi-hormone insulin and those expressing the mono-hormone insulin, can be generated from hPSCs (US2019 / 0359943; US 10253298; US2011 / 0280842; Nostro et al., (2015); D'Amour et al., (2006)).

[0007] However, existing methods do not provide the efficiency and / or reproducibility required for the clinical application of pancreatic β-cells. Therefore, alternative methods are needed to more effectively derive the desired cell type from pluripotent cells. Fully realizing the potential of stem cells faces significant challenges, including: (i) developing in vitro differentiation methods that ensure the generation of enriched cell populations of specific desired cell types; (ii) ensuring the identity and function of in vitro generated cells; and (iii) eliminating contaminating non-desired cell types that may disrupt the function of the desired cell type. The presence of undesired cell types in cultures, including, for example, multi-hormonal pancreatic cells, may, for example, pose safety concerns in promising alternative therapies or negatively impact the results of drug screening or disease modeling.

[0008] Therefore, it is evident from the various parts described in this background that providing a differentiation strategy to overcome the aforementioned drawbacks is desirable. Thus, it is desirable to provide a differentiation strategy to efficiently obtain high-quality islet-like cell aggregates (or islet-like structures) in an in vitro manner in a reproducible manner. Summary of the Invention

[0009] The objective of this disclosure is to provide a differentiation strategy for generating in vitro islet-like cell aggregates that overcomes and / or mitigates the aforementioned or other drawbacks of current strategies.

[0010] The objective of this disclosure is to provide an effective and reproducible in vitro differentiation protocol for generating islet-like cell aggregates that exhibit the desired ability to produce insulin in response to glucose stimulation.

[0011] The objective of this disclosure is to provide an effective and reproducible in vitro differentiation protocol that allows for the large-scale generation of islet-like cell aggregates.

[0012] Furthermore, an objective of this disclosure is to provide an in vitro differentiation protocol that allows for the production of cultures of islet-like cell aggregates exhibiting a high percentage of pancreatic mono-hormone β cells and a low percentage of contaminating cell types. The islet-like cell aggregates also exhibit mono-hormone α cells.

[0013] The objective of this disclosure is also to provide islet-like cell aggregates in which a high percentage of cells exhibit functional characteristics of pancreatic β-cell lineage cells.

[0014] A key objective of this disclosure is to provide islet-like cell aggregates exhibiting a high percentage of pancreatic mono-hormone β cells and a low percentage of contaminating cell types. These islet-like cell aggregates also exhibit mono-hormone α cells. Specifically, the desired cells must be viable and healthy. Such islet-like cell aggregates, or cells derived therefrom, can be used for a variety of applications, including therapeutic and scientific / biotechnological applications, such as in vitro drug development and screening. For example, such islet-like cell aggregates or cells can be used for cell transplantation (in other words, cell replacement therapy) to patients in need.

[0015] These and other objectives, which will be apparent to those skilled in the art from this disclosure, are achieved through various aspects of the invention as claimed in the appended claims and as generally disclosed herein.

[0016] The methods disclosed herein for generating islet-like cell aggregates and / or cells obtainable from said aggregates from pluripotent cells involve using specific culture conditions, such as a combination of soluble factors with environmental conditions and time, which guide a very high proportion of pluripotent cells to differentiate into cells with the desired cell fate.

[0017] This disclosure is based on the surprising realization that providing stage 5 endocrine progenitor cells (EPs) (also referred to herein as endocrine precursor cells) in a single-cell suspension and allowing a population of said EP cells to form a 3D structure, and continuing to culture said EP cells in a 3D structure form under 3D culture conditions that allow differentiation into pancreatic monohormonal β cells to provide islet-like cell aggregates, results in unexpectedly high-quality islet-like cell aggregates. These islet-like cell aggregates contain an unexpectedly high percentage of monohormonal β cells, a low percentage of multihormonal cells, a low percentage of monohormonal α cells (α cells) and / or δ cells, and a low percentage of proliferating cells. The inventors have discovered that culturing pancreatic lineage cells on a 2D substrate, such as adherent culture on a 2D substrate, until the developmental stage of endocrine progenitor cells (EPs) is reached, and dissociating these cells, such as into a single-cell suspension, and then allowing these cells to form a 3D structure, results in the generation of high-quality islet-like cell aggregates. Importantly, as shown in the accompanying examples, the timing of the transition from 2D culture conditions to 3D culture conditions is crucial. Unbound by theory, it appears that the timing of the cell transfer influences the development / differentiation of EP cells into high-quality pancreatic islet cells. Importantly, EP cells in single-cell suspensions, allowed to form 3D structures according to the method disclosed herein, have the potential to develop into mature and functional pancreatic β cells.

[0018] Therefore, in a first aspect of the present invention, a method for generating in vitro islet-like cell aggregates is provided, the method comprising the following steps:

[0019] i) Provide a population of endocrine progenitor cells (EPs), such as EP cells characterized by expression of NEUROD1; for example, EP cells characterized by expression of NKX6.1 and NEUROD1;

[0020] ii) Provide a single-cell suspension of the population of said EP cells;

[0021] iii) Allows the population of said EP cells in a single-cell suspension to form a 3D structure;

[0022] iv) Culture the EP cells in a 3D structure under 3D culture conditions that allow differentiation into pancreatic monohormonal β cells to provide islet-like cell aggregates; and

[0023] v) This generates an aggregate of islet-like cells containing mono-hormone β cells.

[0024] The islet-like cell aggregates described therein comprise at least approximately 25% monohormone β cells.

[0025] As used herein, the term "islet-like cell aggregate" or "islet-like aggregate" refers to an aggregate of pancreatic cells that exhibit characteristics of islets in vivo. Specifically, the aggregates exhibit the following desired properties: a high number of monohormonal β cells, a desired number of monohormonal α cells, a low number of multihormonal cells (including a low number of multihormonal β cells and a low number of multihormonal α cells), a low number of non-endocrine cells, and a low number of proliferating cells. Specifically, it is highly desirable that in vitro islet-like cell aggregates mimic the characteristics of in vivo islets, both in terms of the distribution of cell types present herein and their functional properties.

[0026] Those skilled in the art will understand that the percentages described herein refer to the average percentage exhibited by islet-like cell aggregates. Thus, for example, analyzing 100 aggregates, the average number of said cell type is as shown herein. Therefore, step v) can be rephrased as “thus generating a population of islet-like cell aggregates containing mono-hormone β cells, wherein said population comprises islet-like cell aggregates containing at least 25% mono-hormone β cells.” In this context, the characteristics listed below refer to the average percentage in the population of islet-like cell aggregates according to this disclosure.

[0027] Therefore, in one embodiment, the islet-like cell aggregate comprises, or the population comprises, an islet-like cell aggregate containing, at least about 25%, for example, at least about 30%, for example, at least about 35%, for example, at least about 40%, for example, at least about 45%, for example, at least about 50%, for example, at least about 55%, for example, at least about 60%, for example, at least about 65%, for example, at least about 70% monohormone β cells. In one embodiment, the islet-like cell aggregate comprises, or the population comprises, an islet-like cell aggregate containing, at least 25% to 70%, for example, 30% to 70%, for example, 30% to 70% monohormone β cells, for example, 35% to 70%, 35% to 70%, for example, 40% to 70%, for example, 45% to 70%, for example, 45% to 65%, for example, 45% to 60%, for example, 45% to 55%, for example, about 50% monohormone β cells. In one embodiment, the islet-like cell aggregate comprises, or the population comprises, an islet-like cell aggregate containing approximately 35% to 65%, for example 40% to 65%, or for example 40% to 60% monohormone β cells.

[0028] In one embodiment, the islet-like cell aggregate comprises, or the population comprises islet-like cell aggregates containing, approximately 7% to 25%, for example 7% to 20%, 10% to 20%, for example 15% to 20%, for example approximately 20% monohormone alpha cells.

[0029] In one embodiment, the islet-like cell aggregate comprises, or the population comprises islet-like cell aggregates containing, up to about 10%, for example, up to about 7%, for example, up to about 6%, for example, 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 0.5%, for example, up to about 0.3%, for example, up to about 0.1% of multi-hormone α cells.

[0030] In one embodiment, the islet-like cell aggregate comprises, or the population comprises islet-like cell aggregates containing, up to about 10%, for example, up to about 7%, for example, up to about 6%, for example, 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 0.5%, for example, up to about 0.3%, for example, up to about 0.1% of multi-hormone β cells.

[0031] In one embodiment, the islet-like cell aggregate comprises, or the population comprises islet-like cell aggregates containing, less than 5%, for example less than 4%, for example less than 3%, for example less than 1%, for example less than 1% of delta cells.

[0032] In one embodiment, the islet-like cell aggregate comprises, or the population comprises, an islet-like cell aggregate containing: 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% of proliferating cells, such as proliferating cells expressing Ki-67.

[0033] In one embodiment, the islet-like cell aggregate comprises, or the population comprises, an islet-like cell aggregate containing: at least 40%, for example, at least 50%, monohormone β cells; approximately 15% to 20%, for example, approximately 20%, monohormone α cells; and less than approximately 2%, for example, less than approximately 1%, proliferating cells.

[0034] In one implementation, the composition of islet-like cell aggregates is scored, or in other words, studied, on days 38–42 or later in culture. For example, on days 38, 39, 40, 41, or 42. In other words, the composition of islet-like cell aggregates is scored, or in other words, studied, at the end of phase 6.

[0035] The islets of Langerhans, also known as the pancreatic islets, are regions of the pancreas containing endocrine (hormone-producing) cells. Approximately one million islets are distributed throughout the pancreas of a healthy adult, with each islet having an average diameter of about 0.2 mm. Each islet is separated from the surrounding pancreatic tissue by a thin fibrous connective tissue membrane, which is continuous with the fibrous connective tissue interwoven throughout the rest of the pancreas. Hormones produced by the islets are secreted directly into the bloodstream by at least five types of cells. The types of endocrine cells in the islets include: α cells that produce glucagon, β cells that produce insulin and amylin, PP cells (γ cells or F cells) that produce pancreatic polypeptides, δ cells that produce somatostatin, and ε cells that 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 nerve cells. Technicians are familiar with the composition and cellular structure of the pancreatic islets.

[0036] Endocrine progenitor cells (EPs) can be scored by the expression of a specific marker, NEUROD1, during development along the pancreatic endocrine lineage, as cells enter the developmental EP cell stage (also known as stage 5, see [link]). Figure 1A NEUROD1 is upregulated. Early developmental stage 4 cells (pancreatic progenitor 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 progenitor cells, such as double-positive for NKX6.1 and NEUROD1; or double-positive for NKX6.1 and NGN3. Other markers and combinations thereof can be used to identify EP cells as explained below. Pancreatic endocrine progenitor cells express at least one, two, three, or all four of the following markers: PDX1, NKX6.1, NGN3, and NEUROD1.

[0037] Therefore, in one embodiment of the method disclosed herein, the EP cell population provided in step i) is characterized by expression of NEUROD1 and NKX6.1. In another embodiment, the EP cell population provided in step i) is characterized by expression of NKX6.1 and NGN3 or by expression of NEUROD1 and NGN3. Alternatively, EP cells may also be identified by the expression of: PDX1, NKX6.1 and NGN3; or PDX1, NKX6.1 and NEUROD1; or PDX1, NKX6.1, NGN3 and NEUROD1.

[0038] Those skilled in the art should understand that step ii) of providing a single-cell suspension of a population of EP cells involves dissociating the EP cells from an adherent culture on a 2D substrate into single cells. Such dissociation may involve the use of dissociation agents, such as naturally occurring enzymes, milder non-enzymatic alternatives, or may be performed by chelating calcium to prevent cadherin adhesion, thereby releasing the cells from the surface and from each other. Dissociation can be performed mechanically. Non-limiting examples of dissociation agents include trypsin, collagenase, displases, and dissociation agents such as… Accumax TM And ACS-3010.

[0039] In one embodiment, step ii) of providing a single-cell suspension of a population of EP cells involves dissociating the EP cells from an adherent culture on a 2D substrate into single cells. In another embodiment, step ii) of providing a single-cell suspension of a population of EP cells involves dissociating the EP cells by an enzymatic means, such as using a solution containing enzymes (e.g., proteolytic enzymes and collagenases). For example, such a solution could be... Technicians know the appropriate methods for dissociating EP cells and providing single-cell suspensions for populations of EP cells.

[0040] In one implementation, steps ii) and iii) are performed before subjecting the cells to conditions that allow differentiation into pancreatic mono-hormone β cells, for example, before culturing the cells in a medium that allows differentiation into pancreatic mono-hormone β cells.

[0041] In one embodiment, the dissociation is performed before the cells are subjected to conditions that allow differentiation into pancreatic monohormone β cells, for example, before culturing the cells in a medium that allows differentiation into pancreatic monohormone β cells. In one embodiment, the dissociation is performed at most 96 hours, for example, at most 72 hours, for example, at most 48 hours, or for example, at most 24 hours after the conditions allowing differentiation into endocrine progenitor cells are changed to those allowing differentiation into pancreatic monohormone β cells, as described in the appended embodiments. For example, at most 96 hours, for example, at most 72 hours, for example, at most 48 hours, or for example, at most 24 hours after changing from S5 medium to S6 medium as described in the appended embodiments. In one embodiment, the dissociation is performed 24-48 hours after the conditions allowing differentiation into endocrine progenitor cells are changed to those allowing differentiation into pancreatic monohormone β cells.

[0042] Therefore, in one implementation, steps i), ii) and iii) are performed under conditions that allow differentiation into endocrine progenitor cells, while step iv) is performed under conditions that allow differentiation into pancreatic monohormonal β cells.

[0043] In step iii), EP cells in a single-cell suspension are allowed to form 3D structures. In one embodiment, the 3D culture conditions allow cells to self-aggregate. The aggregation can be forced or induced aggregation, but it can also be spontaneous aggregation.

[0044] In one embodiment, step iii) is performed in the presence of a ROCK inhibitor. The ROCK inhibitor may be H1152 or any analogue or agonist thereof. In one embodiment, the concentration of H1152 ranges from >0 μM to 10 μM. As shown in the embodiments of the invention, and without being bound by theory, the inventors believe that H1152 can promote the survival of S5 EP cells as single cells in suspension.

[0045] Therefore, in one embodiment of the method disclosed herein, the ROCK inhibitor is present in the culture medium for approximately 24 hours during phase iii).

[0046] Unbound by theory, it is envisioned that self-aggregation allows for the selective enrichment of endocrine progenitor cells. As shown in the accompanying embodiments, the formation of 3D structures (also referred to as aggregation) leads to selective enrichment and results in an increased generation of pancreatic mono-hormone β cells in the culture. As explained above, the aggregates exhibit the following desired characteristics: a high number of mono-hormone β cells, a desired number of mono-hormone α cells, a low number of multi-hormone cells (including a low number of multi-hormone β cells and a low number of multi-hormone α cells), and a low number of proliferating cells. In one embodiment, the pancreatic mono-hormone β cells are generated as part of a cell aggregate. In one embodiment, the aggregate comprises mono-hormone β cells. In one embodiment, the aggregate further comprises pancreatic mono-hormone α cells. In one embodiment, the aggregate further comprises pancreatic mono-hormone δ cells, for example, less than 3%, less than 2%, or less than 1% of δ cells. Those skilled in the art will understand that the percentages described herein should be interpreted as relating to the average percentage exhibited by the islet-like cell aggregates. The percentages can be evaluated for the islet-like cell aggregates themselves or for the populations containing islet-like cell aggregates. For clarity, the cell aggregates are referred to herein as islet-like cell aggregates and can be obtained according to the methods defined herein.

[0047] As used herein, the term “differentiation” refers to the process by which unspecialized (“unoriented”) or less specialized (“less oriented”) cells acquire characteristics of specialized cells (“more oriented”) (such as, for example, pancreatic cells). Differentiated cells are those that occupy a more specialized (“oriented”) position in the cell lineage.

[0048] As used in this article, the term "direction" in the context of differentiation refers to a process in which cells have progressed to a point in the differentiation pathway where, under normal circumstances, they would continue to differentiate into a specific cell type or subset of cell types, and under normal circumstances, they cannot differentiate into different cell types or revert to a less differentiated cell type.

[0049] As used in this article, the cellular term "lineage" refers to the heritability of a cell, that is, from which cells it originates and what cells it can produce. Cell lineage places a cell within a genetic program for development and / or differentiation, either in vivo or in vitro.

[0050] As used herein, the term “lineage-specific marker” refers to a phenotype-specific feature of cells of a target lineage and can be used to assess the differentiation of undirected cells into a target lineage.

[0051] Those skilled in the art are familiar with the different developmental stages of pancreatic endocrine lineages (e.g., pancreatic β-cell lineages).

[0052] As used herein, the term "pancreatic β-cell lineage" refers to a genetic program of development and / or differentiation, either in vivo or in vitro, that ultimately results in cells exhibiting characteristic properties of pancreatic mono-hormone β-cells, such as insulin production and expression of at least one of PDX1, NKX6.1, and NEUROD1. Those skilled in the art will understand that cells of a pancreatic β-cell lineage can be any cell at an early developmental stage of said cell.

[0053] As used herein, the term "precursor" refers to pancreatic lineage cells, such as precursors of pancreatic β cells, and means any cell that can differentiate into pancreatic β cells when cultured under conditions suitable and / or allowing the precursor cells to differentiate into pancreatic lineage cells (such as precursors of pancreatic β cells), including, for example, pluripotent stem cells, well-defined endoderm cells, primitive intestinal cells, hindbrain cells, pancreatic progenitor cells, or endocrine progenitor cells.

[0054] Differentiation of cells along the pancreatic β-cell lineage involves differentiation from less oriented to more oriented cell types. In short, the development of insulin-producing pancreatic β-cells represents the culmination of a complex developmental program involving the following in vivo steps: the acquisition of pancreatic characteristics by the foregut cells, the adoption of endocrine fate by the expanded pancreatic primordia, and the becoming capable of generating β-cells by a subset of these precursors. Factors (e.g., transcription factors) that have been shown to be important in the development of pancreatic β-cell lineage cells include, among others, PDX1 (pancreatic and duodenal homeobox 1), PTF1A (pancreatic-specific transcription factor 1a), NGN3 (neuropoietin 3), NEUROD1 (neurogenic differentiation 1), and NKX6.1 (homeobox protein NKX-6.1). This list of factors should be considered non-limiting, and additional factors will be discussed below.

[0055] Furthermore, cell development along the pancreatic β-cell lineage requires signaling from extrinsic factors such as, but not limited to, transforming growth factor-β (TGFβ) and retinoic acid (RA). Recent studies have shown that TGFβ signaling induces the formation of the endoderm in mouse and human embryonic stem (ES) cells, while RA treatment promotes PDX1 expression and pancreatic specialization in ES-derived endoderm. Those skilled in the art understand that in vitro differentiation of pancreatic β-cell lineage cells requires the addition of extrinsic factors to the cell growth medium at appropriate stages of the differentiation process and at suitable / permissible concentrations, which in principle mimic the in vivo development of said cells.

[0056] Therefore, those skilled in the art recognize that, for the listed cell types, "conditions that allow differentiation" refers to conditions that allow cells to exhibit the characteristics of the cell type, and may include the combination of cell culture media, the presence and / or absence of extrinsic factors and their timing.

[0057] The following is a brief summary of the various developmental stages of the pancreas. Technicians will understand that this development can be described in stages. Each developmental stage 0-6 is characterized by the expression of a set of factors (often referred to as biomarkers).

[0058] Those skilled in the art understand that the process of differentiating pluripotent stem cells in vitro into functional pancreatic endocrine cells (e.g., monohormonal pancreatic β cells) can, in some respects, be viewed as progressing through six consecutive stages, as illustrated in the schematic diagram shown in Figure 1, which correspond to in vivo developmental stages. Those skilled in the art are very familiar with these in vivo developmental stages, and these stages are considered to fall within the general knowledge of the art.

[0059] In this progressive development, stage 0 refers to undifferentiated pluripotent cells, such as hES cells or iPS cells; stage 1 refers to cells expressing characteristic markers of defined endoderm (DE) cells; stage 2 refers to cells expressing characteristic markers of primitive intestinal cells (PGT); stage 3 refers to cells expressing characteristic markers of hindbrain cells (PF); stage 4 refers to cells expressing characteristic markers of pancreatic progenitor cells (PP); stage 5 refers to cells expressing characteristic markers of pancreatic endocrine progenitor cells (EP); and stage 6 refers to cells expressing characteristic markers of endocrine islet cells, such as pancreatic β cells. Stage 6 cells, as defined herein, can form in vitro islet-like cell aggregates (in vitro cell aggregates), which mimic the islets found in vivo.

[0060] Technicians understand that not all cells in a given population progress through these stages at the same rate; that is, some cells may progress less or more along the differentiation pathway than most cells in the population.

[0061] The following is a non-exhaustive description of the characteristics associated with different stages of cell culture as described above. Those skilled in the art will understand that the different stages of in vitro culture correspond to developmental stages in vivo. Those skilled in the art should understand that by selectively choosing which protein expression to monitor, one can trace the developmental progress along the pancreatic endocrine lineage (e.g., the pancreatic β-cell lineage). When needed, the expression of more than one protein characteristic of a developmental stage can be evaluated. As cells progress during development, the expression of certain proteins is upregulated and downregulated, making these proteins suitable as markers for different developmental stages.

[0062] As used herein, the term "determined endoderm cell" refers to a cell that exhibits the characteristics of cells produced by the ectoderm during gastrulation and forming the gastrointestinal tract and its derivatives. Determined endoderm cells express at least one, two, or all three of the following markers: CXCR4, FOXA2, and SOX17. Therefore, determined endoderm cells can be identified by the expression of at least one, two, or all three of the following markers: CXCR4, FOXA2, and SOX17. Specifically, determined endoderm cells can be identified by the expression of SOX17.

[0063] As used herein, the term "primitive intestinal cell" refers to cells derived from the fixed endoderm that give rise to all endoderm organs, such as the lung, liver, pancreas, stomach, and intestine. Primitive intestinal cells express at least one or both of the following markers: HNF1β and HNF4α. Therefore, primitive intestinal cells can be identified by the expression of HNF1β, HNF4α, or both HNF1β and HNF4α.

[0064] As used herein, the term "hindbeg cell" refers to the endoderm cells that give rise to parts of the stomach, liver, pancreas, gallbladder, and duodenum. Hindbeg cells express PDX1 or both PDX1 and HNF6. Therefore, hindbeg cells can be identified by the expression of PDX1, HNF6, or both PDX1 and HNF6.

[0065] As used herein, the term "pancreatic progenitor cell" refers to a cell that expresses 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. Specifically, pancreatic progenitor cells co-express PDX1 and NKX6.1.

[0066] 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.

[0067] As used herein, the term "endocrine progenitor cell" refers to pancreatic endoderm cells capable of transforming into pancreatic hormone-expressing cells. Pancreatic endocrine progenitor cells express at least one, two, three, or all four of the following markers: PDX1, NKX6.1, NGN3, and NEUROD1. Endocrine progenitor cells can be identified by the expression of NXK6.1 and NEUROD1. Specifically, endocrine progenitor cells may differ from pancreatic progenitor cells in that they express NEUROD1 and NGN3, while pancreatic progenitor cells do not express these two markers. Endocrine progenitor cells can also be identified by the expression of: PDX1, NKX6.1, and NGN3; or PDX1, NKX6.1, and NEUROD1; or PDX1, NKX6.1, NGN3, and NEUROD1.

[0068] As used herein, the term "islet cell" refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, ghrelin, and pancreatic polypeptide. In addition to these hormones, characteristic markers of pancreatic endocrine cells include one, two, or all three of PDX1, NKX6.1, and NEUROD1. NEUROD1 is expressed in most (e.g., substantially all) endocrine islet cells, while PDX1 and NKX6.1 are specific to stage 6 pancreatic β cells. As used herein, the term "islet-like cell aggregate" or "islet-like aggregate" refers to an aggregate of pancreatic cells that exhibits the characteristics of islets in vivo. Specifically, said aggregates exhibit the properties discussed above.

[0069] Specifically, as used herein, the term "pancreatic β-cell" or "monohormonal pancreatic β-cell" refers to a cell that expresses insulin but not glucagon or somatostatin. The terms "pancreatic β-cell" and "monohormonal pancreatic β-cell" are used interchangeably herein. Therefore, pancreatic monohormonal β-cells can be identified by the expression of insulin and the absence of glucagon and / or somatostatin expression.

[0070] Pancreatic beta cells are monohormone cells; in other words, they express only one hormone, namely insulin. Conversely, pancreatic cells in a cellular context express more than one hormone, such as at least two of insulin, glucagon, and somatostatin.

[0071] For example, as shown in Figure 1, biomarker expression may follow the progression of the aforementioned developmental stages. For instance, progression from stage 0 to stage 1 is associated with downregulation of OCT4, NANOOG, and SOX2 expression, and upregulation of SOX17, FOXA, and CXCR4 expression. Progression from stage 1 to stage 2 is associated with upregulation of HNF1β and HNF4α expression. Progression from stage 2 to stage 3 is associated with upregulation of PDX1 and HNF6 expression.

[0072] Progression from stage 3 to stage 4 is associated with the maintenance of PDX1 and HNF6 expression and the upregulation of NKX6.1, PTF1A, and SOX9 expression. Progression from stage 4 to stage 5 is associated with the downregulation of PTF1A and SOX9 expression, the maintenance of PDX1 and NKX6.1 expression, and the upregulation of NGN3 and NEUROD1 expression. Progression from stage 5 to stage 6 in pancreatic β cells is associated with the downregulation of NGN3 expression, the maintenance of PDX1, NKX6.1, and NEUROD1 expression, and the upregulation of insulin and C-peptide expression. Therefore, for example, the upregulation of NKX6.1 in PDX1+ cells marks progression to stage 4.

[0073] Those skilled in the art should understand that the developmental process of pancreatic lineage cells can be further divided into additional stages, for example, based on the expression levels of biomarkers. For instance, in Verhoeff et al.'s review article (Stem Cell Reviews and Reports (2022):18,2683-2698), seven stages of differentiation into islet-like clusters were summarized, where stages 1-4 and 7 correspond to stages 1-4 and 6 as used in this application. According to Verhoeff, stage 5 is characterized by the expression of NKX6.1 and low expression of NGN3, while according to Verhoeff, stage 6 is characterized by high expression of NGN3 and the expression of endocrine hormones. Stage 5 as defined in this disclosure corresponds to Verhoeff's stages 5 and 6.

[0074] The generated cells are identified or characterized by phenotypic features, morphological features, and / or the expression of cellular markers that are readily understood by those skilled 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 the target cells.

[0075] Non-limiting examples of β-cell lineage markers discussed in this disclosure include: CXCR4, FOXA2, SOX17, HNF1β, HNF4α, PDX1, HNF6, PDX1, PTF1A, NKX6.1, SOX9, NGN3, NEUROD1, and insulin. As mentioned above, combinations of these markers are characteristic of different developmental stages along the β-cell lineage. As discussed above, those skilled in the art will understand that markers can be selected such that the expression or lack of expression of a combination of markers allows differentiation of cells at different developmental stages along the pancreatic endocrine lineage (see Figure 1). The inventors have used the expression of different markers to differentiate cells at different developmental stages, as illustrated in the appended examples. As used herein, when referring to cells within a cell population, the term "characterized by expression of…" should be interpreted as relating to the expression of a given marker or group of markers. Conversely, when referring to cells within a cell population, the terms "characterized by the lack of expression of…" or "lacking expression of…" or "not expressing…" should be interpreted as relating to the absence of expression of a given marker or group of markers. Technicians are very familiar with using biomarker expression as a way to distinguish cells with different characteristics (such as cells at different developmental stages of the pancreatic β-cell lineage).

[0076] Those skilled in the art should understand that when analyzing cell populations using methods limited in the number of biomarkers that can be scored simultaneously, such as due to limitations of the method itself or the reagents used, a subset of biomarkers that allows differentiation between a first and a second cell population can be selected. For example, a cell population of pancreatic progenitor cells characterized by expression of PDX1 and NKX6.1 can be distinguished from a population of endocrine progenitor cells characterized by expression of PDX1, NKX6.1, NEUROD1, and NGN3. For instance, pancreatic progenitor cells may be positive for either PDX1 or NKX6.1 but negative for either NGN3 or NEUROD1. EP cells can be scored as double-positive for NKX6.1 and at least one biomarker not expressed by pancreatic progenitor cells, such as double-positive for NKX6.1 and NEUROD1; or double-positive for NKX6.1 and NGN3. This principle is applied in the Examples section to differentiate cells with different characteristics. Importantly, if the cells are scored with the other biomarkers listed above, the cells will exhibit the full biomarker profile of the specific population. The fact that only a subset of biomarkers is used in an experimental setup should never be interpreted as a lack of expression representative of the remaining characteristic biomarkers. Technicians will understand that biomarker expression can be evaluated at the nucleotide level, such as the mRNA level, or the protein level. Well-known methods for evaluating biomarker expression include, but are not limited to, immunohistochemistry, in situ hybridization, FACS, RNA sequencing, the use of arrays (e.g., microarrays), and quantitative PCR. Technicians are familiar with these and other suitable methods.

[0077] In this context, differential expression refers to an increased level of a positive marker and a decreased level of a negative marker compared to undifferentiated cells or cells at different developmental stages. The detectable levels of the marker nucleic acid or peptide in the target cells are sufficiently high or low compared to other cells, allowing the target cells to be identified and distinguished from other cells using any of a variety of methods known in the art.

[0078] As used herein, a cell is "positive for" or "positive" for a given biomarker when it is adequately detected in the cell (in other words, expressed by the cell). Therefore, a cell characterized by the expression of a given biomarker is positive for that biomarker. Conversely, a cell is "negative for" or "negative" for a given biomarker when it is not adequately detected in the cell. Therefore, a cell characterized by the lack of expression of a given biomarker is negative for that biomarker. The use of "+" or "-" signs associated with biomarkers herein is intended to be understood as indicating positivity or negativity for the biomarker (e.g., NKX6.1). + The cells were positive for the marker NKX6.1.

[0079] Using any in vitro differentiation method, the ability to obtain a population containing cells exhibiting the same or similar properties at a given time point is crucial, as cells at different developmental, maturation, or functional stages may respond differently to external and internal signals. To clarify, factor X may lead to the specialization of cell types A and B during development, while treatment with the same factor X may result in selective cell death of mature cell type A. Therefore, it is important not only to understand the downstream functional consequences of subjecting cells to a given factor, but also to subject the cells to the factor during the time window during which the cells are able to respond to the factor in the desired manner. Thus, those skilled in the art should understand the importance of obtaining a synchronized cell population to ensure the desired response of the cell population as a whole. For example, it is desirable that the majority of cells in the culture are progenitor cells at a given time point.

[0080] Tissue engineering, stem cell, and molecular biology research primarily involves cell culture on flat plastic or glass dishes / cell culture plates. These cultures are considered adherent cultures. This technique is called two-dimensional (2D) cell culture. As used herein, the term "2D" in the context of cell culture refers to culture on flat cell culture plates, where cells adhere directly or indirectly (e.g., via a cell culture substrate) to said culture plate. The plate may be coated with a cell culture substrate. Cell culture substrates fall into two main categories: naturally occurring and synthetic. The most commonly used natural substrates are collagen, fibronectin, and laminin, which form part of the extracellular matrix. Cells interact with these matrix components through cell surface receptors (e.g., integrins). Receptors bind to domains of collagen, fibronectin, and laminin and trigger intracellular signaling pathways that promote the formation of adhesion complexes and may also trigger cell proliferation or differentiation. The most common synthetic substrates for 2D culture include polylysine, a lysine polymer containing positively charged amino groups. The choice of substrate has a significant impact on how cells grow and attach, and is therefore an important parameter to consider.

[0081] 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.

[0082] In one embodiment of the method disclosed herein, cells are cultured on a 2D substrate. In one embodiment, the cells adhere to the 2D substrate. The 2D substrate may comprise one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized filaments (FN filaments), and matrigol. TM For example, it may contain one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, collagen, gelatin, and matrix gel. TM For example, it may contain one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, and matrix gel. TM For example, it may contain one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, and matrix gel. TM Specifically, the fragment may contain a functional domain of the protein. Therefore, those skilled in the art will understand that the fragment may correspond to or contain a functional domain, for example, containing a functional domain and further containing additional N-terminal and / or C-terminal amino acids. Matrigel TMDerived from mouse Engelbreth-Holm-Swarm tumors and containing many unknown components. Laminin is known to be a major component of matrix gel. Avoiding the use of any animal-derived products, in addition to using established culture media, may be beneficial, especially for cell cultures intended for therapeutic purposes, to obtain well-defined and reproducible products and avoid any potential patient safety issues. As used herein, functionalized filaments refer to recombinant fusion proteins containing filament proteins and cell-binding motifs. In some embodiments, the functionalized filament comprises a cell-binding motif selected from the following: 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), TGRGDSPA (SEQ ID NO:10), CTGRGDSPAC (SEQ ID NO:11), and C1X1X2RGDX3X4X5C2 (SEQ ID NO:11). IDNO:12), preferably selected from C1X1X2RGDX3X4X5C2, GRKRK, IKVAV, RGD and CTGRGDSPAC, wherein X1, X2, X3, X4 and X5 are each independently selected from natural amino acid residues other than cysteine; and C1 and C2 are linked by disulfide bonds.

[0083] In one particular embodiment, the functionalized filament comprises a spider silk protein fragment and a cell-binding motif, the cell-binding motif being defined as comprising the amino acid sequence C1X1X2RGDX3X4X5C2 (SEQ ID NO:12), wherein X1, X2, X3, X4 and X5 are each independently selected from natural amino acid residues other than cysteine; and C1 and C2 are linked by a disulfide bond.

[0084] Functionalized fibers have been described in WO 2016 / 207281 and WO 2017 / 137611, and these disclosures are incorporated herein in their entirety. Specifically, the functionalized fibers can be recombinant polypeptides comprising the following amino acid sequence:

[0085] C1X1X2RGDX3X4X5C2(SEQ ID NO:12), where

[0086] X1 is either S or T;

[0087] X2 is G, A, or V;

[0088] X3 is either S or T;

[0089] X4 is G, A, V, or P; and

[0090] X5 is G, A, or V; and

[0091] C1 and C2 are linked by disulfide bonds;

[0092] Furthermore, the spider silk protein fragment contains protein portions REP and CT, wherein REP is a repeating fragment with 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 from 2 to 10;

[0093] Each individual A segment is an amino acid sequence having 8 to 18 amino acid residues, wherein amino acid residues 0 to 3 are not Ala, and the remaining amino acid residues are Ala; each individual G segment is an amino acid sequence having 12 to 30 amino acid residues, wherein at least 40% of amino acid residues are Gly; each individual L segment is a linker amino acid sequence having 0 to 30 amino acid residues; and CT is a fragment having 70 to 120 amino acid residues, having at least 70% identity with SRLSSPSAVSRVSSAVSSLVSNGQVNMAALPNII SNISSSVSASAPGASGCEVIVQALLEVITALVQIVSSSSVGYINPSAVNQITNVV ANAMAQVMG (SEQ ID NO:13). In one embodiment, the CT fragment has at least 70%, such as at least 80%, such as at least 85%, preferably at least 90%, such as at least 95%, identity with SEQ ID NO:13. In some embodiments, the spider silk protein fragment has at least 70%, for example, at least 80%, for example, at least 85%, preferably at least 90%, for example, at least 95% identity with amino acid residues 18-277 of SEQ ID NO:16 or SEQ ID NO:14. In a particular embodiment, the cell-binding motif comprises the amino acid sequence CTGRGDSPAC (SEQ ID NO:11).

[0094] In a particular embodiment, the functionalized filament comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14 and SEQ ID NO:15. In some embodiments, the functionalized filament has at least 70%, for example, at least 80%, for example, at least 85%, preferably at least 90%, for example, at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NO:14 and SEQ ID NO:15.

[0095] Therefore, in one embodiment, the 2D substrate does not contain any animal-derived components. In one embodiment, the 2D substrate is not a matrix adhesive. TM In other words, the components of the 2D substrate can be components generated using recombination technology.

[0096] Therefore, in one embodiment, the 2D substrate may comprise or consist of laminin (LN) and fragments thereof, such as recombinant laminin (LN) and fragments thereof.

[0097] Laminins are high-molecular-weight proteins of the extracellular matrix. They are major components of the basement membrane, which forms the basis of the protein network in most cells and organs. Laminins are important and biologically active parts of the basement membrane, influencing cell differentiation, migration, and adhesion. Therefore, it is important to select laminins for use as cell culture substrates to provide cells with an appropriate chemically and mechanically sensitive microenvironment, thereby providing optimal culture conditions.

[0098] Each laminin isoform consists of three interlocking helical chains—α, β, and γ chains—which exist in five, four, and three genetically distinct variants, respectively. Laminin isoforms are named according to their chain composition. For example, the combination of the α5, β2, and γ1 chains forms laminin 5-2-1 (LN-521). The trimeric protein forms a cross-shaped structure that can bind to other extracellular matrix molecules and various cell membrane receptors.

[0099] Therefore, the selection of laminin or its fragments used as 2D substrates is important and will be influenced by the cell type being cultured. Furthermore, differences between cell lines (e.g., different ES or iPS cell lines or primary cells) may affect the selection of the optimal 2D substrate. Full-length laminins can be used as cell culture substrates; however, fragments can also be used for cell culture. Different domains of laminins have been identified, mediating various activities such as cell attachment and effects on cell proliferation, differentiation, and motility.

[0100] In one embodiment of the method disclosed herein, 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 or the group consisting of LN-511 and its fragments.

[0101] In one embodiment, the laminin 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 511; for example, the laminin and its fragments are LN-521 or for example, the laminin and its fragments are LN-511.

[0102] Laminin E8 fragments are known to be truncated proteins composed of the C-terminal regions of the α, β, and γ chains. These laminin fragments contain an active integrin-binding site comprising the laminin globular 1–3 domain of the α chain and a glutamate residue in the C-terminal tail of the γ chain, but lack other activities, such as heparin / heparan sulfate binding activity associated with full-length laminin. The E8 fragment represents a functionally minimal form that retains the full ability to bind to α6β1 integrin. In one embodiment, one or more of these fragments are E8 fragments.

[0103] Therefore, in one embodiment, the laminin and fragments comprise the E8 fragment of laminin, for example, an E8 fragment selected from the group consisting of: the E8 fragment of LN-511, the E8 fragment of LN-521, the E8 fragment of LN-332, the E8 fragment of LN-421, the E8 fragment of LN-121, and the E8 fragment of LN-111; for example, the group consisting of: the E8 fragment of LN-511, LN- E8 fragments of 521, LN-332, 421, and LN-121; for example, the group consisting of: E8 fragments of LN-511, LN-521, and LN-332; for example, the group consisting of: E8 fragments of LN-511 and LN-521; for example, the E8 fragment of LN-511 or LN-521.

[0104] It should be understood that the EP cells provided in step i) of this method can be generated using various differentiation protocols (e.g., those previously described). It should be understood that providing a large number of EP cells in step i) is beneficial. Therefore, it may be advantageous to generate the EP cells using the methods disclosed herein. For clarity, the usefulness of the methods described in steps i)-v) herein is by no means limited to EP cells generated by the methods disclosed herein.

[0105] 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 expressing NEUROD1. For example, the EP cells provided in step i) can be obtained in step c+1) disclosed herein. Thus, the population of EP cells in step i) can be a portion of the total cell population that includes a portion of cells that do not express characteristic markers of EP cells. In one embodiment disclosed herein, the population of EP cells in step i) is a population containing EP cells. In one embodiment disclosed herein, a method is provided in which, in step i), a population of EP cells is provided, wherein 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, such as EP cells characterized by expressing NKX6.1 and at least NEUROD1, such as endocrine progenitor cells characterized by expressing NKX6.1 and at least NEUROD1. In one embodiment as disclosed herein, in step i), the population containing EP cells comprises more than about 30%, for example more than about 40%, for example more than about 45%, for example more than about 50% of EP cells, such as EP cells characterized by expression of NKX6.1 and at least NEUROD1, such as endocrine progenitor cells characterized by expression of NKX6.1 and at least NEUROD1. As discussed and illustrated above, other combinations of two or more of the markers PDX1, NKX6.1, NEUROD1, and NGN3 can be used as characteristics of EP cells.

[0106] In one embodiment of the method disclosed herein, step ii) is performed when more than 15%, for example, more than 20%, for example, more than 25%, for example, more than 30%, for example, more than 35%, for example, more than 40%, for example, more than 45%, for example, more than 50% of the total cell population are EP cells characterized by expressing NEUROD1 or NGN3. In one embodiment, the EP cells are characterized by expressing NEUROD1 and NGN3. In one embodiment, step ii) is performed when more than 15%, for example, more than 20%, for example, more than 25%, for example, more than 30%, for example, more than 35%, for example, more than 40%, for example, more than 45%, for example, more than 50% of the total cell population are EP cells characterized by expressing NKX6.1 and NEUROD1. In one embodiment, step ii) is performed when the cells do not exhibit hormone expression, for example, do not exhibit expression. In one embodiment, step ii) is performed approximately when the cells do not exhibit hormone expression, for example, do not exhibit expression of insulin and / or glucagon. In this context, "not exhibiting expression" should be understood as not exhibiting any detectable expression, such as expression that can be detected by the methods or means disclosed in this embodiment section. In one embodiment, step ii) is performed before the cells exhibit hormone expression (e.g., insulin and / or glucagon expression).

[0107] In one embodiment, a method for generating in vitro islet-like cell aggregates as defined herein is provided, wherein step iv) of culturing a population of said EP cells in a 3D structural form under 3D culture conditions that allow differentiation into pancreatic monohormone β cells to provide islet-like cell aggregates includes culturing in a medium suitable for culturing endocrine progenitor cells under conditions that allow differentiation into monohormone β cells. Non-limiting examples of the culture medium for step 6 (S6) are defined in this embodiment. Those skilled in the art will understand that other suitable culture media may be used. The culture medium in step iv) may be supplemented with other factors specified herein.

[0108] Trolox (6-hydroxy-2,5,7,8-tetramethylchromo-2-carboxylic acid) is a water-soluble analog of vitamin E with potent antioxidant activity. Trolox is also a potent inhibitor of membrane damage. In one embodiment, the culture medium in step iv) contains approximately 5 μM to 15 μM of Trolox, for example, approximately 10 μM of Trolox. As those skilled in the art will understand, derivatives or agonists thereof may be used instead of Trolox.

[0109] N-acetyl-L-cysteine ​​is a cell culture component, such as the intestinal basal medium used for culturing mouse intestinal stem cells, and can also be used as a component of expansion media. In one embodiment, the medium in step iv) contains approximately 0.5 mM to 3 mM of N-acetyl-L-cysteine, for example, approximately 1 mM of N-acetyl-L-cysteine. As those skilled in the art will understand, derivatives or agonists thereof can be used instead of N-acetyl-L-cysteine.

[0110] H1152 is a Rho kinase inhibitor and is a cell-permeable, highly specific, reversible, potent, and ATP-competitive Rho-associated kinase (ROCK) inhibitor. As understood by those skilled in the art, its derivatives or agonists can be used in place of H1152.

[0111] GC-1 is a thyroid hormone receptor (TR) agonist and is more effective than thyroid hormone T3. Thyroid hormone T3 is important for β-cell development and will be discussed in more detail below. In one embodiment, the culture medium in step iv) contains approximately 0.5 μM to 3 μM of GC-1, for example, approximately 1 μM of GC-1. As those skilled in the art will understand, derivatives or agonists thereof can be used instead of GC-1.

[0112] In one embodiment, step iv) of the method disclosed herein includes culturing an EP cell population in a medium containing H1152, GC-1, Trolox, and N-acetyl-L-cysteine. In another embodiment, step iv) of the method disclosed herein includes culturing an EP cell population in a medium containing H1152, GC-1, Trolox, and N-acetyl-L-cysteine ​​for approximately 3 weeks, followed by culturing in a medium containing approximately GC-1, Trolox, and N-acetyl-L-cysteine ​​but not H1152.

[0113] In one embodiment, step iv) of the method disclosed herein includes culturing an EP cell population in a medium containing approximately 10 μM H1152, approximately 1 μM GC-1, approximately 10 μM Trolox, and approximately 1 mM N-acetyl-L-cysteine. In one embodiment, step iv) of the method disclosed herein includes culturing an EP cell population for approximately 3 weeks in a medium containing approximately 10 μM H1152, approximately 1 μM GC-1, approximately 10 μM Trolox, and approximately 1 mM N-acetyl-L-cysteine, followed by culturing in a medium containing approximately 1 μM GC-1, approximately 10 μM Trolox, and approximately 1 mM N-acetyl-L-cysteine ​​but without H1152.

[0114] In one implementation, the culture in step iv) is on a shaker, such as a fixed-track shaker.

[0115] As discussed in the context of this method, 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 with β cells and exhibit low percentages of other monohormonal cells, low percentages of multihormonal cells and low percentages of proliferating cells.

[0116] In one embodiment of the method, cells are not transferred from a 2D substrate culture to a 3D substrate culture until they exhibit expression of characteristic markers of endocrine progenitor cells. Endocrine progenitor cells are characterized by expression of at least one of PDX1, NKX6.1, and NEUROD1 and NGN3, as discussed above. As mentioned above, EP cells do not express pancreatic hormones. In one embodiment, step ii) is performed approximately 24 hours after the EP cells begin to express NEUROD1 and / or NGN3, for example, 24 hours after 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 culture begin to express NEUROD1 and / or NGN3. In one embodiment, step ii) is performed within 6 days after the EP cells begin to express NEUROD1 or NGN3, for example, within 5 days, for example, within 1-4 days, for example, within 1-3 days, for example, within 1-2 or 2-3 days. Step ii) is performed before insulin and / or glucagon expression.

[0117] As discussed above, in one embodiment, the formation of the 3D structure in step iii) is a spontaneous formation of the 3D structure. For example, such spontaneous formation of the 3D structure can occur through self-aggregation. Alternatively, the formation of the 3D structure in step iii) is a forced (also called assisted) formation of the 3D structure. Non-limiting examples of forced (or assisted) formation of the 3D structure include culture conditions in which cells are forced close together by the shape of a cell culture flask or bottle. In one embodiment, 3D culture conditions allow for cell self-aggregation. Without being bound by theory, this embodiment shows that self-aggregation allows for the selective enrichment of endocrine progenitor cells that develop into stage 6 cells. In addition, the selective enrichment results in an increased generation of pancreatic monohormone β cells in the culture. In one embodiment, the pancreatic monohormone β cells are generated as part of a cell aggregate. In one embodiment, the aggregate comprises monohormone β cells. In one embodiment, the pancreatic monohormone β cells are generated as part of an in vitro islet-like cell aggregate. In one embodiment, the in vitro islet-like cell aggregate also includes pancreatic monohormone α and / or δ cells.

[0118] In one embodiment, the in vitro islet-like cell aggregates are generated from human ES cells and contain at least 40%, for example, at least 45%, for example, at least 50%, for example, at least 55% β cells, for example, at least 60% β cells, for example, at least 65% monohormone β cells. In one embodiment, the in vitro islet-like cell aggregates are generated from human iPS cells and contain at least 40%, for example, at least 45%, for example, at least 50%, for example, at least 55% β cells, for example, at least 60% β cells, for example, at least 65% monohormone β cells. In a particular embodiment, the iPS cells are C7 cells, and the islet-like cell aggregates contain at least 60% β cells, for example, at least 65% monohormone β cells. In one embodiment, the monohormone β cells are characterized by insulin expression.

[0119] Therefore, the inventors have demonstrated that the present invention is equally applicable to the differentiation of ES and iPS cells, and its beneficial effects are not limited to specific cell lines.

[0120] In one embodiment, the in vitro islet-like cell aggregate contains 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% proliferating cells, such as proliferating cells expressing Ki-67. In one embodiment, the in vitro islet-like cell aggregate contains 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% proliferating cells, such as proliferating cells expressing Ki-67. In one embodiment, the in vitro islet-like cell aggregate contains 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% proliferating cells, such as proliferating cells expressing Ki-67. In one embodiment, the Ki-67 is scored one day after aggregate formation. In one embodiment, the Ki-67 is scored on day 15 of culture. Therefore, the evaluation can be performed approximately 4 weeks after step iv).

[0121] In one embodiment, the in vitro islet-like cell aggregate comprises at least 50%, for example, at least 55%, for example, at least 60%, for example, at least 65%, for example, at least 70%, for example, at least 75% of cells expressing NEUROD1 and NKX6.1. In one embodiment, the in vitro islet-like cell aggregate comprises approximately 60% to 90%, for example, approximately 60% to 80%, for example, approximately 65% ​​to 80% of cells expressing NEUROD1 and NKX6.1. In one embodiment, the in vitro islet-like cell aggregate comprises at least 50%, for example, at least 80%, for example, at least 85%, for example, at least 87%, for example, at least 90% of cells expressing NEUROD1. In a particular embodiment, the in vitro islet-like cell aggregate is generated from human ES cells. In one embodiment, the expression of NEUROD1 and / or NKX6.1 is scored on day 15 of culture. Therefore, the evaluation can be performed after the aggregate formation in step iv). In one embodiment, step iv) includes culturing the cell population for about 2 weeks or longer, such as about 3 weeks or longer, such as about 3 to 5 weeks, such as about 4 weeks.

[0122] As used herein, the term "monohormone" refers to a cell that expresses only one hormone. For example, monohormone β cells express only insulin and do not express other hormones expressed by pancreatic islet cells, such as glucagon or somatostatin. As used herein, the term "multihormone" refers to a cell that expresses at least two different hormones.

[0123] In vivo β cells are monohormonal cells, and advantageously, the population obtained by the method of the present invention exhibits the characteristics of in vivo natural β cells, in other words, the characteristics of in vivo endogenous β cells, such as the characteristics of in vivo healthy natural β cells, or the characteristics of in vivo healthy endogenous β cells.

[0124] Therefore, in one embodiment of the method of the present invention disclosed herein, the islet-like cell aggregate containing the β cells generated in step v) comprises monohormone β cells. In one embodiment of the method of the present invention, the monohormone β cells in step v) express insulin. In one embodiment, the monohormone β cells in step v) express C-peptide upon glucose stimulation.

[0125] Specifically, the mono-hormone β-cells in step v) do not express glucagon or somatostatin.

[0126] In one embodiment, the monohormone β-cells in step v) are characterized by expressing insulin. The monohormone β-cells may also express at least one of NKX6.1, PDX1, and NEUROD1.

[0127] In one embodiment, the monohormone β-cell is characterized by expressing insulin and PDX1. In one embodiment, the monohormone β-cell is characterized by expressing insulin and NKX6.1. In one embodiment, the monohormone β-cell is characterized by expressing insulin and NEUROD1. In one embodiment, the monohormone β-cell is characterized by expressing: insulin and two of NKX6.1, PDX1, and NEUROD1; for example, insulin, NKX6.1, and PDX1; or insulin, NKX6.1, and NEUROD1; or insulin, PDX1, and NEUROD1. In one embodiment, the monohormone β-cell is characterized by expressing insulin, PDX1, NKX1, and NEUROD1.

[0128] In one embodiment, a method is provided for generating in vitro islet-like cell aggregates as defined herein, wherein the islet-like cell aggregates in v) comprise at least about 25%, for example, at least about 30%, for example, at least about 35%, for example, at least about 40%, for example, at least about 45%, for example, at least about 50%, for example, at least about 55%, for example, at least about 60%, monohormonal β cells, for example, at least about 65%. In one embodiment, the in vitro islet-like cell aggregates in v) comprise about 25% to 70%, for example, about 30% to 70%, for example, about 40% to 70%, for example, about 40% to 60%, monohormonal β cells.

[0129] In one embodiment, the in vitro islet-like cell aggregate in v) comprises at least 40%, for example, at least 45%, for example, at least 50%, for example, at least 55%, for example, at least 60%, for example, at least 65%, for example, at least 70% of β-cell monohormonal β-cells. In one embodiment, the monohormonal β-cells are characterized by insulin expression.

[0130] In one embodiment, the in vitro islet-like cell aggregate in v) comprises up to about 20%, for example, up to about 18%, for example, up to about 16%, for example, up to about 13%, for example, up to about 10% monohormone α cells. In one embodiment, the monohormone α cells are characterized by expressing glucagon.

[0131] In one embodiment, a method is provided for generating in vitro islet-like cell aggregates as defined herein, wherein the islet-like cell aggregates in v) comprise monohormonal β cells, and up to 5% α cells, and 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%, for example up to about 4%, 3%, 2%, or 1%, multihormonal cells. In one embodiment of the method, the islet-like cell aggregates in v) comprise up to about 5%, for example up to about 4%, 3%, 2%, or 1%, non-endocrine cells.

[0132] In one implementation, the in vitro islet-like cell aggregates in v) are scored at the end of S6, for example, on days 38-42 of culture, such as day 38, day 39, day 40, day 41, day 42 or later.

[0133] Furthermore, the inventors have demonstrated that a shorter culture time for posterior foregut cells (PF) yields a greater number of endocrine progenitor cells (EP) (also referred to herein as endocrine precursor cells) under conditions that allow differentiation into pancreatic progenitor cells (PP), even if the number of PP cells obtained is lower than that obtained in corresponding methods with longer culture times. As used herein, the terms “endocrine progenitor cells (EP)” and “endocrine precursor cells (EP)” are used interchangeably. Without being bound by theory, it appears that PP cells obtained by the method of the present invention, including the aforementioned short culture time, are capable of developing / differentiating into EP cells. Importantly, EP cells obtained by the method disclosed herein have the potential to develop into mature and functional pancreatic β cells, such as monohormone β cells, which are capable of responding to glucose stimulation by expressing C-peptide. As used herein, the term “corresponding method” refers to a method in which all steps are identical except for those specifically indicated. Thus, a corresponding method should be interpreted as the same method but differing in the indicated steps, for example, a step that may be related to the culture time, such as step b-1); or b); or the culture time between both b-1) and b.

[0134] Therefore, in one embodiment, a method for generating in vitro islet-like cell aggregates is provided, wherein prior to step i), the method comprises the following steps a)-c):

[0135] a) Provide a population of hindbrain cells, which are, for example, hindbrain cells characterized by expression of PDX1;

[0136] b) The cell population of the posterior foregut cells is cultured under conditions that allow differentiation into pancreatic progenitor cells for no more than approximately 78 hours, for example, no more than approximately 72 hours; and

[0137] c) This generates a population of pancreatic progenitor cells, which are, for example, pancreatic progenitor cells characterized by the expression of both PDX1 and NKX6.1.

[0138] To clarify, the cell population of hindforegut cells (PF) characterized by PDX1 expression provided in step a) does not express NKX6.1; in other words, it lacks NKX6.1 expression.

[0139] Therefore, in one embodiment of the method disclosed herein, the cell population of hindforegut cells (PF) provided in step a) is characterized by the expression of PDX1 and the lack of NKX6.1 expression. In one embodiment, the cell population of PF cells provided in step a) is further characterized by the expression of HNF6. Therefore, it should be understood that the hindforegut cells may be characterized by the expression of HNF6, PDX1, or co-expression of PDX1 and HNF6.

[0140] In one embodiment of the first aspect disclosed herein, a method is provided in which, in step b), the cell population is cultured for no more than about 75 hours, for example, no more than about 72 hours, for example, no more than about 66 hours, for example, no more than about 60 hours, for example, no more than about 48 hours. In another embodiment, a method is provided in which, in step b), the cell population is cultured for a period of approximately 42 to 78 hours, for example, a period of approximately 44 to 76 hours, for example, a period of approximately 46 to 74 hours, for example, a period of approximately 48 to 72 hours. In one embodiment, the cell population is cultured for a period of approximately 40 to 78 hours, for example, a period of approximately 42 to 76 hours, for example, a period of approximately 44 to 74 hours, for example, a period of approximately 48 to 72 hours. In one embodiment, the cell population is cultured for a period of approximately 42 to 54 hours, for example, a period of approximately 44 to 52 hours, for example, a period of approximately 46 to 50 hours, for example, a period of approximately 48 hours.

[0141] In one embodiment of the method disclosed herein, the pancreatic progenitor cell population in step c) is, for example, a pancreatic progenitor cell population characterized by expression of PDX1 and NKX6.1, further characterized by expression of at least one biomarker selected from the group consisting of PTF1A, SOX9, HNF6, and CPA, for example, biomarkers selected from the group consisting of SOX9 and PTF1A. In one embodiment of the method disclosed herein, the pancreatic progenitor cell population in step c) is further characterized by expression of PTF1A and SOX9.

[0142] As discussed above, the term "conditions allowing differentiation" refers to conditions that allow cells to develop (in other words, differentiate) so that they exhibit characteristics of the cell type, and may include a combination of cell culture media, the presence and / or absence of extrinsic factors, and their timing. In one embodiment, the culture medium in step b) is suitable for culturing foregut cells under conditions allowing differentiation into pancreatic progenitor cells. Non-limiting examples of the culture medium in stage 4 (S4) are as defined in the appended examples. The culture medium in step b) may be supplemented with other factors as specified herein. Those skilled in the art will understand that other suitable culture media may be used. Specifically, culture under conditions allowing differentiation into pancreatic progenitor cells as disclosed herein may involve culturing a cell population in a culture medium in the presence of specific extrinsic factors, such as epidermal growth factor (EGF) and nicotinamide (NIC) or derivatives or agonists thereof. Therefore, in one embodiment of the method disclosed herein, the conditions for allowing differentiation into pancreatic progenitor cells include culturing the cell population in a culture medium in the presence of an effective amount of epidermal growth factor (EGF) (e.g., 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 method disclosed herein, step b) includes culturing the cell population in a culture medium in the presence of an effective amount of EGF (e.g., human EGF) and an effective amount of NIC.

[0143] Epidermal growth factor (EGF) is a protein that stimulates cell growth and differentiation by binding to its receptor, EGFR. Human EGF is a 6-kDa protein with 53 amino acid residues and three intramolecular disulfide bonds. Binding to the receptor stimulates ligand-induced dimerization, activating intrinsic protein tyrosine kinase activity, thereby initiating a signal transduction cascade that leads to various intracellular biochemical changes—increased intracellular calcium levels, increased glycolysis and protein synthesis, and increased expression of certain genes, including the EGFR gene—ultimately resulting in DNA synthesis and cell proliferation. EGF is a member of the EGF protein family. Members of this protein family share highly similar structural and functional characteristics. In addition to EGF itself, other family members include heparin-bound EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), bimodal protein (AR), epithelial regulatory protein (EPR), Epigen, cytidine (BTC), neuroregulatory protein-1 (NRG1), neuroregulatory protein-2 (NRG2), neuroregulatory protein-3 (NRG3), and neuroregulatory protein-4 (NRG4).

[0144] Those skilled in the art will understand that, as used herein, the term "epidermal growth factor (EGF) or its derivatives or agonists" is intended to include factors that enhance or substitute for EGF signaling. These factors may be involved in downstream signaling of EGF or are 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. Therefore, in one embodiment of this aspect, the EGF or its derivatives or agonists are selected from the group consisting of EGF, TGF-α, BTC, HB-EGF, AR, EPR, and Epigen, for example, EGF. In a particular embodiment, the EGF is human EGF.

[0145] Nicotinamide (NIC), also known as NAM, is a form of vitamin B. The structure of nicotinamide consists of a pyridine ring with a primary amide group attached meta-positioned to it, and this primary amide group is an amide of nicotinic acid. Nicotinamide is a well-known cell culture supplement used for the differentiation of embryonic stem cells and induced pluripotent stem cells, and has been shown to regulate stem cell differentiation in a variety of applications, including pancreatic cell differentiation. Those skilled in the art will understand that, as used herein, the term "nicotinamide (NIC) or a derivative or agonist thereof" is intended to include factors that enhance or substitute for NIC signaling. Non-limiting examples of such derivatives or agonists include NIC, nicotinic acid (nicotinic acid), nicotinamide nucleoside, NAD / NADP, and tryptophan as a precursor to NIC. Therefore, in one embodiment of this aspect, said NIC or a derivative or agonist thereof is selected from the group consisting of NIC, nicotinic acid, nicotinamide nucleoside, NAD / NADP, and tryptophan, for example, wherein said NIC or a derivative or agonist thereof is NIC.

[0146] In one embodiment of the method, in step b), the effective amount of the EGF or its derivatives or agonists 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.

[0147] In one embodiment of the method, in step b), the effective amount of the NIC or its derivatives or agonists 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.

[0148] Step b) of the method disclosed herein may include culturing a cell population in a culture medium containing additional factors, such as one or more factors selected from: KGF and its derivatives and agonists; ActA 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, such as one of a variety of factors selected from KGF, ActA, retinoic acid, SANT-1, PDBu, and LDN. In one embodiment, step b) includes culturing the cell population in a culture medium further containing: KGF or its derivatives or agonists; ActA or its derivatives or agonists; retinoic acid or its derivatives or agonists; SANT-1 or its derivatives or agonists; PDBu or its derivatives or agonists; and LDN or its derivatives or agonists, for example, a culture medium also containing KGF, ActA, retinoic acid, SANT-1, PDBu, and LDN. It should be understood that the culture medium may contain a mixture of factors and their derivatives and / or agonists.

[0149] Technicians should understand that cell culture media and the factors contained therein can be adjusted by replacing the factors with their derivatives and / or agonists (such as those described below).

[0150] Keratinocyte growth factor (KGF), also known as fibroblast growth factor 7 (FGF7), is a member of the FGF protein family. It is a bioactive protein commonly used in cell culture applications. KGF binds to fibroblast growth factor receptor 2b (FGFR2b). KGF induces the proliferation of many epithelial cells but not fibroblasts and endothelial cells. It 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 replaced by its derivatives or agonists in the cell cultures described herein. Non-limiting examples of such agonists include other factors that bind to FGFR2b and signal through said receptor (e.g., FGF10).

[0151] In one embodiment, the KGF or its derivatives or agonists are selected from the group consisting of KGF and FGF10, for example, FGF10.

[0152] In one embodiment, the culture medium in step b) contains 25 ng / mL to 75 ng / mL KGF or a derivative or agonist thereof, such as 50 ng / mL KGF or a derivative or agonist thereof. In another embodiment, the culture medium in step b) contains approximately 25 ng / mL to 75 ng / mL KGF, such as approximately 50 ng / mL KGF.

[0153] ActA, or activin A, is a member of the TGF-β superfamily. Both activins and Nodal ligands can signal to regulate transcription through the same receptors and effectors. In many cases, the effects of Nodal and activin-mediated signaling are indistinguishable; therefore, they are referred to as activin / Nodal. Activin / Nodal binds to type II activin receptors (ActRII / IIB), leading to the recruitment, phosphorylation, and activation of type I activin receptors (activin receptor-like kinases, or ALKs, including ALK1-7), particularly ALK4. The serine / threonine kinase receptors ActRII / IIB and ALK4 / 7 then trigger the phosphorylation of Smad transcription factors Smad2 and Smad3.

[0154] It is known in the art that TGFβ signaling is involved in embryogenesis, cell differentiation and apoptosis, as well as other functions. The activin / Nodal and TGFβ pathways share downstream effectors Smad2 and Smad3.

[0155] It has been reported that activin / Nodal is involved in maintaining the pluripotency of stem cells; however, activin / Nodal signaling is also essential for endoderm differentiation.

[0156] Those skilled in the art will understand that ActA can be replaced by its derivatives or agonists in the cell cultures described herein. Examples of such agonists include Nodal, which signals via the receptor, downstream effector molecules (e.g., Smad 2 and 3), and TGFβ, which signals via the same effector molecules. Other derivatives or agonists include, but are not limited to, TGFβ1-3 (TGFβ1, TGFβ2, TGFβ3), Nodal, activin A, GDF-1, GDF-8, and GDF-11, all of which activate the Smad2 / 3 / 4 complex.

[0157] In one embodiment, the ActA or its derivatives or agonists are selected from the group consisting of ActA and GDF-8, Nodal, TGFβ1-3 (TGFβ1, TGFβ2, TGFβ3). In one embodiment, the culture medium in step b) contains approximately 1 to 10 ng / mL, for example 2.5 to 7.5 ng / mL of ActA, for example approximately 5 ng / mL of ActA.

[0158] Retinoic acid (RA) is a metabolite of vitamin A and mediates the function of vitamin A required for growth and development. RA is known to participate in defining the position along the anterior-posterior axis of the embryo, also known as pattern formation, and is also known to play a role in later stages of pancreatic development to promote the generation and differentiation of pancreatic endocrine progenitor cells into islets and β cells.

[0159] Retinoic acid exerts its effects by binding to the retinoic acid receptor (RAR), which, as a heterodimer with the retinoid X receptor (RXR), binds to DNA in a region called the retinoic acid response element (RARE). The binding of the retinoic acid ligand to the RAR alters the conformation of the RAR, thereby affecting the binding of other proteins that induce or inhibit the transcription of nearby genes. Those skilled in the art will understand that the term "retinoic acid or its derivatives or agonists" is intended herein to include factors that enhance or substitute for retinoic acid signaling. These factors may be involved in downstream signaling of retinoic acid or are small molecule agonists. A non-limiting list of retinoic acid derivatives or agonists includes all-trans retinoic acid, synthetic retinoids EC23, Ch55, TTNPB, fenivel-Amine, RAR agonists (e.g., the RARA agonist and the RARB agonist AC261066), adapalene, AC55649, AM80, AM580, BMS753, tazarotene, and Ro 41-5253. Therefore, in one embodiment of this aspect, the retinoic acid or its derivatives or agonists are selected from the group consisting of: retinoic acid, all-trans retinoic acid, synthetic retinoids EC23, Ch55, TTNPB, fenivel-Aminamide, RAR agonists (e.g., RARA agonists and RARB agonists AC261066), adapalene, AC55649, AM80, AM580, BMS 753, tazarotene, and Ro 41-5253.

[0160] In one embodiment, the retinoic acid (RA) or its derivative or agonist is selected from the group consisting of retinoic acid, all-trans retinoic acid, and synthetic retinol ec23. In one embodiment, the retinoic acid or its derivative or agonist is selected from the group consisting of retinoic acid and all-trans retinoic acid. In one embodiment, the retinoic acid or its derivative or agonist is all-trans retinoic acid. In one embodiment, the culture medium in step b) contains approximately 50 nM to 150 nM of RA or its derivative or agonist, for example, approximately 100 nM of RA or its derivative or agonist.

[0161] In one embodiment, the culture medium in step b) contains approximately 50 nM to 150 nM of RA, for example, approximately 100 nM of RA.

[0162] Hedgehog (HH or Hh) signaling is known to play a crucial role in regulating vertebrate organogenesis, such as in the growth of limbs (fingers and toes) and brain tissue. The vertebrate hedgehog protein family consists of the sound hedgehog factor (SHH), the Indian hedgehog factor (IHH), and the desert hedgehog factor (DHH), which signal through similar pathways and share many functional characteristics. The sound hedgehog factor (SHH) is a key negative regulator of pancreatic development; therefore, inhibiting SHH at the onset of pancreatic development is crucial, and hedgehog inhibition must be maintained to ensure normal pancreatic development.

[0163] In short, Hh signals by interacting with a Hh receptor complex containing two components: patched (Ptc) and smoothed (Smo), which transduces Hh signals into the cell. Ptc is thought to inhibit Hh signaling by binding to Smo in the cell membrane. This inhibition is lifted in the presence of Hh ligands, allowing Smo to signal. In vertebrates, zinc finger proteins Gli1, Gli2, and Gli3 are downstream mediators of Hh signaling and participate in controlling transcriptional responses of target genes in an Hh-dependent manner.

[0164] SANT-1 is an inhibitor of hedgehog (Hh) signaling and exerts its effect by antagonizing smoothed activity. Non-limiting examples of hedgehog signaling inhibitors include cyclopamine, IHR 1, IHR-Cy3, itraconazole, veratrine, M25, MRT 10, PF 04449913 maleate, PF 5274857 hydrochloride, SANT-1, and SANT-2.

[0165] In one embodiment, the SANT-1 or its derivatives or agonists are selected from the group consisting of: cyclopamine, IHR 1, IHR-Cy3, itraconazole, veratrine, M 25, MRT 10, PF 04449913 maleate, PF 5274857 hydrochloride, SANT-1 and SANT-2.

[0166] In one embodiment, the culture medium in step b) contains approximately 0.10 μM to 0.50 μM of SANT-1, for example, approximately 0.25 μM of SANT-1.

[0167] 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 as a tumor promoter, activating a variety of cellular responses, including proliferation. PDBu can be substituted with other protein kinase C activators, such as, but not limited to, phorbol-12-myristate 13-acetate (PMA) or TPPB. In one embodiment, the PDBu or its derivatives or agonists are selected from the group consisting of PDBu, PMA, and TPPB. In one embodiment, the culture medium in step b) contains approximately 0.25 μM to 0.75 μM of PDBu, for example, approximately 0.5 μM of PDBu.

[0168] LDN193189 (referred to herein as LDN) is a bone morphogenetic (BMP) inhibitor and works by inhibiting ALK2 and ALK3. LDN primarily works by blocking the phosphorylation of Smad1, Smad5, and Smad8. LDN is an analogue of dosomorphin and noggin, which may replace LDN. In one embodiment, the LDN or its derivatives or agonists are selected from the group consisting of LDN, noggin, and dosomorphin. In one embodiment, the culture medium in step b) contains approximately 100 nM to 300 nM of LDN, for example, 100 nM-250 nM of LDN, for example, 150 nM-250 nM of LDN, for example, approximately 200 nM of LDN.

[0169] In one embodiment, step b) of the method disclosed herein includes culturing a cell population in a culture medium containing approximately 50 ng / mL KGF, approximately 5 ng / mL ActA, approximately 100 nM retinoic acid, approximately 0.25 mM MSANT-1, approximately 500 nM PDBu, approximately 200 nM LDN, approximately 100 ng / mL EGF, and approximately 10 mM NIC.

[0170] As explained above, this disclosure also describes how a shorter culture time for posterior foregut cells (PF) produces a greater number of endocrine progenitor cells (EP) under conditions that allow differentiation into pancreatic progenitor cells (PP), compared to corresponding methods with longer culture times, even if the number of PP cells obtained is lower than that in corresponding methods with longer culture times.

[0171] Therefore, in one embodiment of this aspect, a method is provided in which at least about 70%, for example at least about 75%, for example at least about 80%, of the posterior foregut cells in a) differentiate into pancreatic progenitor cells in c).

[0172] In one embodiment, a method as disclosed herein is provided, wherein in step c), at least about 80%, for example, about 80% to 85%, for example, about 80% to 90% of the total cell population expresses PDX1. In one embodiment, a method as disclosed herein is provided, wherein in step c), at most about 10%, for example, at most about 8%, for example, at most about 7%, for example, at most about 5%, for example, at most about 3% of the total cell population expresses NEUROD1. In one embodiment, a method as disclosed herein is provided, wherein in step c), at most about 10%, for example, at most about 8%, for example, at most about 7%, for example, at most about 5%, for example, at most about 3% of the total cell population expresses NEUROD1 and does not express NKX6.1. In one embodiment, a method as disclosed herein is provided, wherein in step c), about 25% to 50%, for example, about 25% to 48%, for example, about 25% to 45% of the total cell population expresses NKX6.1. In one embodiment, a method as disclosed herein is provided, wherein in step c), approximately 30% to 50%, for example, approximately 35% to 45%, or for example, approximately 40% to 45%, of the total cell population expresses NKX6.1. In one embodiment, in step c), approximately at least 40%, for example, at least 50%, or for example, at least 60%, of the cells do not express NEUROD1 and NKX6.1.

[0173] In one embodiment, the hindbrain cells are characterized by the expression of PDX1 and the lack of NKX6.1 expression in a) the foregut cells.

[0174] In one embodiment of the method for generating in vitro islet-like cell aggregates as disclosed herein, prior to step i), the method includes the following steps a-1)–c-1):

[0175] a-1) Provide a population of primitive intestinal cells, which are, for example, primitive intestinal cells characterized by the expression of HNF1β and / or HNF4α;

[0176] b-1) The cell population of the primitive intestinal cells shall be cultured for no more than approximately 54 hours under conditions that allow differentiation into hindforegut cells; and

[0177] c-1) This generates a population of hindbrain cells, which are, for example, hindbrain cells characterized by the expression of PDX1. It should be understood that the hindbrain cells may also be characterized by the expression of HNF6 or the co-expression of PDX1 and HNF6.

[0178] In one embodiment of the method, step a-1)-c-1) is performed before step a)-c).

[0179] In one embodiment of the method for generating in vitro islet-like cell aggregates as disclosed herein, prior to step i), the method includes the following steps a-1)-c-1) and steps a) and c):

[0180] a-1) Provide a population of primitive intestinal cells, which are, for example, primitive intestinal cells characterized by the expression of HNF1β and / or HNF4α;

[0181] b-1) The cell population of the primitive intestinal cells was cultured for no more than approximately 54 hours under conditions that allowed differentiation into hindforegut cells;

[0182] c-1) thereby generating a population of hindbrain cells, which are, for example, hindbrain cells characterized by expression of PDX1.

[0183] a) Provide a cell population of hindbrain cells generated in step c-1), which are, for example, hindbrain cells characterized by expressing PDX1;

[0184] b) The cell population of the posterior foregut cells is cultured under conditions that allow differentiation into pancreatic progenitor cells for no more than approximately 78 hours, for example, no more than approximately 72 hours; and

[0185] c) This generates a population of pancreatic progenitor cells, which are, for example, pancreatic progenitor cells characterized by the expression of both PDX1 and NKX6.1.

[0186] As used herein, each method step is represented by a letter code, such as a), b), c), etc. A complete method set for culturing a cell population at a developmental stage under appropriate conditions to allow it to differentiate into a population at the next developmental stage includes steps a), b), c), etc. Each step can also be represented by a letter code with an associated numerical indicator. The numerical indicator indicates whether each step in the complete method set precedes (negative numerical indicator) or follows (positive numerical indicator) the complete method set for culturing foregut cells (PF) to generate pancreatic progenitor cells (PP). To illustrate the method steps associated with the complete method set for culturing a cell population at a developmental stage under appropriate conditions to allow it to differentiate into a population at the next developmental stage, they are shown in Table 1 below:

[0187]

[0188]

[0189] Table 1. Summary of method groups and steps. In this paper, steps a+2) to c+2) are also referred to as steps i)-v) (in other words, steps i), ii), iii), iv) and v).

[0190] In one embodiment of the method disclosed herein, the population of primitive intestinal cells provided in step a-1) is characterized by expressing HNF1β, HNF4α, or both HNF1β and HNF4α.

[0191] In one embodiment, the population of hindbrain cells is characterized by expression of PDX1 or HNF6. In another embodiment, the population of hindbrain cells is characterized by expression of both PDX1 and HNF6. In one embodiment of the method disclosed herein, the cell population of hindbrain cells in step c-1) is characterized by expression of PDX1 and lack of expression of NKX6.1.

[0192] In one embodiment, the population of hindbrain cells in c-1) is characterized by the expression of PDX1 and HNF6 and the lack of NKX6.1 expression. The population of hindbrain cells does not express NKX6.1, PTF1A, and SOX9.

[0193] Without being bound by theory, it is conceivable that step b-1) of culturing the cell population of the primitive intestinal cells under conditions allowing differentiation into hindbrain cells is advantageous for no more than 54 hours in order to obtain a population of hindbrain cells capable of developing into later stages of the β-cell lineage. It is conceivable that primitive intestinal cells cultured for longer periods than 54 hours under conditions allowing differentiation into hindbrain cells are less capable of developing 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 no more than approximately 52 hours, for example, no more than approximately 50 hours, for example, no more than approximately 48 hours, for example, no more than approximately 44 hours, for example, no more than approximately 40 hours, for example, no more than approximately 36 hours, for example, no more than approximately 32 hours, for example, no more than approximately 28 hours, for example, no more than approximately 26 hours, for example, no more than approximately 24 hours.

[0194] In one embodiment of the method disclosed herein, the cell population in step b-1) is cultured for a period of approximately 18 to 54 hours, for example, approximately 20 to 52 hours, for example, approximately 22 to 50 hours, for example, approximately 24 to 48 hours. In one embodiment, the cell population in step b-1) is cultured for a period of approximately 42 to 54 hours, for example, approximately 44 to 52 hours, for example, approximately 46 to 50 hours, for example, approximately 48 hours. In one embodiment, the cell population in step b-1) is cultured for a period of approximately 18 to 30 hours, for example, approximately 20 to 28 hours, for example, approximately 22 to 26 hours, for example, approximately 24 hours.

[0195] As discussed above, "conditions that allow differentiation" refers to conditions that allow cells to develop / differentiate to exhibit the characteristics of the cell type, and may include the combination of cell culture media, the presence and / or absence of extrinsic factors and their timing. These factors, as well as their derivatives and agonists, have been discussed in detail with respect to step b) above, and will not be repeated here for the sake of brevity.

[0196] In one embodiment, the culture medium described in step b-1) is suitable for culturing primitive intestinal cells under conditions that allow differentiation into hindgut cells. Non-limiting examples of the culture medium for stage 3 (S3) are defined in this embodiment. Those skilled in the art will understand that other suitable culture media may be used. The culture medium described in step b-1) may be supplemented with other factors specified herein.

[0197] Therefore, in one embodiment, a method is provided, wherein 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, such as 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, such as one or more factors selected from KGF and retinoic acid, such as both KGF and retinoic acid. In one embodiment, step b-1) comprises culturing the cell population in a culture medium containing: KGF or its derivatives or agonists; retinoic acid or its derivatives or agonists; SANT-1 or its derivatives or agonists; PDBu or its derivatives or agonists; and LDN or its derivatives or agonists, such as a culture medium containing KGF, retinoic acid, SANT-1, PDBu, and LDN. It should be understood that the culture medium may contain a mixture of factors and their derivatives and / or agonists.

[0198] In one embodiment, the culture medium in step b-1) contains 25 ng / mL to 75 ng / mL KGF or a derivative or agonist thereof, such as 50 ng / mL KGF or a derivative or agonist thereof. In one embodiment, the culture medium in step b-1) contains approximately 25 ng / mL to 75 ng / mL KGF, such as approximately 50 ng / mL KGF. In one embodiment, the culture medium in step b-1) contains approximately 1 μM to 3 μM RA, such as approximately 2 μM RA.

[0199] In one embodiment, the culture medium in step b-1) comprises from about 0.10 μM to 0.50 μM of SANT-1, such as about 0.25 μM of SANT-1.

[0200] In one embodiment, the culture medium in step b-1) comprises from about 250 nM to 750 nM of PDBu, such as about 500 nM of PDBu.

[0201] In one embodiment, the culture medium in step b-1) comprises from about 100 nM to 300 nM of LDN, such as 100 nM - 250 nM of LDN, such as 150 nM - 250 nM of LDN, such as about 200 nM of LDN.

[0202] In one embodiment, step b-1) of the method as disclosed herein comprises culturing a 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.

[0203] In another embodiment of the method as disclosed herein, the method comprises steps a+1)-c+1) after steps a)-c).

[0204] a+1) Providing a cell population of pancreatic progenitor cells generated in step c;

[0205] b+1) Culturing the cell population of pancreatic progenitor cells under conditions permissive for differentiation into endocrine progenitor cells; and

[0206] c+1) Thereby generating a population of endocrine progenitor cells that are, for example, characterized by expression of NEURO D1, such as endocrine progenitor cells characterized by expression of NKX6.1 and NEURO D1.

[0207] In one embodiment, there is provided a method as disclosed herein, wherein prior to step i), the method comprises the following steps [[ID=!26]]

[0208] a-1) Providing a cell population of primitive gut tube cells that are, for example, characterized by expression of HNF1β and / or HNF4α; ]>

[0209] b-1) Culturing the cell population of primitive gut tube cells under conditions permissive for differentiation into posterior foregut cells for no more than about 54 hours;

[0210] c-1) Thereby generating a population of posterior foregut cells that are, for example, characterized by expression of PDX1;

[0211] It should be noted that there seems to be an error in the original text where the tag [[ID=!26]] is likely incorrect. It should probably be for proper formatting and processing.a) Provide a cell population of the posterior foregut cells generated in step c-1), which are posterior foregut cells characterized by, for example, expressing PDX1;

[0212] b) Culture the cell population of the posterior foregut cells under conditions allowing differentiation into pancreatic progenitor cells for no more than about 78 hours, such as no more than about 72 hours;

[0213] c) Thereby generate a cell population of pancreatic progenitor cells, which are pancreatic progenitor cells characterized by, for example, expressing both PDX1 and NKX6.1;

[0214] a+1) Provide a cell population of the pancreatic progenitor cells generated in step c;

[0215] b+1) Culture the cell population of the pancreatic progenitor cells under conditions allowing differentiation into endocrine progenitor cells; and

[0216] c+1) Thereby generate a population of endocrine progenitor cells, which are endocrine progenitor cells characterized by, for example, expressing NEUROD1, such as endocrine progenitor cells characterized by expressing NKX6.1 and NEUROD1.

[0217] In one embodiment, the population of endocrine progenitor cells is further characterized by expressing at least one of NKX6.1 and NGN3. Those skilled in the art should understand that different combinations of two markers can also be used for endocrine progenitor cells, such as NKX6.1 and NGN3, NKX6.1 and NEUROD1, or NKX6.1 and NGN3, in order to distinguish the cells from pancreatic progenitor cells. For example, the population of endocrine progenitor cells in step c+1) can be characterized by the expression of: PDX1, NKX6.1 and NEUROD1; PDX1, NKX6.1 and NGN3; or PDX1, NKX6.1, NEUROD1 and NGN3. It should be understood that the expression of NGN3 or NEUROD1 in PDX1+ / NKX6.1+ cells (in other words, cells that are positive for both PDX1 and NKX6.1) indicates that pancreatic progenitor cells adopt an endocrine progenitor cell fate. Those skilled in the art understand that due to experimental limitations of the staining protocol, the expression of NGN3 or NEUROD1 in cells stained for PDX1 or NKX6.1 can be evaluated. In one embodiment of the method disclosed herein, the cell population of endocrine progenitor cells in step c+1) is characterized by expressing: PDX1, NKX6.1 and NEUROD1; PDX1, NKX6.1 and NGN3; or PDX1, NKX6.1, NEUROD1 and NGN3.

[0218] In one embodiment of the method as disclosed herein, the cell population of pancreatic progenitor cells provided in step a+1) is characterized by the expression of PDX1 and NKX6.1, in other words, the co-expression of PDX1 and NKX6.1. In another embodiment, the cell population of the pancreatic progenitor cells in step a+1) is further characterized by the expression of one or more of the markers PTF1A, SOX9, and HNF6, such as the expression of two of the markers PTF1A, SOX9, and HNF6, such as the expression of all three of PTF1A, SOX9, and HNF6. In another embodiment, the cell population of the pancreatic progenitor cells in step a+1) is further characterized by the expression of one or both of the markers PTF1A and SOX9.

[0219] In an embodiment of the method as disclosed herein, the cell population in step b+1) is cultured for about 3 to 5 days, such as about 3 to 4 days or about 4 to 5 days, such as about 4 days. In one embodiment, the cell population in step b+1) is cultured for about 5 days. This culture time is considered sufficient to generate a population of endocrine progenitor cells in step c+1).

[0220] Similar to the description in steps a)-c), steps a+1)–c+1) are carried out in 2D culture on a 2D substrate. It should be understood that the discussion of the 2D substrate related to steps a)–c) is equally relevant to steps a+1)–c+1) and is not repeated here merely for the sake of brevity. Similar to the description in steps a)-c), steps a-1)–c-1) are carried out in 2D culture on a 2D substrate. It should be understood that the discussion of the 2D substrate related to steps a)–c) is equally relevant to steps a-1)–c-1) and is not repeated here merely for the sake of brevity.

[0221] Thus, in one embodiment method as disclosed herein, the cells in step b+1) are cultured on a 2D substrate. Thus, in one embodiment method as disclosed herein, the cells in step b-1) are cultured on a 2D substrate.

[0222] In one embodiment, the cells adhere to the 2D substrate. The 2D substrate may comprise 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 TM , such as 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, and Matrigel TM, for example, selected from the group consisting of: laminin (LN) and its fragments, vitronectin and its fragments, fibronectin and its fragments, collagen, gelatin, and Matrigel TM , for example, selected from the group consisting of: laminin (LN) and its fragments, vitronectin and its fragments, fibronectin and its fragments, and Matrigel TM .

[0223] In one embodiment, the 2D substrate may comprise or consist of: laminin (LN) and its fragments, such as recombinantly produced laminin (LN) and its fragments.

[0224] In one embodiment of the method as disclosed herein, 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;

[0225] 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;

[0226] for example, the group consisting of: LN-521 and its fragments, LN-511 and its fragments, and LN-332 and its fragments; '

[0227] for example, the group consisting of LN-521 and its fragments or the group consisting of LN-511 and its fragments. In one embodiment, the laminin 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, wherein the laminin and its fragments are LN-521 or wherein the laminin and its fragments are LN-511.

[0228] In one embodiment, the one or more fragments thereof are E8 fragments. In one embodiment, the laminin and fragment 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 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, and an E8 fragment of LN-121; such as the group consisting of: an E8 fragment of LN-511, an E8 fragment of LN-521, and an E8 fragment of LN-332; such as the group consisting of: 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.

[0229] As discussed above, the "conditions permitting differentiation" refer to conditions that permit the cells to exhibit the characteristics of the cell type, and may include combinations of cell culture media, the presence and / or absence of extrinsic factors, and their temporal settings. The factors and their derivatives and agonists have been discussed in detail with respect to steps b) and b-1) above, and the discussion will not be repeated here for the sake of brevity. Thus, in one embodiment, a method is provided wherein step b+1) comprises culturing the cell population in a medium comprising one or more factors selected from the following: BTC and its derivatives and agonists, retinoic acid and its derivatives and agonists, Alk5 inhibitors (such as Alk5i II) and its derivatives and agonists, retinoic acid and its derivatives and agonists, γ-secretase inhibitors (such as GSI-XX) and its derivatives and agonists, GC-1 and its derivatives and agonists, LDN and its derivatives and agonists, retinoic acid and its derivatives and agonists, and SANT-1 and its derivatives and agonists; for example, one or more factors selected from the following: BTC, Alk5 inhibitors (such as Alk5i II), γ-secretase inhibitors (such as GSI-XX), GC-1, LDN, retinoic acid, and SANT-1. Thus, in one embodiment, a method is provided wherein step b+1) comprises culturing the cell population in a medium comprising the following: BTC or its derivatives or agonists; Alk5 inhibitors (such as Alk5i II) or its derivatives or agonists: γ-secretase inhibitors (such as GSI-XX) or its derivatives or agonists; GC-1 or its derivatives or agonists; LDN or its derivatives or agonists; retinoic acid or its derivatives or agonists: and SANT-1 or its derivatives or agonists; for example, BTC, Alk5 inhibitors (such as Alk5i II), γ-secretase inhibitors (such as GSI-XX), GC-1, LDN, retinoic acid, and SANT-1. Thus, in one embodiment, a method is provided wherein step b+1) comprises culturing the cell population in a medium comprising the following: BTC and / or its derivatives and / or agonists; Alk5 inhibitors (such as Alk5i II) and / or its derivatives and / or agonists; γ-secretase inhibitors (such as GSI-XX) and / or its 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 should be understood that the medium may comprise a mixture of factors and their derivatives and / or agonists. In one embodiment of the methods disclosed herein, step b+1) comprises culturing the cell population in a medium comprising BTC, Alk5i II, GSI-XX, GC-1, LDN, retinoic acid, and SANT-1.Specifically, in one embodiment, a method is provided, wherein step b+1) comprises culturing the cell population in a culture medium comprising at least an Alk5 inhibitor or its derivatives or agonists and a γ-secretase inhibitor and its derivatives and agonists (such as Alk5i II and GSI-XX).

[0230] BTC, also known as betacellulin, is a member of the EGF growth factor family and induces the differentiation of β cells and can also induce the differentiation of other cell types. As used herein, the term "BTC and its derivatives and agonists" refers to EGFR ligands / EGF family growth factors.

[0231] Examples of Alk5 inhibitors are Alk5i II (ALK5 inhibitor II), which is a cell-permeable and selective inhibitor of the TGF-β1 type activin-like kinase receptor ALK5. Non-limiting examples of Alk5 inhibitors include Alk5i II, LY2157299, LY364947, Repsox, SB525334, A83-01, GW788388, LY-2109761, SB-505124, and D4476. In one embodiment, the Alk5 inhibitor or its derivatives or agonists are selected from the group consisting of Alk5i II, LY2157299, LY364947, Repsox, SB525334, A83-01, GW788388, LY-2109761, SB-505124, and D4476.

[0232] GSI-XX, also known as γ-secretase inhibitor XX, is a cell-permeable dibenzazepine compound that inhibits γ-secretase. γ-secretase is a multi-subunit protease complex (which itself is an integral membrane protein) that cleaves single 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.

[0233] GC-1 is a thyroid hormone receptor (TR) agonist and is more potent than the thyroid hormone T3. The thyroid hormone T3 is important for the development of β cells.

[0234] 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.

[0235] In one embodiment, the medium in step b+1) is a medium suitable for culturing pancreatic progenitor cells under conditions that permit differentiation into endocrine progenitor cells. Non-limiting examples of stage 5 (S5) medium are defined in this Example. Those skilled in the art will understand that other suitable media may be used. The medium in step b+1) may be supplemented with other factors specified herein.

[0236] In one embodiment, the medium in step b+1) contains from about 10 ng / mL to 30 ng / mL of BTC, such as about 20 ng / mL of BTC.

[0237] In one embodiment, the medium in step b+1) contains from about 5 μM to 15 μM of Alk5i II, such as about 10 μM of Alk5i II.

[0238] In one embodiment, the medium in step b+1) contains from about 50 nM to 150 nM of GSI-XX, such as about 100 nM of GSI-XX.

[0239] In one embodiment, the medium in step b+1) contains from about 0.5 μM to 1.5 μM of GC-1, such as about 1 μM of GC-1.

[0240] In one embodiment, the medium in step b+1) contains from about 50 nM - 150 nM of LDN, such as 75 nM - 125 nM of LDN, such as about 100 nM of LDN.

[0241] In one embodiment, the medium in step b+1) contains from about 50 nM to 150 nM of RA, such as about 100 nM of RA.

[0242] In one embodiment, the medium in step b+1) contains from about 0.10 μM to 0.50 μM of SANT-1, such as about 0.25 μM of SANT-1.

[0243] In one embodiment, step b+1) of the method as disclosed herein includes culturing a cell population in a medium comprising approximately 20 ng / mL of BTC, approximately 10 μM of Alk5i II, approximately 100 nM of GSI-XX, approximately 1 μM of GC-1, approximately 100 nM of LDN, approximately 100 nM of RA, and approximately 0.25 μM of SANT-1.

[0244] As discussed above, in one embodiment, a method of generating in vitro islet-like cell aggregates as disclosed herein is provided, wherein the population of endocrine progenitor cells are endocrine progenitor cells characterized, for example, by the expression of NEUROD1. In one embodiment, the EP cells are characterized by the expression of NGN3. In one embodiment, the EP cells are characterized by the expression of NGN3 and NEUROD1. In another embodiment, the EP cells are characterized by the expression of NKX6.1 and NEUROD1. In one embodiment, the EP cells are further characterized by the expression of at least one of PDX1 and NGN3.

[0245] In one embodiment, in step b+1), the cell population of pancreatic progenitor cells is cultured for approximately 3 to 5 days, such as approximately 3 to 4 days or approximately 4 to 5 days, such as approximately 4 days or such as approximately 5 days. In one embodiment, the pancreatic progenitor cells are cultured on a 2D substrate until at least endocrine progenitor cells are generated in step c+1).

[0246] During the differentiation protocol, the cells can be cultured on a 2D substrate, such as adherently cultured on a 2D substrate. It should be understood that the above discussion related to the characteristics of the 2D substrate is equally relevant in the context of steps a)–c+1), and is not repeated here for the sake of brevity.

[0247] It should be understood that the endocrine progenitor cells obtained in step c+1) can be further differentiated into pancreatic monohormonal β cells. The inventors have found that it is beneficial to transfer the cells from 2D culture conditions to 3D culture conditions at this step.

[0248] In one embodiment of the method, the cells are not transferred from culturing on a 2D substrate to culturing on a 3D substrate until the expression of characteristic markers of endocrine progenitor cells is exhibited. Endocrine progenitor cells are characterized by the expression of PDX1, NKX6.1, and at least one of NEUROD1 and NGN3, as discussed above.

[0249] In one embodiment, a method as disclosed herein is provided, which further comprises steps i)-v) as defined herein after steps a+1)–c+1), including transferring a population of the endocrine progenitor cells (e.g., endocrine progenitor cells characterized by expressing NKX6.1 and NEUROD1) from culture on a 2D substrate to 3D culture conditions;

[0250] Culturing the population of the endocrine progenitor cells under conditions permitting differentiation into pancreatic monohormonal β-cells; and resulting in the generation of a population of pancreatic monohormonal β-cells, which are pancreatic monohormonal β-cells characterized by expressing insulin, for example. The monohormonal β-cells may also express at least one of NKX6.1, PDX1, and NEUROD1.

[0251] As discussed in detail above, 3D culture conditions permit cell self-aggregation.

[0252] In one embodiment, steps i)–v) are after steps a+1)–c+1), for example immediately after steps a+1)–c+1). It should be understood that the endocrine progenitor cells can be cryopreserved for a desired period of time before performing steps i)-v). In this case, steps i)-v) are after the cryopreservation step and subsequent thawing of the cryopreserved cells. Those skilled in the art are familiar with the process of thawing cryopreserved cells, which generally includes rapidly thawing the cells in a 37 °C water bath, removing the cells from the cryopreservation medium by gentle centrifugation and / or dilution with growth medium, and inoculating the cells in complete growth medium in a culture vessel. Thus, in one embodiment, steps i)-v) are after the cryopreservation and subsequent thawing of the EP cells obtained in c+1).

[0253] In one embodiment, step iv) includes culturing the population of the endocrine progenitor cells for about 3 weeks or longer, such as about 3 to 5 weeks, such as about 4 weeks.

[0254] As used herein, the term “monohormonal” refers to cells that express only one hormone. For example, monohormonal β-cells express only insulin and do not express other hormones expressed by islet cells, such as glucagon or somatostatin. As used herein, the term “multihormonal” refers to cells that express at least two different hormones.

[0255] In vivo monohormonal β-cells are monohormonal cells, and advantageously, the population obtained by the method of the present invention exhibits the characteristics of in vivo natural β-cells or in vivo endogenous β-cells, such as the characteristics of in vivo healthy natural β-cells or in vivo healthy endogenous β-cells.

[0256] Thus, in one embodiment of the method disclosed herein, the population of the single-hormone β cells generated in step v) is single-hormonal. Specifically, the population of the single-hormone β cells generated in step v) does not express glucagon or somatostatin. Specifically, the population of the single-hormone β cells generated in step v) does not express both glucagon and somatostatin.

[0257] It should be understood that, compared to the prior art methods in which step b) is culturing the cell population of the posterior foregut cells for about 96 hours or longer under conditions permitting differentiation into pancreatic progenitor cells, the increased proportion and / or number of EP cells obtained by the method according to steps a-1) to c+1) can be translated into higher quality islet-like cell aggregates, such as those containing a higher proportion of single-hormone β cells as defined herein. Moreover, the islet-like cell aggregates may contain a low percentage of multi-hormonal cells, a low percentage of single-hormonal cells that are not β cells, and / or a low percentage of proliferating cells.

[0258] In one embodiment disclosed herein, a method is provided wherein in step c+1), more than about 30%, such as more than about 40%, 40%, such as more than about 45%, such as more than about 50% of the total cell population are endocrine progenitor cells, such as endocrine progenitor cells characterized by expressing NKX6.1 and at least NEUROD1. As discussed and illustrated above, other combinations of two or more of the markers PDX1, NKX6.1, NEUROD1, and NGN3 can be used to characterize endocrine progenitor cells. In one embodiment, in step c+1), more than about 30%, such as more than about 40%, such as more than about 45%, such as more than about 50% of the total cell population are endocrine progenitor cells, such as endocrine progenitor cells characterized by expressing NKX6.1 and NEUROD1.

[0259] In one embodiment, a method as disclosed herein is provided wherein the number of endocrine progenitor cells in step c+1) is higher compared to the number of endocrine cells obtained using a corresponding method in which step b) is culturing the cell population of the posterior foregut cells for about 24 hours or shorter and / or about 96 hours or longer. Thus, according to the present invention, the culturing time of step b) results in an increase in the number of EP cells. As shown in the appended examples, the number of EP cells obtained by the method as disclosed herein is significantly higher compared to methods with longer or shorter culturing times in step b). Specifically, this is surprising and unexpected because the number of PP cells is less compared to the number of PP cells when step b) is about 96 hours or longer.

[0260] In one embodiment, the method produces at least about 10%, such as at least about 15%, such as at least about 20%, such as at least about 30%, such as at least about 40%, such as at least 50%, such as at least 60% more endocrine progenitor cells than the corresponding method, in which step b) is culturing the cell population of the posterior foregut cells for about 24 hours or less and / or about 96 hours or more under conditions permitting differentiation into pancreatic progenitor cells.

[0261] In a specific embodiment of the method as disclosed herein, the number of endocrine progenitor cells in step c+1) is higher compared to the number of endocrine cells obtained using the corresponding method when step b-1) is culturing the cell population of the primitive gut tube cells for no more than 54 hours under conditions permitting differentiation into posterior foregut cells to obtain a population of posterior foregut cells, in which step b-1) is culturing the cell population of the primitive gut tube cells for about 56 hours or more under conditions permitting differentiation into posterior foregut cells. Specifically, the combination of the culture time of step b-1) and the culture time of step b) is considered particularly beneficial with respect to the number of EP cells obtained in step c+1).

[0262] This effect is demonstrated in the appended examples and particularly in Figure 4, in which the number of endocrine progenitor cells is scored by co-expression of NKX6.1 and NEUROD1. Importantly, this effect is demonstrated in cell cultures of different human stem cell lines, including human ESC lines and IPSC lines.

[0263] As explained above, it should be understood that the increased proportion and / or number of EP cells obtained by the present method can be translated into a higher proportion of single-hormone β cells as defined herein compared to prior art methods in which step b) is culturing the cell population of the posterior foregut cells for about 96 hours or more under conditions permitting differentiation into pancreatic progenitor cells. It should be understood that the increased proportion and / or number of EP cells obtained by the method of the present invention can be translated into a higher proportion of single-hormone β cells as defined herein compared to prior art methods in which step b-1) is 56 hours or more and step b) is 96 hours or more.

[0264] In one embodiment of the method disclosed herein, a method is provided, wherein in step v), more than about 40%, such as about 40% to 50%, such as about 40% to 60%, such as about 40% to 70% of the total cells in the islets are single-hormone β cells, such as single-hormone β cells characterized by the expression of insulin. In one embodiment, the method produces at least 30%, such as at least about 35%, such as at least about 40% more single-hormone β cells, such as single-hormone β cells characterized by the expression of insulin, than the corresponding method in which step b) is culturing the cell population of the posterior foregut cells for about 96 hours under conditions permitting differentiation into pancreatic progenitor cells. In one embodiment, the method produces at least 30%, such as at least about 35%, such as at least about 40% more single-hormone β cells, such as single-hormone β cells characterized by the expression of insulin, than the corresponding method in which step b-1) is 56 hours or longer and step b) is 96 hours or longer. The single-hormone β cells may be characterized by insulin and at least one of NKX6.1, PDX1, and NEUROD1, such as the expression of insulin and NKX6.1.

[0265] In one embodiment, the single-hormone β cells express insulin and may further be characterized by the expression of NKX6.1, PDX1, and / or NEUROD1. In one embodiment, the single-hormone β cells are characterized by the expression of: insulin and NKX6.1; insulin and PDX1; or insulin and NEUROD1.

[0266] In one embodiment, the single-hormone β cells are characterized by the expression of: insulin and two of NKX6.1, PDX1, and NEUROD1; such as insulin, NKX6.1, and PDX1; or insulin, NKX6.1, and NEUROD1; or insulin, PDX1, and NEUROD1. In one embodiment, the single-hormone β cells are characterized by the expression of insulin, PDX1, NKX6.1, and NEUROD1.

[0267] In a particular embodiment, the method produces at least 2-fold more pancreatic single-hormone β cells, such as pancreatic single-hormone β cells characterized by the expression of insulin, than single-hormone pancreatic α cells characterized by the expression of glucagon. Thus, the islet-like cell aggregates obtained by the method of the present invention contain at least 2-fold more pancreatic single-hormone β cells than α cells.

[0268] Skilled artisans understand that single-hormone pancreatic β-cells not only express insulin and characteristic markers of mature pancreatic β-cells, but also that the single-hormone β-cells are functional pancreatic β-cells and are thus capable of responding to glucose stimulation. The result of glucose stimulation can be scored by insulin production and / or by the expression of C-peptide. C-peptide is released simultaneously with insulin, and for each insulin molecule produced, there is one C-peptide molecule produced by the β-cells. C-peptide itself does not affect blood glucose, but C-peptide is a useful marker of insulin production because C-peptide tends to remain in the blood longer than insulin.

[0269] Thus, in one embodiment, there is provided a method as described herein, wherein the single-hormone β-cells, such as the single-hormone β-cells of the islet-like cell aggregates, are functional pancreatic β-cells, such as functional pancreatic β-cells scored by the expression of C-peptide after glucose stimulation.

[0270] The aim of the method is to provide islet-like cell aggregates containing single-hormone β-cells, such as human islet-like cell aggregates containing single-hormone β-cells, by means of in vitro differentiation.

[0271] The source cells can be pluripotent cells, such as cell lines of pluripotent cells, such as embryonic stem cells or induced pluripotent stem cells. Thus, the cells can be from an established cell line, or alternatively, the cells can be primary cells directly derived 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 cells (IPS) or a culture of embryonic stem cells (ES). In one embodiment, the cell population in step a) or a-1) or a+1) is a primary cell population directly derived from a patient. Alternatively, the cell population in step a) or a-1) or a+1) can be derived from a culture of unipotent cells, such as a cell line that has been restricted to the endodermal lineage. The cells can be human cells.

[0272] In one embodiment, the cell population in step a) or a-1) is a mammalian cell population, such as a human cell population. Thus, this also applies to the cell population in step i). Thus, the cell population in step i) can be derived from a culture of pluripotent stem cells, such as a culture of induced pluripotent stem cells (IPS) or a culture of embryonic stem cells (ES). The cell population in step i) can be a primary cell population directly derived from a patient, or derived from a culture of unipotent cells, such as a cell line that has been restricted to the endodermal lineage. The cells can be human cells.

[0273] As used herein, the term "derived from" refers to the source of the cell, and the cell in step i) can be derived via one or several differentiation steps prior to step i) (e.g., one of several in a-1–c-1); a-c); and a+1-c+1) as defined herein).

[0274] It is known in the art that stem cells are undifferentiated cells defined by their ability to self-renew and differentiate at the single cell level. Stem cells can give rise to progeny cells, including self-renewing progenitors, non-renewing progenitors, 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 also give rise to tissues of multiple germ layers after transplantation and make a substantial contribution to most, if not all, tissues after injection into the blastocyst.

[0275] Stem cells are classified according to their developmental potential into: (1) totipotent, meaning able to give rise to all embryonic and extra-embryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system; (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage.

[0276] As explained above, differentiation is the process by which non-specialized ("undirected") or less specialized cells acquire the characteristics of specialized cells (such as, for example, nerve cells or muscle cells). Differentiated cells or cells induced to differentiate are cells that occupy a more specialized ("directed") position in the lineage of the cell. The term "directed" when applied to the process of differentiation means that the cell has progressed in the differentiation pathway to a point where it would normally continue to differentiate into a specific cell type or subset of cell types and cannot normally differentiate into a different cell type or revert to a less differentiated cell type. "Dedifferentiation" is the process by which a cell reverts to a less specialized (or directed) position within the cell lineage.

[0277] It should be noted that the teachings of the present invention can be practiced using any human pluripotent embryonic stem cells, including human pluripotent embryonic stem cells derived without destroying a human embryo, such as those derived from parthenogenetically activated oocytes. In certain embodiments, the cell population in step i) is derived from a population of human embryonic stem cells. In one embodiment, the cell population in step i) is derived from a population of human embryonic stem cells, which is obtained without destroying a human embryo. In certain embodiments, the cell population in step a) or step a-1) or step a+1) is derived from a population of human embryonic stem cells. In one embodiment, the cell population in step a) or step a-1) or step a+1) is derived from a population of human embryonic stem cells, which is obtained without destroying a human embryo.

[0278] In a particular embodiment, the cell population in step a), a-1), a+1) or i) is derived from a population of human embryonic stem cells, such as a population of human embryonic stem cells selected from the group consisting of the embryonic stem cell lines composed of HS980 cells, H1 cells and H9 cells, such as the embryonic stem cell line group composed of H1 cells and HS980 cells, or the embryonic stem cell line group composed of H1 and H9 cells, or the embryonic stem cell line group composed of HS980 cells and H9 cells. In one embodiment, the cell is an H1 cell. In one embodiment, the cell is an H9 cell. In one embodiment, the cell is an HS980 cell. In one embodiment, the population of human embryonic stem cells is a population obtained without destroying an embryo. Only for the purpose of complying with European patent practice, the above embodiments related to HS980, H1 and / or H9 cells will be regarded as reference examples in the European jurisdiction.

[0279] In a particular embodiment, the cell population in step a), a-1), a+1) or i) is derived from iPS cells, such as human iPS cells.

[0280] In a particular embodiment, the iPS cells are selected from the group consisting of patient-derived iPS cells and iPS cell lines. In a particular embodiment, the iPS cell line is, for example, CTRL-7-II (C7). C7 was described in Kele M et al. 2016.

[0281] As discussed above, the method may also include a step of cryopreserving cells, particularly it may be suitable for cryopreserving endocrine progenitor cells. Thus, in one embodiment, a method is provided that includes a step of cryopreserving endocrine progenitor cells. In one embodiment, the cryopreservation is of the endocrine progenitor cells generated in step c+1). In one embodiment, the method as disclosed herein includes cryopreserving EP cells prior to step i). In one embodiment, the population of EP cells provided in step i) is a population of cryopreserved EP cells or a population of EP cells that have been cryopreserved previously. As shown in the appended examples, cryopreservation does not have a negative impact on the generation of the islet-like cell aggregates or single-hormone β cells.

[0282] In a second aspect of the present disclosure, isolated islet-like cell aggregates or populations of isolated islet-like cell aggregates obtainable by the method as described herein are provided. Cells obtainable from the islet-like cell aggregates are also provided, as discussed in the third aspect below. Specifically, the islet-like cell aggregates contain single-hormone β cells. 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 islets in vivo, both in terms of the distribution of cell types present herein and in terms of their functional properties. Those skilled in the art will recognize that

[0283] Specifically, the isolated islet-like cell aggregates or populations of isolated islet-like cell aggregates are characterized in that they exhibit the following desirable properties: a high number of single-hormone β cells, a desirable number of single-hormone α cells, a low number of multi-hormone cells (including a low number of multi-hormone β cells and a low number of multi-hormone α cells), a low number of non-endocrine cells, and a low number of proliferating cells.

[0284] Thus, in one embodiment, the isolated islet-like cell aggregates contain 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%, such as at least about 50%, such as at least about 55%, such as at least about 70% single-hormone β cells. In one embodiment, the islet-like cell aggregates contain from about 25% to 70%, such as from about 30% to 70%, such as from about 40% to 70%, such as from about 40% to 60% single-hormone β cells.

[0285] In one embodiment, the isolated islet-like cell aggregates contain at least 40%, such as at least 45%, such as at least 50%, such as at least 55% β cells, such as at least 60% of β cells, such as at least 65%, such as at least 70% single-hormone β cells. In one embodiment, the single-hormone β cells are characterized by insulin expression.

[0286] In one embodiment, the islet-like cell aggregate comprises up to about 20%, for example, up to about 18%, for example, up to about 16%, for example, up to about 13%, for example, up to about 10% of monohormone α cells. In one embodiment, the monohormone α cells are characterized by expressing glucagon.

[0287] In one embodiment, the isolated islet-like cell aggregates comprise monohormonal β cells and α cells, and contain less than about 5%, for example less than about 4%, 3%, 2%, or 1%, of 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%, for example up to about 4%, 3%, 2%, or 1%, of multihormonal cells. In one embodiment of the method, the islet-like cell aggregates in v) comprise up to about 5%, for example up to about 4%, 3%, 2%, or 1%, of non-endocrine cells. Therefore, in one embodiment, the isolated islet-like cell aggregates comprise, or the isolated population comprises islet-like cell aggregates containing at least about 25%, for example at least about 30%, for example at least about 35%, for example at least about 40%, for example at least about 45%, for example at least about 50%, for example at least about 55%, for example at least about 60%, for example at least about 65%, for example at least about 70%, of monohormonal β cells. In one embodiment, the isolated islet-like cell aggregates comprise, or the isolated population comprises islet-like cell aggregates containing, approximately 25% to 70%, for example 30% to 70%, for example 30% to 70% monohormone β cells, for example 35% to 70%, 35% to 70%, for example 40% to 70%, for example 45% to 70%, for example 45% to 65%, for example 45% to 60%, for example 45% to 55%, for example approximately 50% monohormone β cells. In another embodiment, the islet-like cell aggregates comprise, or the population comprises islet-like cell aggregates containing, approximately 35% to 65%, for example 40% to 65%, for example 40% to 60% monohormone β cells.

[0288] In one embodiment, the islet-like cell aggregate comprises, or the population comprises islet-like cell aggregates containing, approximately 7% to 25%, for example 7% to 20%, 10% to 20%, for example 15% to 20%, for example approximately 20% monohormone alpha cells.

[0289] In one embodiment, the islet-like cell aggregate comprises, or the population comprises islet-like cell aggregates containing, up to about 10%, for example, up to about 7%, for example, up to about 6%, for example, 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 0.5%, for example, up to about 0.3%, for example, up to about 0.1% of multi-hormone α cells.

[0290] In one embodiment, the islet-like cell aggregate comprises, or the population comprises islet-like cell aggregates containing, up to about 10%, for example, up to about 7%, for example, up to about 6%, for example, 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 0.5%, for example, up to about 0.3%, for example, up to about 0.1% of multi-hormone β cells.

[0291] In one embodiment, the islet-like cell aggregate comprises, or the population comprises islet-like cell aggregates containing less than 5%, for example less than 4%, for example less than 3%, for example less than 1% of delta cells.

[0292] In one embodiment, the islet-like cell aggregate comprises, or the population comprises, islet-like cell aggregates containing: 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% of proliferating cells, for example proliferating cells expressing Ki-67.

[0293] In one embodiment, the isolated in vitro islet-like cell aggregates are scored at the end of S6, for example, on days 38–42 of the culture described herein, such as on days 38, 39, 40, 41, 42 or later.

[0294] It should be understood that the isolated islet-like cell aggregates, populations thereof, or cells derived therefrom can be used in therapies, such as cell replacement therapy, as well as in drug development or other research applications. Specifically, it should be understood that providing a population exhibiting a high percentage or high fraction of the desired cell type is advantageous when no further cell selection or sorting is required. For clarification, the term "isolated" in relation to isolated islet-like cell aggregates, populations thereof, or cells derived from such aggregates refers to the removal (in other words, isolation) of cells from their natural environment (e.g., the in vivo environment). It should be understood that the isolated islet-like cell aggregate populations, populations thereof, or cells derived from islet-like cell aggregates as disclosed herein can be part or all of a cell aggregate formed during cell culture. Such aggregates (also referred to as islet-like cell aggregates) may contain, in addition to pancreatic β cells, cells such as pancreatic α cells and / or δ cells, or cells from one or more pancreatic α and / or δ cell lineages.

[0295] Therefore, in one embodiment, the isolated islet-like cell aggregates or populations of islet-like cell aggregates are provided, wherein the cells constituting the islet-like cell aggregates have not yet undergone enrichment for a desired phenotype, for example, not yet undergone enrichment by manual intervention or by machine (in other words, automated sorting). In one embodiment, the cells have not undergone enrichment before forming 3D structures in step iii). Therefore, in one embodiment of the isolated islet-like cell aggregates or populations of isolated islet-like cell aggregates, the cells constituting the islet-like cell aggregates have not yet undergone enrichment for a desired phenotype, for example, not yet undergone sorting for a desired phenotype, such as sorting based on the expression of a desired marker or, for example, sorting based on FACS for a desired phenotype. Those skilled in the art will understand that selection / sorting may be based on the presence of a desired phenotype (e.g., based on marker expression) or on the absence of an undesired phenotype, in which case undesired cells are removed from the population, and thus the population is enriched with cells exhibiting the desired phenotype. In one embodiment, the cells have not undergone sorting prior to forming the 3D structure in step iii). For clarity, as used herein, the term "enrichment" refers to enriching cells through intervention (manual or machine-based), such as sorting cells based on cell characteristics, rather than a process that occurs naturally in cell culture. Therefore, in one embodiment, the cells constituting the islet-like cell aggregates (in other words, the cells of the islet-like cell aggregates) have not been sorted for a desired phenotype, such as based on the expression of a desired biomarker. In one embodiment, the cells constituting one or more of the islets have not undergone FACS-based sorting for a desired phenotype. In one embodiment, the cells containing one or more of the islets have not undergone enrichment by removing undesirable phenotypes, such as based on biomarker expression, such as via FACS.

[0296] The islet-like cell aggregates of the present invention are characterized by a high percentage of cells with desired properties obtained through their own differentiation methods. Therefore, in one embodiment of this aspect, isolated islet-like cell aggregates are provided, wherein more than about 40%, for example about 40 to 60%, or about 40 to 50%, of the total cell population of one or more said islets are monohormonal β cells, such as monohormonal β cells characterized by insulin expression. In one embodiment, the population comprises at least two times (e.g., three, four, or five times) more monohormonal pancreatic β cells characterized by insulin expression than monohormonal pancreatic α cells characterized by glucagon expression.

[0297] The monohormone β cells can be characterized as described above, for example by expressing insulin and at least one or more, such as two, of the markers NKX6.1, PDX1, and NEUROD1. The monohormone β cells may be characterized by expressing insulin and NKX6.1, for example, by expressing insulin, NKX6.1, and at least one of PDX1 and NEUROD1. In one embodiment, the monohormone β cells may be characterized by expressing insulin, NKX6.1, PDX1, and NEUROD1. In one embodiment, the monohormone β cells may comprise portions of cell aggregates, for example, also comprising portions of cell aggregates of pancreatic monohormone α and / or δ cells.

[0298] In a related third aspect, cells obtained from isolated islet-like cell aggregates as defined herein, these portions being obtained by dissociating one or more of the islets, are provided. Thus, populations of such cells are provided. For the sake of brevity, such cells are referred to in this context as “cells derived therefrom”.

[0299] As mentioned above, it is envisioned that isolated islets or isolated islet-like cell aggregates, or cells derived therefrom, as disclosed herein, could be used to treat and / or prevent diabetes.

[0300] Therefore, in a third aspect of this disclosure, isolated islet-like cell aggregates, isolated populations of islet-like cell aggregates, or cells derived therefrom, as disclosed herein, are provided for use in a therapy (in other words, as a medicine).

[0301] It should be understood that, for the purpose of cell replacement therapy, the islet-like cell aggregates or cells derived therefrom may be transplanted into patients in need. The replacement therapy may be for the purpose of providing pancreatic β cells to patients who do not have endogenous β cells or only have non-functional β cells, or to patients who require more pancreatic β cells (because their endogenous population is reduced or functional below what is needed for a healthy patient state).

[0302] The islet-like cell aggregates or cells may be derived from a donor and are 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 β-cell lineage. Non-limiting examples include hES cell lines, non-embryonic stem cell lines (e.g., pluripotent, oligopotent, or unipotent cell lines) with the potential to develop along the endocrine lineage, iPS cell lines, and cell lines derived from iPS cells capable of developing along the pancreatic β-cell lineage. The cells may also be patient-endogenous, such as derived from iPS cells obtained from the patient or other patient-derived cells capable of developing along the pancreatic β-cell lineage.

[0303] It is believed that islet-like cell aggregates or cells obtained by the methods disclosed herein will be advantageous for use in therapy because the total percentage of the desired cell type is higher than that obtained by the previously described culture methods. Therefore, islet-like cell aggregates or cells obtained by the disclosed methods, compared to those generated by methods known in the art, will require at least a lower degree of exposure to cell sorting or similar stress and potentially destructive techniques to obtain a homogeneous population with a high percentage of the desired cell type. Therefore, treatments according to this disclosure, such as transplantation (e.g., in the form of cell replacement therapy), are expected to be performed with healthier and more vigorous populations of islets or cells compared to those generated 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 considered beneficial to patients in terms of fewer potential adverse side effects and better clinical outcomes.

[0304] In a fourth aspect, isolated islet-like cell aggregates, isolated populations of islet-like cell aggregates, or cells derived therefrom, as disclosed herein, are provided for use in the treatment, prevention, and / or improvement of diabetes (e.g., type 1 or type 2 diabetes). In one embodiment, the diabetes is type 1 diabetes. In one embodiment, the type 2 diabetes is insulin-deficient type 2 diabetes.

[0305] In one embodiment, isolated islets, isolated islet-like cell aggregates, or cells derived therefrom, as disclosed herein, are provided for use in a therapeutic treatment, wherein the isolated islets, isolated islet-like cell aggregates, or cells derived therefrom, as disclosed herein, have been generated by methods as defined herein.

[0306] In one embodiment, isolated islet-like cell aggregates, isolated populations of islet-like cell aggregates, or cells derived therefrom are provided for use in a therapy (in other words, as a drug), wherein said use includes the steps of generating isolated islet-like cell aggregate cells, isolated populations of islet-like cell aggregates, or cells obtained therefrom according to methods as defined herein; and

[0307] Administer a therapeutically effective amount of the islet-like cell aggregates or cells to the patient.

[0308] In one embodiment, the use also includes isolating cells from the patient, such as isolating cells from the patient for generating iPS cells, or isolating stem cells, and using the cells to generate islet-like cell aggregates or cells according to methods as defined herein.

[0309] In one embodiment, isolated islet-like cell aggregates or cells derived therefrom are provided for use in the treatment, prevention, and / or improvement of diabetes (e.g., type 1 or type 2 diabetes), wherein the islet-like cell aggregates or cells have been generated by methods as defined herein.

[0310] In another embodiment, isolated islet-like cell aggregates or cells derived therefrom are provided for use in the treatment, prevention, and / or improvement of diabetes (e.g., type 1 or type 2 diabetes), wherein the use includes the following steps:

[0311] The islet-like cell aggregates or cells are generated according to the methods defined herein; and

[0312] A therapeutically effective amount of the cells is administered to the patient. In one embodiment, the islet-like cell aggregates may be administered in the form of islet-like cell aggregates, in other words, in the form of cell aggregates, as defined above. Alternatively, the islet-like cell aggregates may be dissociated and these cells may be administered in the form of cell suspensions (e.g., single-cell suspensions). In one embodiment, the use further includes isolating cells from the patient, such as cells for generating iPS cells, or isolating stem cells from the patient, and using the cells to generate the islet-like cell aggregates or cells derived therefrom according to the methods defined herein. Similar to what has been discussed with respect to the third aspect, other cell types are considered useful in this regard, including allogeneic cells and endogenous cells.

[0313] Therefore, in one embodiment, the use includes the step of generating isolated islet-like cell aggregates according to the method defined herein; and

[0314] The patient is given a therapeutically effective amount of the islet-like cell aggregate.

[0315] Therefore, in one embodiment, the use includes the step of generating isolated islet-like cell aggregates according to the method defined herein;

[0316] The islet-like cell aggregates are dissociated, and a therapeutically effective amount of the dissociated islet cells is administered to the patient.

[0317] It should be understood that the cell replacement therapy may involve the transplantation of islet-like cell aggregates (cell aggregates) or cells derived therefrom, which then produce insulin in the patient and have the ability to respond to glucose stimulation in the patient.

[0318] Therefore, in one embodiment, the use includes transplanting an aggregate of said cells into a patient in need. In another embodiment, the use includes transplanting all cells or a subset of said cells (e.g., monohormonal pancreatic β cells) obtained from the dissociated islet-like cell aggregates into a patient in need.

[0319] It is anticipated that the isolated islet-like cell aggregates or cells derived therefrom will be usable as pharmaceutical compositions. As used herein, the term "pharmaceutical composition" encompasses compositions derived from cell therapy. Therefore, in a relevant fifth aspect of this disclosure, a pharmaceutical composition is provided comprising isolated islet-like cell aggregates or cells derived therefrom as disclosed herein and at least one pharmaceutically acceptable excipient or carrier.

[0320] It is also envisioned that it may be advantageous to administer, together with isolated islet-like cell aggregates or cells derived therefrom, or pharmaceutical compositions as disclosed herein, with a loading substrate that promotes the growth, proliferation, and / or optional differentiation of said cell populations. Therefore, in the fourth and sixth aspects of this disclosure, kits are provided comprising isolated islet-like cell aggregates or cells derived therefrom, or pharmaceutical compositions as disclosed herein, and a suitable loading substrate. The suitable loading substrate may be a 3D scaffold.

[0321] In one embodiment, a kit is provided in which the loading substrate is a 3D substrate and wherein said cells are monohormonal β cells. In other embodiments, the kit comprises a 2D substrate. A non-limiting example of such a 2D substrate is the substrate discussed herein.

[0322] It should be understood that one or more isolated islets or cells derived therefrom, as disclosed herein, can have numerous uses in biological research, such as for drug screening, e.g., in vitro drug screening. It should be understood that one or more isolated islets or cells derived therefrom, as disclosed herein, would be advantageous because the percentage of the desired cell type in the total cells would be higher than the percentage in islet-like cell aggregates of cell populations obtained by the previously described culture methods. Therefore, compared to islet-like cell aggregates or cells generated according to the prior art, the islet-like cell aggregates or cells derived therefrom of the present invention, if necessary, would require at least a lower degree of exposure to cell sorting or similar stress and potentially destructive techniques to obtain a homogeneous population with a high percentage of the desired cell type. A homogeneous population with a high percentage of the desired cell type is likely to generate data in drug screening assays that are less or unaffected by one or more potential responses from other irrelevant or contaminating cell types. Therefore, in the seventh aspect of this disclosure, the use of isolated islet-like cell aggregates or cells derived therefrom, as disclosed herein, in drug screening (e.g., in vitro drug screening).

[0323] This document provides a method for in vitro drug screening, comprising the steps of: generating isolated islet-like cell aggregates or cells derived therefrom according to a method as defined herein; and exposing the islet-like cell aggregates or cells to at least one candidate drug compound. The method may further include the step of evaluating the response of the isolated islet-like cell aggregates or cells derived therefrom to the candidate drug compound.

[0324] In one embodiment, an in vitro drug screening method is provided, the method comprising the steps of: generating isolated islet-like cell aggregates according to a method defined herein; and exposing the islet-like cell aggregates to at least one candidate drug compound.

[0325] In one embodiment, an in vitro drug screening method is provided, the method comprising the steps of: generating isolated islet-like cell aggregates according to a method defined herein; dissociating the islet-like cell aggregates; and exposing at least a portion of the dissociated islet cells to at least one candidate drug compound.

[0326] In an eighth aspect, a method for treating a patient in need is provided, the method comprising administering to the patient a therapeutically effective amount of isolated islet-like cell aggregates or cells derived therefrom as disclosed herein. Furthermore, a method for treating diabetes in a patient in need is provided, the method comprising the steps of: generating isolated islet-like cell aggregates or cells derived therefrom according to a method as defined herein; and

[0327] The isolated islet-like cell aggregates or cells derived therefrom are administered to the patient in a therapeutically effective amount. In one embodiment, the isolated islet-like cell aggregates or cells derived therefrom are allogeneic, and in another embodiment, the isolated islet-like cell aggregates or cells derived therefrom are endogenous to the patient.

[0328] In one implementation, the method is used to treat diabetes, such as type 1 or type 2 diabetes. Therefore, the patient may have type 1 or type 2 diabetes.

[0329] Therefore, a method for treating diabetes in a patient in need is provided, the method comprising administering to said patient a therapeutically effective amount of isolated islet-like cell aggregates or cells derived therefrom as disclosed herein. Furthermore, a method for treating a patient in need is provided, the method comprising the steps of: generating isolated islet-like cell aggregates or cells derived therefrom according to a method as defined herein; and

[0330] The method involves administering a therapeutically effective amount of the isolated islet-like cell aggregate or cells derived therefrom to the patient. The method may further include isolating cells from the patient, such as cells for generating iPS cells or stem cells, and using the cells to generate the isolated islet-like cell aggregate or cells derived therefrom according to methods as defined herein. The islet-like cell aggregate or cells may be administered via transplantation.

[0331] Therefore, in one embodiment, the administration includes transplanting the islet-like cell aggregates or cells into the patient.

[0332] In a related ninth aspect, the use of isolated islet-like cell aggregates or cells derived therefrom as described herein for the manufacture of a medicament for the treatment of diabetes in patients in need is provided. In one embodiment, the manufacture of said medicament comprises generating isolated islet-like cell aggregates or cells derived therefrom by methods as defined herein. In one embodiment, said isolated islet-like cell aggregates or cells derived therefrom are patient-endogenous, in other words, patient-specific islet-like cell aggregates or cells. In one embodiment, said isolated islet-like cell aggregates or cells derived therefrom are allogeneic islet-like cell aggregates or cells.

[0333] It should be understood that any discussion relating to the third and fourth aspects of this disclosure and its embodiments is equally relevant to these eight aspects and the related ninth aspect, and will not be repeated here for the sake of brevity only. In one embodiment, a method for treating diabetes in a patient in need, as disclosed herein, is provided, wherein the patient has type 1 or type 2 diabetes. In one embodiment, the administration comprises transplanting the population into the patient.

[0334] In yet another related aspect, a method for generating in vitro islet-like cell aggregates is provided, the method comprising the following steps:

[0335] a-1) Provide a population of primitive intestinal cells, which are, for example, primitive intestinal cells characterized by the expression of HNF1β and / or HNF4α;

[0336] b-1) The cell population of the primitive intestinal cells was cultured for no more than approximately 54 hours under conditions that allowed differentiation into hindforegut cells;

[0337] c-1) thereby generating a population of hindbrain cells, which are, for example, hindbrain cells characterized by expression of PDX1.

[0338] a) Provide a cell population of hindbrain cells generated in step c-1), which are, for example, hindbrain cells characterized by expressing PDX1;

[0339] b) The cell population of the posterior foregut cells is cultured under conditions that allow differentiation into pancreatic progenitor cells for no more than about 78 hours, for example, no more than about 72 hours.

[0340] c) This generates a population of pancreatic progenitor cells, which are, for example, pancreatic progenitor cells characterized by the expression of both PDX1 and NKX6.1;

[0341] a+1) provides the cell population of pancreatic progenitor cells generated in step c);

[0342] b+1) Culture the pancreatic progenitor cell population under conditions that allow differentiation into endocrine progenitor cells;

[0343] c+1) thereby generating a population of endocrine progenitor cells, which are, for example, endocrine progenitor cells characterized by the expression of NEUROD1, such as those characterized by the expression of NKX6.1 and NEUROD1;

[0344] i) Provide a population of endocrine progenitor cells (EPs) generated in step c+1), which are, for example, EP cells characterized by expression of NEUROD1; for example, EP cells characterized by expression of NKX6.1 and NEUROD1;

[0345] ii) Provide a single-cell suspension of the population of said EP cells;

[0346] iii) Allows the population of said EP cells in a single-cell suspension to form a 3D structure;

[0347] iv) Culture the EP cells in a 3D structure under 3D culture conditions that allow differentiation into pancreatic monohormonal β cells to provide islet-like cell aggregates; and

[0348] v) This generates an aggregate of islet-like cells containing mono-hormone β cells.

[0349] The islet-like cell aggregates described therein contain at least 25% monohormone β cells.

[0350] Similar to the first aspect, step v) relevant to this aspect can be rephrased as “thereby generating a population of islet-like cells comprising mono-hormone β cells, wherein the population comprises islet-like cells containing at least 25% mono-hormone β cells.” It should be understood that the features disclosed in the context of the first aspect, particularly in terms of the percentage of different cells, are equally relevant here and will not be repeated here solely for the sake of brevity.

[0351] As used herein, the phrase “a population of…” is intended to be interpreted as a population containing a certain type of cell when referring to a particular cell type. For example, the phrase “a population of EP cells” should be understood as a population containing EP cells. This population may also include other cells, such as, but not limited to, cells from early developmental stages, such as, for example, PP cells.

[0352] As used herein, when the terms “about” or “approximately” are used with respect to numerical values, they should be interpreted as a range of ±10%, such as ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%. For example, when a value is stated as about 10, this means that the value is actually in the range of 9 to 11, such as in the range of 9.9 to 10.9, 9.8 to 10.8, 9.7 to 10.7, 9.6 to 10.6, 9.5 to 10.5, 9.4 to 10.4, 9.3 to 10.3, 9.2 to 10.2, or 9.1 to 10.1.

[0353] Those skilled in the art know that numerical values ​​related to measurement are affected by measurement errors, thus limiting their accuracy. For this reason, the general convention in scientific and technical literature applies: the last decimal place of a numerical value indicates its accuracy. In the absence of other error tolerances, the maximum tolerance is determined by applying rounding conventions to the last decimal place; for example, for a measurement of 3.5 cm, the error tolerance is 3.45–3.54. Those skilled in the art interpret numerical ranges in patent specifications on the same basis.

[0354] While the invention has been described with reference to various exemplary aspects and embodiments, those skilled in the art will understand that various changes can be made and elements can be substituted with equivalents without departing from the scope of the invention. Furthermore, many modifications can be made to adapt particular situations, culture conditions, or cell populations to the teachings of the invention without departing from the essential scope of the invention. Therefore, it is intended that the invention be limited to any particular embodiment contemplated, but rather that the invention encompass all embodiments falling within the scope of the appended claims. Attached Figure Description

[0355] Figure 1 is a schematic diagram of the developmental stages along the pancreatic β-cell lineage, including the expression of characteristic markers for each developmental stage. (A) Pancreatic endocrine cell types can be generated from human pluripotent embryonic stem cells (hES) or iPS cells by reproducing embryonic pancreatic development in a Peyer's dish. Pancreatic differentiation can be divided into several stages, including the fixed endoderm (DE), primitive gut (PGT), hindbrain (PF), pluripotent pancreatic progenitor cells (PP), endocrine progenitor cells (EP), and islets of Langerhans (ISL). Key markers expressed at each stage are shown in the figure. (B) Figure 1B This is a schematic diagram of a long differentiation protocol based on existing technology and a short differentiation protocol as disclosed herein. The effects of the long and short durations of stages 3 and 4 (S3+S4) on subsequent endocrine differentiation were analyzed. The expression of stage-specific markers was examined at the end of stage 2 (S2), at the end of stage 3 (S3), at the end of stage 4 (S4), four days into stage 5 (S5), and at the end of stage 6 (S6). 2D LN-521 is an example of a 2D substrate and can be replaced by other 2D substrates as disclosed herein.

[0356] 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 protocol. The expression of key progenitor cell markers PDX1, NKX6.1, and NEUROD1 was examined by flow cytometry at the end of S4 or 4 days into S5. Representative dot plots of HS980 cells in (A) stage 4 and (B and C) stages 5 are shown. Bar charts representing the results for HS980 and H1 cells are shown. Results were calculated based on three independent samples. The percentages of PDX1+ / NKX6.1+ and NKX6.1+ / NEUROD1+ cells in (A and B) and (C) substrates were compared using an unpaired two-tailed t-test in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001.

[0357] Figure 3 shows a comparison of different culture lengths of primitive intestinal cells (PGT) and hindbeg cells (PF). HS980 and H1 cells differentiated into S3 on LN-521-coated plates. The expression of the PF marker PDX1 was analyzed by immunocytochemistry (ICC) and flow cytometry at the end of S2 or at day 1 or 2 before entering S3. Representative confocal micrographs and dot plots of HS980 cells are shown. (A) HS980 cells. (B) H1 cells.

[0358] Figure 4 shows the evaluation results of the effect of S4 duration on endocrine differentiation. HS980, H1, and H9 cells differentiated on LN-521-coated plates. The duration of S4 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 at four days into S5. The expression of endocrine markers insulin (INS) and glucagon (GCG) was examined at the end of S6. Results were calculated based on multiple independent experiments. To compare the percentage of different cell populations at different S4 durations, an unpaired two-tailed t-test was performed in Microsoft Excel. (A) Schematic diagram of different S4 durations. (B) Representative dot plot of progenitor markers NKX6.1 and NEUROD1 expression analysis in HS980 cells at S4 and S5. (C) Representative dot plot of progenitor markers NKX6.1 and NEUROD1 expression analysis in H1 and H9 cells at S5. (D) Bar chart showing the calculated percentages of NKX6.1+ / NEUROD1+ and NKX6.1+ / NEUROD1- cells. Results show the percentages of H980, H1, and H9 cells. (E) Bar chart 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 plot of INS and GCC expression analysis in HS980, H1, and H9 cells at S6. (G) Bar chart showing the calculated percentages of total INS+ / GCG- monohormone β cells and GCG+ / INS- monohormone α cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001.

[0359] Figure 5 shows the assessment of pancreatic differentiation on different coated substrates. Using a short protocol, HS980, H1, and H9 cells differentiated toward S5EP cells on plates coated with different recombinant human laminin isoforms (LN) or Matrigel. H1 cells also differentiated on plates coated with fibronectin (FN) or villin (VTN). NKX6.1 and NEUROD1 expression was measured by flow cytometry at day four of S5. LN-332, LN-511, LN-521, and HS980 cells on Matrigel further differentiated toward S6 islet cells in 3D suspension. INS and GCG expression was measured by flow cytometry at the end of S6. A representative dot plot of S5 EP cells is shown below. Figure 5A-1 and 5A-2 As shown, a representative dot plot of S6 pancreatic islet cells is as follows: Figure 5CAs shown. To compare the percentage of NKX6.1+ / NEUROD1+ cells between laminin isotypes and matrix gel, a paired 2-tailed t-test was performed in Microsoft Excel. (B) Percentage of NKX6.1+ / NEUROD1+ cells. *p<0.05, **p<0.01.

[0360] Figure 6 illustrates the effect of 3D suspensions of S4 and S5 progenitor cell cultures on islet formation. (A) Schematic diagram shows the timeline of HS980 cell differentiation on LN-521 using the short protocol. Cells dissociated into single cells at the end of S4 (top timeline) or at S54 or 6 days (bottom timeline). 4 x 10 cells per well 6 S4 and S5 cells were maintained in a 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 a bar chart and are calculated based on 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.

[0361] Figure 7 shows the enrichment of S5 EP cells during aggregate formation in the suspension. HS980 and H1 cells differentiated toward S5 on a 2D surface coated with LN-521. Upon entering S5, cells were dissociated into single cells and then maintained in suspension for one day to generate 3D aggregates. The expression of NKX6.1, NEUROD1, and Ki-67 before and after aggregate formation was examined by flow cytometry. To investigate the effect of the ROCK inhibitor H1152 on aggregate formation, different concentrations of H1152 (0 to 10 μM) were added to single-cell suspensions, as shown in the figure. On the second day, the expression of markers and the number of islet cells were measured. Representative dot plots are shown in (A), (B), and (C). The percentages of NKX6.1+ / NEUROD1+ and NEUROD1+ cells were calculated based on three independent experiments, as shown in the bar chart in (A). From 10 of the 3D suspension... 6 The number of pancreatic islet cells generated per single cell was calculated based on three independent samples and is shown in the bar chart in (D). An 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.

[0362] Figure 8 shows the comparison results of the short and long pancreatic differentiation protocols. HS980 and H1 cells were differentiated into S5 EP cells on LN-521-coated plates using the short protocol as disclosed herein and the comparative long protocol. At day four of S5, cells dissociated into single cells and further matured into islet-like cell aggregates in 3D suspension. As shown in the figures (S3-S6), the expression of key markers was examined by flow cytometry at each stage. In vitro glucose-stimulated insulin C-peptide secretion was examined at the end of S6. Islet-like aggregates were counted under a microscope and then dissociated into single cells for cell counting. Results were calculated based on multiple independent samples. Unpaired 2-tailed t-tests were performed in Microsoft Excel. (A) Representative dot plot of NKX6.1 and NEUROD1 expression in HS980 cells during S3-5. (B) Quantitative bar charts showing NKX6.1+ / NEUROD1+ and NKX6.1+ / NEUROD1- obtained using the short and long protocols. (C) Representative dot plots of INS and GCG expression in HS980 and H1 cells at S6, obtained using the short and long protocols, and bar charts showing their quantification. (D) Bar charts comparing the results of in vitro glucose-stimulated insulin secretion in cells obtained using the short and long protocols, and bar charts showing the number of islet-like aggregates and islet cells per well in cultures using the short and long protocols. HS980 cell aggregates were analyzed at S6. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001.

[0363] Figure 9 shows the immunocytochemical analysis of stage-specific marker expression in cell cultures. HS980 cells differentiated using short and long protocols were analyzed using specific antibodies against key markers expressed during S4–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 for cell visualization.

[0364] Figure 10 shows a comparison between spontaneous aggregation in 3D suspension and forced aggregation using microplates. H1 cells differentiated toward S5 on LN-521-coated 2D surfaces. Four days into S5, cells were dissociated into single cells and then maintained in 3D suspension (free), 96-well plates (96 wells), or AggreWell plates (AggreWell). Expression of NKX6.1, NEUROD1, and Ki-67 was examined by flow cytometry before and one day after aggregate formation at S5. Representative scatter plots are shown in Figure (A). The percentages of S5 NKX6.1+ / NEUROD1+ EP cells and Ki-67+ proliferating cells were calculated from multiple independent samples and are shown in the bar chart in (B). Differentiation toward S6 islet cells was carried out in suspension and AggreWell plates. Expression of INS, GCG, and Ki-67 was examined by flow cytometry at the end of S6. Representative scatter plots are shown in Figure (C). Perform an unpaired two-tailed t-test in Microsoft Excel. *p<0.05, **p<0.01.

[0365] Figure 11 This is a schematic diagram of the in vitro differentiation protocol disclosed in this paper. The factors and duration of each stage are shown. Cells on the LN-521-coated surface dissociated into single cells on day 14 and were then maintained in a 3D suspension.

[0366] Figure 12 shows pancreatic differentiation in human ESC and iPSC lines. Cells were differentiated using a short protocol. Expression of key markers was examined by flow cytometry at four days into 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 based on multiple independent experiments. Unpaired 2-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). (A) shows a bar chart (left) showing quantification of INS+ / GCG-, INS- / GCG+, and INS+ / GCG+ cell populations and a bar chart (right) showing the results of in vitro glucose-stimulated insulin secretion. (A) Differentiation of hESC line. (B) Differentiation of human iPSC line C7.

[0367] Figure 13 shows a comparison between the short protocol and two published scRNA sequencing protocols. H1 cells were differentiated using the short protocol and scRNAseq was performed at the end of S6. (A) Description of the short protocol and the two published protocols. The duration, factors, and 2D / 3D culture system for each stage are shown. (B) UMAP and cell type prediction (top) and expression levels of the biomarker genes INS and GCG in the predicted cell types (bottom).

[0368] Figure 14 Frozen S5 EP cells were shown to generate S6 islet-like aggregates. HS980 and H1 cells differentiated toward S5 on a 2D surface coated with LN-521. After four days in S5, cells dissociated into single cells and were then maintained in a 3D suspension to generate S6 aggregates. Alternatively, single cells were frozen and stored in liquid nitrogen. To generate 3D aggregates, frozen S5 cells were thawed and cultured in a 3D suspension. The expression of the markers INS and GCG, as well as in vitro glucose-stimulated insulin C-peptide release, were examined at the end of S6. A bar graph (left) showing the quantification of INS+ / GCG-, INS- / GCG+, and INS+ / GCG+ cell populations and a bar graph (right) showing the results of in vitro glucose-stimulated insulin secretion are shown. Results were calculated based on multiple independent experiments. Unpaired 2-tailed t-tests were performed in Microsoft Excel. *p<0.05, **p<0.01. Example

[0369] Overview: This embodiment demonstrates the method of the present invention disclosed herein, which includes culturing a cell population of foregut cells for no more than approximately 78 hours, such as 72 hours or 48 hours, under conditions that allow differentiation into pancreatic progenitor cells, resulting in an increase in the number of endocrine precursor cells that have the ability to develop into mature pancreatic β cells.

[0370] Materials and methods

[0371] hESC culture

[0372] hESC line HS980 was derived under heterologous and defined conditions as previously described (Rodin, S., et al., 2014). hESC lines WA01 / H1 and WA09 / H9 were obtained from the Wisconsin Cell Research Institute (Madison, Wisconsin). Human iPSC line CTRL-7-II (C7) is described in Kele M et al., 2016. hESC lines were maintained in a Sarstedt multiporous cell culture plate coated with 10 μg / mL human recombinant laminin (LN)-521 (BioLamina, Sweden; LN-521) at 37°C in an incubator with 5% CO2, 5% O2, and 100% humidity on NutriStem hPSC XF medium (Biological Industries, Israel; 05-100-1A). Every 3-5 days, at 12,000-24,000 cells / cm² 2 Perform enzymatic passage of hESC.

[0373] For routine passage, hESC cultures on LN-521 are cultured in a Ca-free environment. 2+ and Mg 2+ Briefly wash in D-PBS (Thermo Fisher Scientific; 14190169) and incubate with Gibco TrypLE Select (Thermo Fisher Scientific; A1285901) at 37°C for 5 minutes. Collect cells into fresh NutriStem hPSC XF medium by gently pipetting 5-10 times using a P1000 pipette, centrifuge at 300g for 4-5 minutes, resuspend in fresh NutriStem hPSC XF medium, and seed onto newly coated cell culture plates.

[0374] In vitro pancreatic differentiation of hESC

[0375] The stepwise pancreatic differentiation protocol described here is a modification of previously published protocols (Pagliuca et al., 2014; Rezania et al., 2014; Millman et al., 2016; Vegas et al., 2016).

[0376] hESC lines H1, H9, and HS980 were cultured at a concentration of 24,000 cells / cm³. 2 Cells were seeded into NutriStem hPSC XF medium on LN-521-coated cell culture plates. Pancreatic differentiation began after four days, reaching 95%-100% confluence. The differentiated cell cultures were maintained in an incubator at 37°C, 5% CO2, 20% O2, and 100% humidity.

[0377] Differentiation can be divided into six stages, from S1 to S6, and the culture medium used in each stage is as follows:

[0378] S1 medium: MCDB131 (Thermo Fisher Scientific; 10372019) + 25mM NaHCO3 (Sigma; S6297) + 1X GlutaMAX (Thermo Fisher Scientific; 35050038) + 50U / mL penicillin-streptomycin (Thermo Fisher Scientific; 15140122) + 2.5mM D-glucose (8mM final concentration, Sigma; G8769) + 0.2% or 0.5% fatty acid-free bovine serum albumin (FAF-BSA, Sigma; A8806).

[0379] S2 medium: MCDB131 + 25mM NaHCO3 + 1X GlutaMAX + 50U / mL penicillin-streptomycin + 2.5mM D-glucose (8mM final concentration) + 0.2% or 0.5% FAF-BSA + 0.25mM vitamin C (Sigma; A4544).

[0380] S3 medium: MCDB131 + 25mM NaHCO3 + 1X GlutaMAX + 50U / mL penicillin-streptomycin + 2.5mM D-glucose (8mM final concentration) + 0.5% FAF-BSA + 0.25mM vitamin C + 1:200 ITS-X (Thermo Fisher Scientific; 51500056).

[0381] S5 medium: MCDB131 + 25mM NaHCO3 + 1X GlutaMAX + 50U / mL penicillin-streptomycin + 14.5mM MD-glucose (20mM final concentration) + 0.5% FAF-BSA + 1:200ITS-X + 10μM ZnSO4 (Sigma-Aldrich; Z0251) + 10μg / mL heparin (Sigma-Aldrich; H3149).

[0382] S6 medium: CMRL (Thermo Fisher Scientific; 11530037) + 14mM NaHCO3 + 1X GlutaMAX + 50U / mL penicillin-streptomycin + 14.5mM D-glucose (20mM final concentration) + 1% FAF-BSA + 1:200 ITS-X (for three weeks) + 10μM ZnSO4 + 10μg / mL heparin + 1X NEAA (Thermo Fisher Scientific; 11140035).

[0383] Short differentiation scheme

[0384] Stage 1: Determining the endoderm (3 days): Undifferentiated H1, H9, and HS980 cells are treated with a solution containing Ca... 2+ and Mg 2+ Cells were washed once with D-PBS (Thermo Fisher Scientific; 14040091) and then induced for 24 hours in S1 medium with 5 μM CHIR99021 (Tocris; 4423) and 100 ng / ml activin A (Research & Development Corporation; 338-AC). For the next two days, cells were fed daily with S1 medium containing only 100 ng / ml activin A. The concentration of FAF-BSA in S1 medium was 0.2% for HS980 cells and 0.5% for H1 and H9 cells.

[0385] Phase 2 primitive gut (3 days): Cells were induced for three days with 50 ng / ml KGF (Research and Development Corporation; 251-KG) in S2 medium. The FAF-BSA concentration was 0.2% for HS980 cells and 0.5% for H1 and H9 cells.

[0386] Phase 3 foregut (1 day): Cells were induced for 24 hours in S3 medium with 50 ng / ml KGF, 2 μM retinoic acid (Sigma-Aldrich; R2625), 0.25 μM SANT-1 (Sigma-Aldrich; S4572), 0.5 μM PDBu (Torches; 4153) and 200 nM DN193189 (Torches; 6053).

[0387] Phase 4 pancreatic progenitor cells (2 or 3 days): Cells were induced for 3 days in S3 medium with 50 ng / ml KGF, 100 ng / ml EGF (Research and Development Corporation; 236-EG), 5 ng / ml activin A, 10 mM nicotinamide (Sigma-Aldrich; N0636), 100 nM retinoic acid, 0.25 μM SANT-1, 0.5 μM PDBu, and 200 nM LDN193189. To analyze the effect of Phase 4 duration, cells were also differentiated for 1–5 days during this step.

[0388] Phase 5 Endocrine Progenitor Cells (5 days): Cells were induced in S5 medium with 20 ng / ml cytidine (Research and Development Company; 261-CE), 100 nM retinoic acid, 0.25 μM SANT-1, 100 nM GSI-XX (Sigma-Aldrich; 565789), 10 μM ALK5 inhibitor II (Cayman Chemical; 14794), 1 μM GC-1 (Torchs; 4554), and 100 nM LDN193189.

[0389] Four days into stage 5, the cells were treated with calcium-free... 2+ and Mg 2+ Wash once with DPBS, treat with Accutase at 37°C for 10 minutes, and then dissociate into single cells in S5 medium by multiple pipettings using a P1000 pipette. The single cells are pelleted by centrifugation at 300g for 5 minutes, and then separated into single cells at 1.0–1.5 x 10⁻⁶. 6 Cells were resuspended at 10 μM H1152 (Tocris; 2414) and other factors in S5 medium. To generate islet-like aggregates, cells were transferred to ultra-low attachment 6-well plates (Corning; 3471) at a total of 4-6 x 10⁶ cells / ml. 4 Cells were incubated overnight in an incubator at 95 rpm on a fixed-track shaker (Infors HT Celltron).

[0390] To investigate the effects of the ROCK inhibitor H1152 on cell survival and aggregate formation, different concentrations of H1152 (0 μM to 10 μM) were added to single-cell suspensions for one day.

[0391] Phase 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). After three weeks, ITS-X and H1152 were removed from the medium. The aggregates were maintained in an incubator on a track-mounted shaker at 95 rpm.

[0392] Change the culture medium daily from stage 1 to stage 5, and every 2-3 days during stage 6.

[0393] Long-term differentiation scheme:

[0394] hESCs were induced to differentiate into endocrine progenitor cells using the same factors and culture medium as the short protocol described above, but the durations of phases 3 and 4 were 2 days and 5 days, respectively.

[0395] Stage 5 Cryopreservation of Endocrine Progenitor Cells

[0396] Four days into S5, the cells dissociated into single cells as described above. These single cells were precipitated by centrifugation at 300g for 5 minutes, followed by centrifugation at 1x10⁻⁶ cells / day. 7Cells / ml were resuspended in cold STEM-CELLBANKER GMP grade solution (amsbio, 11924). The cell suspension was dispersed into Nunc cryogenic tubes (Thermo Fisher Scientific; 377267), 1–1.5 ml per tube, and cooled to -80°C using Mr. Frosty cryovials (Thermo Fisher Scientific; 5100-0001). For controlled cooling, a programmable refrigeration device was used to cool the cells at 1°C per minute. For long-term cryopreservation, the cryogenic tubes were transferred to liquid nitrogen storage.

[0397] Phase 5 Thawing of Endocrine Progenitor Cells

[0398] Remove the frozen S5 cells from the cryovials from liquid nitrogen storage and thaw rapidly at 37°C. Dilute each 1 ml cell suspension with 5 ml of preheated complete S5 medium. The cells are pelleted by centrifugation at 300 g for 5 minutes, then centrifuged at 1.0–1.5 x 10⁻⁶. 6 Cells / ml were resuspended in complete S5 medium. Further differentiation procedures were performed as described above.

[0399] Aggregate formation in micropores

[0400] To generate islet-like aggregates in micropores, S5 cells were dissociated into single cells as described above. These single cells were then spaced at 1.5 x 10⁻⁶ mm. 6 Cells were resuspended at 100 cells / ml and then seeded into AggreWell 400 6-well plates (StemCell Technologies; 34421), 4 ml per well for a total of 6 x 10 cells / ml. 4 10 cells. Alternatively, 10 cells in 50 μl. 4 Single cells were seeded into each well of a 96-well Microtest plate (Sardor, 82.1583.001). The cells were then incubated overnight in an incubator.

[0401] As described above, the cells differentiated into S6 islet-like cell aggregates. The aggregates were held in AggreWell 400 6-well plates without shaking.

[0402] Aggregate and cell count

[0403] S6 islet-like aggregates were counted under a bright-field microscope. The aggregates were then examined in a Ca-free environment. 2+ and Mg 2+ Wash once with D-PBS and incubate with Accutase at 37°C for 15 minutes. Dissociate the aggregates into single cells by multiple pipettings using a P1000 pipette, centrifuge at 300g for 4-5 minutes, and resuspend in Ca-free solution. 2+and Mg 2+ Cells were counted in D-PBS using an ORFLOMOXI Z mini automated cell counter (ORFLO Corporation; MXZ001).

[0404] Flow cytometry

[0405] Cells were dissociated into single cells by treatment with Accutase at 37°C for 15 minutes. The cells were then washed twice and distilled at 10... 6 Cells / ml were resuspended in Mg2+- and Ca2+-free D-PBS. Cells were incubated on ice for 30 min using a live / dead fixable dead cell staining kit (Thermo Fisher Scientific; L34963 and L34965). 2+ and Ca 2+ After washing twice with D-PBS, cells were fixed on ice for 20 minutes with BD Bioscience Cytofix / Cytoperm buffer (554722). Cells were then washed twice with 1X BD Bioscience permeabilization / wash buffer (554723) and incubated on ice for 30 minutes with conjugated antibody diluted in 1X BD Bioscience permeabilization / wash buffer. After washing twice with 1X BD Bioscience permeabilization / wash buffer, cells were resuspended in FACS buffer (D-PBS without Ca2+ or Mg2+, containing 2% fetal bovine serum (Thermo Fisher Scientific; 10082147) and 1 mM EDTA (Thermo Fisher Scientific; 15575020)) 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 Biosciences: 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).

[0406] Immunofluorescence

[0407] Cells were fixed with 4% (wt / vol) paraformaldehyde at room temperature (RT) for 20 minutes, and then with Mg-free paraformaldehyde.2+ and Ca 2+ Wash three times with D-PBS. For immunostaining, use D-PBS (Mg-free) containing 0.3% (vol / vol) Triton X-100 (Sigma; T9284). 2+ and Ca 2+ Cells were blocked for 1 hour at RT with 5% normal donkey serum (Merck Millipore; S30-100 mL), and then in D-PBS (Mg-free) containing 0.1% (vol / vol) Triton X-100 or Tween 20 (Sigma; P9416) and 5% normal donkey serum. 2+ and Ca 2+ The primary antibody diluted in the solution was incubated overnight at 4°C. The next day, the cells were incubated with the secondary antibody at room temperature for 1 hour. After each incubation step, the cells were rinsed with a Mg-free solution. 2+ and Ca 2+ Cells were washed three times with D-PBS. The primary antibodies used were as follows: goat anti-human PDX-1 (1 / 300, Research & Development Corporation; AF2419), mouse anti-Nkx6.1 (1 / 100, DSHB; F55A12-s), sheep anti-human neuropoietin 3 (NGN3) (1 / 100, Research & Development Corporation; AF3444), goat anti-human / mouse NeuroD1 (1 / 100, Research & Development Corporation; 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-Aldrich; G2654), and rabbit anti-somatostatin (1 / 500, Sigma-Aldrich; 332A-1).

[0408] In vitro glucose stimulation

[0409] At the end of Phase 6, twenty to thirty hESC-derived aggregates were incubated overnight in S6 medium without ITS-X and additional glucose (finally 5 mM). The next day, the aggregates were transferred to 24-well ultra-low adhesion plates (Corning; 3473) and washed twice with 2 ml Krebs buffer (129 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO2, 1 mM Na2HPO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM HEPES, and 0.1% FAF-BSA). The aggregates were then pre-incubated for 2 hours in 2 ml Krebs buffer containing 2 mM glucose to remove residual insulin. Afterward, the aggregates were washed twice with 2 ml Krebs buffer and then incubated for 30 min in 2 ml Krebs buffer containing 2 mM glucose. 500 μl of supernatant sample (low glucose sample) was collected after incubation. The aggregates were washed once with 2 mL of Krebs buffer and then incubated for 30 min in 2 mL of Krebs buffer containing 20 mM glucose. After incubation, 500 μL of supernatant was collected (high glucose sample). The aggregates were washed twice with 2 mL of Krebs buffer and then incubated again for 30 min in 2 mL of Krebs buffer containing 2 mM glucose. 500 μL of supernatant was collected (low glucose sample). The aggregates were washed once with 2 mL of Krebs buffer and then incubated for 30 min in 2 mL of Krebs buffer containing 2 mM glucose and 30 mM KCl (polarization stimulation). 500 μL of supernatant was collected (KCl-stimulated sample). After KCl stimulation, the aggregates were dispersed into single cells by treatment with Accutase for 15 min, and the total cell count was performed using an ORFLO MOXI Z cell counter. The collected supernatant sample containing secreted insulin was processed using a Human C-peptide ELISA kit (Research and Development Corporation; DICP00). Human insulin C-peptide measurements were normalized to total cell count and expressed as a fraction of 10. 3 pmol c peptide released by each cell. If ELISA is not performed on the same day, store the sample at -80°C.

[0410] scRNA sequencing sample preparation

[0411] At the end of S6, hESC-derived islets were collected for scRNA sequencing. Islet-like aggregates were then subjected to Ca2+-free... 2+ and Mg 2+Wash twice with D-PBS and incubate with TrypLE on a track-mounted shaker at 37°C for 15-20 minutes. Dissociate the aggregates into single cells by multiple pipettings using a P1000 pipette, centrifuge at 300g for 4-5 minutes, and resuspend in D-PBS (containing 0.04% FAF-BSA without Ca) 2+ and Mg 2+ The cells were then filtered using a 40 μm cell filter (VWR, 732-2760). To determine the total cell count and viable cell ratio, the single-cell suspensions were stained with 0.4% trypan blue solution (Thermo Fisher Scientific, 15250061) and counted using a hemocytometer.

[0412] Single-cell RNA sequencing analysis

[0413] scRNA sequencing libraries were constructed using 3000 cells. RNA sequencing was performed on an Illumina Nextseq 2000 instrument using the 10x Genomics Chromium Next GEM Single Cell 3′ Kit v3.1 (10x Genomics, CG000315 or CG000388), sometimes in conjunction with a cell multiplex oligonucleotide labeling protocol (10x Genomics, CG000391). Analysis steps were performed in Cell Ranger 3.1.0 to generate FastQ files and a feature barcode matrix. Unified manifold approximation and projection (UMAP) was performed for dimensionality reduction, cell type identification, and differential gene expression analysis.

[0414] Example 1

[0415] In this embodiment, the ability of different hESC lines to support pancreatic differentiation in cell culture substrates was compared. Pancreatic differentiation was evaluated based on the expression of key biomarkers in stage 4 (S4) pancreatic progenitor cells (PP) and stage 5 (S5) endocrine progenitor cells (EP).

[0416] Protocols for in vitro pancreatic differentiation of hPSCs cultured on 2D surfaces coated with feeder cells or Matrigel have been established (Pagliuca et al. 2014; D'Amour et al. 2006; Kroon et al. 2008). However, for the clinical application of hPSC-derived islets, chemically defined coating substrates free of heterologous material are highly preferred. Therefore, we compared the ability of recombinant human LN-511 and LN-521 to support pancreatic differentiation in the Matrigel setting. hESC lines HS980 and H1 were differentiated on these three substrates using a long differentiation protocol (see upper figure). Figure 1BThe expression of key progenitor cell markers PDX1, NKX6.1 and NEUROD1 was measured by flow cytometry at the end of S4 and four days into S5.

[0417] result The results showed that the percentage of S4 PDX1+ / NKX6.1+ pancreatic progenitor cells (PP) was comparable across all basal cells in both the HS980 and H1 cell lines. Figure 2A However, the level of PDX1+ / NKX6.1+ cells decreased significantly on matrix gel, with a smaller decrease on LN-511 cells, but remained relatively stable on LN-521 cells as S4 cells further differentiated into S5 cells. Figure 2B The percentage of S5 NKX6.1+ / NEUROD1+ endocrine progenitor cells (EPs) was significantly higher on LN-521 compared to that on Matrigel, while the percentage was also slightly higher on LN-511. Figure 2C ).

[0418] In summary, the data indicate that Mattergel, LN-511, and LN-521 can support differentiation into S4 PP cells, especially LN-521, which provides a permissive surface environment for the endocrine differentiation of S4 PP cells. Therefore, we decided to continue all our experiments on LN-521 instead of Mattergel. Since the percentage of S5EP cells on LN-521 is only 20%-30%, modifications are needed to optimize the differentiation protocol.

[0419] Example 2

[0420] In this embodiment, the effect of the duration of stage 4 on endocrine differentiation was investigated.

[0421] Evaluation of different lengths in Phase 3

[0422] It has been previously reported that short-duration S3 promotes S4 PDX1 + / NKX6.1 + PP cell populations and suppressed immature multi-hormone cell populations (Nostro et al., 2015). However, it was unclear whether the duration of S4 would affect later differentiation stages in a similar manner. Therefore, we decided to analyze / optimize the durations of S3 and S4. To identify the shortest duration of S3, HS980 and H1 cells were differentiated into foregut cells (PF) after S3, and the expression of the key PF marker PDX1 was measured at the end of S2 and 1 or 2 days into S3.

[0423] resultThe results showed that PDX1 was expressed in most cells 1 day after entering S3, and as indicated by immunocytological analysis and observation of PDX1 levels on days 6, 7, and 8 of culture. + As shown by representative dot blots of cell number, this expression only increased slightly after 2 days (Fig. 3).

[0424] Therefore, the data shows that the duration of one day in S3 is sufficient for S2 PGT cells to differentiate into S3 PDX1. + PF cells. The two-day duration also resulted in a large number of S3 PDX1 cells. + PF cells. Therefore, it is concluded that the duration of S3 should be at most 48 hours, for example 24 hours.

[0425] The effect of stage 4 duration on differentiation into endocrine progenitor cells

[0426] To analyze the effect of S4 duration on S5 EP differentiation, HS980, H1, and H9 cells were induced for 1 day under S3, followed by induction under S4 for 1, 2, 3, 4, or 5 days (e.g., ...). Figure 4A (As shown in the schematic diagram).

[0427] result Expression of NKX6.1 and NEUROD1 was measured at the end of S4 and then four days into S5 (representative pilot plots are shown below). Figure 4B (See the upper and lower subplots). The results showed that when the S4 duration of HS980 cells increased from 2 days to 5 days, S4 NKX6.1 + The percentage of PP cells increased significantly. Conversely, S5 NKX6.1 + / NEUROD1 + The percentage of EP cells decreased with increasing S4 duration. Figure 4B Similar inhibitory effects were observed in H1 and H9 cells. When the duration exceeded 2–3 days, NKX6.1… + / NEUROD1 + The percentage of EP cells began to decline. Figure 4C The experiment was repeated several times, and the results showed that 2-3 days under S4 conditions were crucial for subsequent differentiation into S5 NKX6.1. + / NEUROD1 + EP cells are the best. Figure 4D Conversely, the increase in duration under S4 resulted in a significantly higher percentage of NKX6.1 under S5. + / NEUROD1 - cell( Figure 4D NKX6.1 + / NEUROD1 -The cell is most likely actually S4 NKX6.1 + PP cells, due to prolonged inhibitory effects, failed to differentiate further into S5 NKX6.1. + / NEUROD1 + EP cells ( Figure 4D The inhibitory effect of prolonged S4 duration was particularly pronounced in H9 cells, and decreased from 5 days to 2 days, which reduced total NEUROD1 levels. + The percentage of endocrine cells increased to similar levels in HS980 and H1 cells. Figure 4E The results also indicated that S4 was insufficient to induce NKX6.1 ( ). Figure 4E ).

[0428] Therefore, it can be concluded that a 2-3 day S4 duration allows for a higher percentage of NKX6.1. + / NEUROD1 + EP cells, and therefore beneficial for differentiation into S5 EP cells. Optimal S4 duration also minimizes cell line variation during pancreatic differentiation.

[0429] The effect of stage 4 duration on pancreatic islet cell maturation

[0430] The effect of S4 duration on S6 islet cell maturation was also analyzed. To generate S6 islet-like cell aggregates, S5 EP cells from different S4 durations were dissociated into single cells and then maintained in suspension as described above. The expression of key markers insulin (INS) and glucagon (GCG) was analyzed at the end of S6.

[0431] result The results showed that when the duration of S4 became longer than 3 days, INS + The percentage of single hormone β cells decreased, and GCG + The percentage of single hormone α cells increased significantly, as shown in the representative dot plot ( Figure 4E ) and bar chart ( Figure 4F As shown in the figure. It is noteworthy that a 2-day duration of S4 resulted in a high percentage of EP cells in both H1 and H9 cell cultures, while a 3-day duration of S4 resulted in a high percentage of EP cells in the H980 culture. Figure 4D However, a duration of 3 days resulted in the highest percentage of S6INS+β cells. Figure 4F ).

[0432] In summary, these results indicate that a longer S4 duration promotes differentiation into S4NKX6.1. + PP cells, but inhibited further differentiation into S5 NKX6.1 + / NEUROD1 + EP cells. On the other hand, shorter S4 durations generate fewer S4NKX6.1 cells. + PP cells, but with increased S5NKX6.1 + / NEUROD1 + EP cells. The results also indicated that S4 duration had opposite effects on α and β cell populations. S4 duration of 3 days promoted INS. + β cells, with a 5-day duration, strongly promote GCG. + α cells. Importantly, the results were confirmed in three different hESC lines (H1, H9, and HS980).

[0433] Example 3

[0434] In this embodiment, different cell culture coating substrates for pancreatic differentiation were evaluated.

[0435] Cell culture substrates with defined chemical compositions and free of heterologous substances (such as LN-521) can provide more consistent and reliable conditions for the expansion and differentiation of pluripotent stem cells.

[0436]

[0437]

[0438] Table 2: Cell adhesion to 2D surfaces coated with different matrix proteins. HS980, H1, and H9 cells were passaged onto plates coated with different recombinant human laminin isoforms (LN) or matrix gel, as shown in the table. Cell adhesion and survival were examined under a microscope after 3–4 days. + indicates that cells adhered to the bottom and formed a monolayer, - indicates that cells failed to adhere to the coated surface.

[0439] To determine whether hESCs could also differentiate into pancreatic endocrine cells on other matrix proteins, HS980, H1, and H9 cells were passaged into cell culture plates coated with either Matrigel (1 / 100, Corning; 354277) or recombinant human LN-111, LN-121, LN-211, LN-221, LN-332, LN-411, LN-421, LN-511, or LN-521 (all obtained from Peore Leminer). HS980, H1, and H9 cells attached 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 protocol, and the expression of NKX6.1 and NEUROD1 was analyzed at day 4 of stage 5 (Figure 5A). The results showed that LN-332, LN-521, and LN-511 were more effective than Matrigel in supporting differentiation into S5 NKX6.1+ / NEUROD1+ EP cells (Figure 5A). Figure 5B It is noteworthy that when the short protocol was used instead of the long protocol, LN-511 enhanced S5 EP differentiation of HS980 and H1 cells as effectively as LN-521 (Figure 5A). Figure 5B H1 cells also differentiated on plates coated with 10 μg / mL fibronectin (FN, Sigma; F0895) or vitrin (VTN, Sigma; 5051). The results showed that FN and VTN supported S5 EP differentiation. Figure 5B ).

[0440] S5 EP cells generated on LN-332, LN-511, LN-521, and Matricella substrates were selected for further differentiation toward S6 islet cells as described above. INS and GCG expression were measured at the end of S6. The results showed that S5 EP cells from these substrates were capable of generating the S6 monohormone INS. + β cells and GCG + α cells ( Figure 5C ).

[0441] In summary, the results show that the short scheme is highly effective for a variety of substrates without heterologous material.

[0442] Example 4

[0443] In this embodiment, it was investigated whether S4 or S5 cells were more suitable for in vitro formation of pancreatic islets.

[0444] In previous reports, purified S4 PP cells have been used to generate islets (Cogger et al., 2017; Ameri et al., 2017; Kelly et al., 2011). To determine which cell population was more suitable for islet formation, HS980 cells were dissociated into single cells at the end of S4 or at the beginning of S54. The cells were then kept in suspension to generate islet-like aggregates (…). Figure 6A At the end of S6, the expression of key endocrine markers INS and GCG, the number of islet-like aggregates, and the number of islet cells were analyzed.

[0445] result The results showed that aggregates generated from S5 EP cells had a much higher percentage of INS than aggregates from S4 PP cells. + β cells and GCG + α cells (both) Figure 6B S5 EP cells also produce more than 10 times more islet-like aggregates and islet cells than S4 PP cells. Figure 6B ).

[0446] To determine whether a longer S5 duration improved islet formation, S5 EP cells were dissociated into single cells at day 6 of S5 and then maintained in a 3D suspension. However, the percentages of α and β cells remained unchanged, and the number of aggregates / cells actually decreased at S6. Figure 6B ).

[0447] In summary, these data indicate that S5 EP cells, rather than S4 PP cells, effectively form islet-like aggregates in 3D suspensions, and that a 4-day duration under S5 is sufficient for aggregate formation in suspensions.

[0448] Example 5

[0449] In this embodiment, the effect of cell culture in 3D single-cell suspension was studied.

[0450] To determine whether the 3D single-cell suspension selectively promotes aggregate formation from S5 EP cells, HS980 and H1 cells were differentiated into S5 EP cells on LN-521 cells and then dissociated into single cells in the 3D suspension. The expression of NKX6.1, NEUROD1, and the cell proliferation marker Ki-67 was analyzed before and after aggregate formation in the 3D suspension.

[0451] result The results showed that, compared with 2D cells on day 14, the newly formed aggregates contained S5NKX6.1 on day 15. + / NEUROD1 + NEUROD1+ The percentage of cells increased significantly, and NEUROD1 was present in the 3D aggregates. - The percentage of non-endocrine cells decreased significantly. Figure 7A The results also showed that S5 NEUROD1 + EP cells are non-proliferating Ki-67 cells. - Cells, and most of them are proliferating Ki-67 cells. + / NEUROD1 - Non-proliferative Ki-67 - / NEUROD1 - Cells are removed during the formation of aggregates in the 3D suspension. Figure 7B ).

[0452] To determine whether ROCK inhibitors were required for S5 EP cell survival and aggregate formation, different concentrations of H1152 (0 μM to 10 μM) were added to 3D single-cell suspensions for one day. One day after aggregate formation in the 3D suspensions, the expression of NKX6.1, NEUROD1, and Ki-67, as well as the number of aggregated cells, were measured.

[0453] result The results showed that the concentration of H1152 had no effect on the percentage of S5 EP cells in the newly formed aggregates. Figure 7C However, in the presence of low concentrations of H1152, the number of islet cells increased significantly, indicating that H1152 promotes the survival of S5 EP cells as single cells in suspension. Figure 7D ).

[0454] In summary, these results indicate that S5 EP cells selectively enrich due to the formation of 3D aggregates from single cells, consistent with previous studies on fetal pancreatic development (Gouzi et al., 2017).

[0455] Example 6

[0456] In this embodiment, the short pancreas differentiation protocol and the long pancreas differentiation protocol were compared by comparing the percentages of pancreatic progenitor cells (PP) and endocrine progenitor cells (EP) obtained.

[0457] Next, we compared the short and long protocols. Using the short and long differentiation protocols described above, HS980 cells on LN-521 were differentiated into S5 EP cells (e.g., Figure 1B (As shown). The expression of key progenitor cell markers was analyzed at the end of S3 and S4 and four days into S5 (days 7, 10, and 14 of differentiation for the short protocol, and days 8, 13, and 17 of differentiation for the long protocol).

[0458] result The results show that the short and long solutions have opposite effects. The short solution generates S4 NKX6.1. + The percentage of PP cells was lower than in the long protocol, but S5 NKX6.1 + / NEUROD1 + The percentage of EP cells was higher than in this long protocol ( Figure 8A and Figure 8B (S5), the short-scheme NKX6.1 + / NEUROD1 - The percentage of non-endocrine cells was lower than in the long protocol ( Figure 8A and 8B These results collectively indicate that a short S3+S4 duration has a positive impact on S5EP differentiation.

[0459] To determine whether the duration of S3+S4 also affects islet maturation during S6, S5 EP cells were dissociated into single cells and then maintained in suspension as described above. At the end of S6, the expression of key endocrine markers INS and GCG, the number of islet-like aggregates and islet cells, and in vitro glucose-stimulated insulin secretion were analyzed. Figure 8C and Figure 8D ).

[0460] result The results show that short-path induced INS + The percentage of single-hormone β cells was higher than with the long protocol, but GCG + The percentage of single hormone alpha cells was lower than in the long protocol ( Figure 8C A similar effect on the duration of S3+S4 was also observed in H1 cells. Figure 8C The short protocol also generated more islet-like aggregates and islet cells than the long protocol. Figure 8D In vitro glucose stimulation experiments confirmed that the aggregates at the end of S6 were functional and could respond to high glucose concentrations by increasing insulin secretion tenfold. However, aggregates generated using the short protocol secreted more insulin at high glucose levels. Figure 8D In summary, these results indicate that a short S3+S4 duration has a strong positive impact on islet maturation during S6.

[0461] Immunocytochemistry (ICC) was also used to analyze the expression of stage-specific markers (Figure 9). The long protocol induced a very dense layer at S4, which mainly contained PDX1. + / NKX6.1 + PP cells ( Figure 9A Conversely, the shorter protocol induces less S4PDX1. + / NKX6.1 +PP cells, some of which still maintain NKX6.1 - ( Figure 9A Both schemes observed NKX6.1 at S4. - Premature induction of NEUROD1 in cells ( Figure 9A Further differentiation into S5 EP cells showed the opposite effect. The short protocol generated more S5 NKX6.1 cells than the long protocol. + / NEUROD + EP cells ( Figure 9B The results also showed that aggregates generated using the short and long protocols had different ratios between β and α cells. Figure 9C The short protocol induced more insulin C-peptide (CPEP) positive β cells, while the long protocol induced more GCG. + α cells ( Figure 9C Some somatostatin (SST) positive delta cells were also observed. Figure 9C In conclusion, the ICC results support the conclusions drawn from the flow cytometry analysis described above.

[0462] Example 7

[0463] In this embodiment, the effect of spontaneous aggregation of S5 cells into 3D cultures on the production of monohormonal pancreatic β cells was investigated compared with forced aggregation using AggreWell 400 plates and 96-well plates.

[0464] For spontaneous aggregation, H1 cells dissociated into single cells at S5 and were maintained in a 3D suspension as described above. For forced aggregation in microwells, S5 cells were transferred to AggreWell or 96-well plates. The identity of the generated cells was studied by flow cytometry one day after 3D aggregation and again at the end of S6.

[0465] result: The results showed that the aggregates formed in the micropores mainly contained NKX6.1. + / NEUROD1 + / Ki-67 - EP cells, because aggregates spontaneously generate in 3D suspension culture ( Figure 10A , Figure 10B Proliferative Ki-67 + Cells are removed from aggregates in the micropores, just like in a 3D suspension. Figure 10A , Figure 10B At the end of S6, the aggregates formed in the micropores contained INS similar to those of spontaneously generated aggregates. + β cells and GCG + α-cell percentage ( Figure 10COnly a very small number of Ki-67 remain in these aggregates. + Proliferating cells ( Figure 10C ).

[0466] In summary, these results indicate that S5 EP cells can separate from other non-endocrine cell types and then spontaneously aggregate.

[0467] Example 8

[0468] In this embodiment, cell line variability was evaluated in multiple hPSC lines differentiated using a short pancreatic differentiation protocol.

[0469] hESC lines HS980, H1, and H9 differentiated into S6 pancreatic islet cells as described above. At the end of S6, the expression of key biomarkers INS and GCG, as well as in vitro glucose-stimulated insulin secretion, was analyzed. To determine whether the short differentiation protocol also applies to human iPSC lines, C7 cells were differentiated on LN-521 cells, and the expression of key biomarkers was examined at the end of S5 and S6.

[0470] Results: The results showed that in all three hESC lines, the single-hormone INS... + β cells and GCG + The percentage of alpha cells is comparable, and the aggregates are functional and can respond in a similar manner to increased insulin secretion at high glucose concentrations. Figure 12A The results also showed that the human iPSC lineage could successfully differentiate into S5NKX6.1. + / NEUROD1 + EP cells, which subsequently differentiate into S6 mono-hormone INS cells. + β cells and GCG + α cells ( Figure 12B ).

[0471] In summary, these results show relatively low cell line variability among multi-person ESC and iPSC lines differentiated using a short pancreatic differentiation protocol.

[0472] Example 9

[0473] In this embodiment, the short pancreatic differentiation protocol was compared with two previously published protocols (Augsornworawat et al. 2020 and Balboa et al. 2022) using a dataset from a single-cell RNA sequencing (scRNAseq) experiment. Figure 13A ).

[0474] H1 cells were differentiated into S6 islet cells using the short pancreatic differentiation protocol described above. At the end of the S6 differentiation period, the gene expression profile of the islet cells was investigated by single-cell RNA sequencing. The obtained dataset was processed and visualized using UMAP projection. Figure 13B The expression of cell type identity and key marker genes in each cell population was analyzed. Figure 13B The analysis also included two publicly available datasets (Augsornworawat et al. 2020 and Balboa et al. 2022).

[0475] result: The results showed that S6 islets generated using the short differentiation protocol contained two mature mono-hormone cell populations: β cells expressing INS and α cells expressing GCG. Figure 13B (Left). The pancreatic islets generated by Augsornworawat et al. also contain two major populations of endocrine cells ( Figure 13B (In the middle). However, most cells co-express both INS and GCG, and are therefore immature multi-hormonal β and α cells. In contrast, the islets of Langerhans from Balboa et al. contain both endocrine and non-endocrine cell types, although the predicted β and α are monohormonal ( Figure 13B ,right).

[0476] In summary, these results indicate that islets generated via the short pancreas differentiation protocol primarily contain mature monohormonal β and α cells. In contrast, the two recently published protocols generated islets containing immature multihormonal cells or non-endocrine cells.

[0477] Example 10

[0478] In this embodiment, the ability of frozen and unfrozen S5 EP cells to generate 3D islets was compared.

[0479] As described above, HS980 and H1 cells differentiate towards S5. Cells are dissociated into single cells in a 3D suspension and then further differentiate towards S6. Alternatively, S5 single cells are frozen and stored in liquid nitrogen. The frozen cells are then thawed and differentiate towards S6 as described above. At the end of S6, the identity of the islet cells and in vitro glucose-stimulated insulin release are examined.

[0480] result: The results showed that the freezing / thawing process at S5 did not alter the INS levels in HS980 and H1 cells. + Percentage of β cells ( Figure 14 (Left). GCG in HS980 cells after freeze / thaw procedure. + The percentage of α cells decreased significantly, but the percentage of H1 cells did not ( Figure 14(Left). The results also showed that islets differentiated from S5 EP cells (both frozen and unfrozen) were functional and could respond to increased insulin release in response to high glucose levels. Figure 14 ,right).

[0481] In summary, these results indicate that S5 EP cells can be cryopreserved, and that frozen cells are an ideal source for the in vitro production of functional islets.

[0482] References:

[0483] 1. Rodin, S., et al., Clonal culturing of human embryonic stem cells on laminin-521 / E-cadherin matrix in a defined and xeno-free environment. Nat Commun, Nature Communications, 2014.5: p. 3195.

[0484] 2. Pagliuca, FW, et al., Generation of functional human pancreatic β-cells in vitro. Cell, 2014, 159(2): 428-39.

[0485] 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): 1121-33.

[0486] 4. Millman, JR, et al., Generation of stem cell-derived β-cells from patients with type 1 diabetes. NatCommun, 2016.7: 11463.

[0487] 5. Vegas, AJ, et al., Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. NatMed, 2016, 22(3): 306-11.

[0488] 6. D'Amour, KA, et al., Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol, 2006, 24(11): 1392-401.

[0489] 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): 443-52.

[0490] 8. Nostro, MC, et al., Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines. Stem Cell Reports, 2015, 4(4): 591-604.

[0491] 9. Cogger, KF, et al., Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors. NatCommun, 2017.8(1): 331.

[0492] 10. Ameri, J., et al., Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2. Cell Reports, 2017, 19(1): 36-49.

[0493] 11. Gouzi, M., et al., Neurogenin 3 initiates stepwise delamination of differentiating endocrine cells during pancreas development. Dev Dyn, 2011, 240(3): 589-604.

[0494] 12. Augsornworawat, P., et al., Single-Cell Transcriptome Profiling Reveals β-Cell Maturation in Stem Cell-Derived Islets after Transplantation. Cell Reports, 2020, 32(8): 1-13.

[0495] 13. Balboa, D., et al., Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nat Biotechnol, 2022, 40(7): 1042-55.

[0496] 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. Nov 2016; 17(3): 474-478.

[0497] Implementation Plan List

[0498] 1. A method for generating in vitro islet-like cell aggregates, the method comprising the following steps:

[0499] i) Provide a population containing endocrine progenitor cells (EPs), such as a population of EP cells, which are, for example, EP cells characterized by expression of NEUROD1; for example, EP cells characterized by expression of NKX6.1 and NEUROD1;

[0500] ii) Provide a single-cell suspension of the population of said EP cells;

[0501] iii) Allows the population of said EP cells in a single-cell suspension to form a 3D structure;

[0502] iv) Culture the EP cells in a 3D structure under 3D culture conditions that allow differentiation into pancreatic monohormonal β cells to provide islet-like cell aggregates; and

[0503] v) This generates an aggregate of islet-like cells containing mono-hormone β cells.

[0504] The islet-like cell aggregates described herein contain at least approximately 25%, for example at least approximately 30%, for example at least approximately 35%, for example at least approximately 40%, for example at least approximately 45% of monohormonal β cells.

[0505] 2. The method for generating in vitro islet-like cell aggregates as described in Project 1, wherein the population of EP cells in step i) is an adherent culture of EP cells on a 2D substrate.

[0506] 3. The method for generating in vitro islet-like cell aggregates as described in Project 2, wherein the 2D substrate comprises one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized filaments (FN filaments), and matrix gel. TMFor example, one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, and matrix gel. TM .

[0507] 4. The method for generating in vitro islet-like cell aggregates as described in Project 3, 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, and LN-511 and its fragments; for example, the group consisting of LN-521 and its fragments or the group consisting of LN-511 and its fragments.

[0508] 5. The method for generating in vitro islet-like cell aggregates as described in item 3 or 4, 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, wherein the laminin and its fragments are LN-521 or wherein the laminin and its fragments are LN-511.

[0509] 6. The method for generating in vitro islet-like cell aggregates as described in item 3 or 4, wherein the laminin (LN) and fragments comprise the E8 fragment of laminin; for example, the E8 fragment of laminin selected from the group consisting of: the E8 fragment of LN-511, the E8 fragment of LN-521, the E8 fragment of LN-332, the E8 fragment of LN-421, the E8 fragment of LN-121, and the E8 fragment of LN-111; for example, the group consisting of: LN E8 fragments of LN-511, LN-521, LN-332, LN-421, and 121; for example, the group consisting of: E8 fragments of LN-511, LN-521, and LN-332; for example, the group consisting of: E8 fragments of LN-511 and LN-521; for example, E8 fragments of LN-511 or LN-521.

[0510] 7. A method for generating in vitro islet-like cell aggregates as described in any one of items 1-6, wherein in step i), more than 15%, for example more than 20%, for example more than 25%, for example more than 30%, for example more than 35%, for example more than 40%, for example more than 45%, for example more than 50% of the total cell population are EP cells characterized by expressing NEUROD1;

[0511] or

[0512] In vitro, in step i), more than 15%, for example more than 20%, for example more than 25%, for example more than 30%, for example more than 35%, for example more than 40%, for example more than 45%, for example more than 50% of the total cell population are EP cells characterized by expressing NKX6.1 and NEUROD1.

[0513] 8. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-7, wherein step ii) is performed when more than 15%, for example more than 20%, for example more than 25%, for example more than 30%, for example more than 35%, for example more than 40%, for example more than 45%, for example more than 50% of the total cell population are EP cells characterized by expressing NEUROD1;

[0514] or

[0515] Step ii) involves providing a single-cell suspension of the EP cell population when more than 15%, for example, more than 20%, for example, more than 25%, for example, more than 30%, for example, more than 35%, for example, more than 40%, for example, more than 45%, for example, more than 50% of the total cell population are EP cells characterized by expressing NKX6.1 and NEUROD1.

[0516] 9. An in vitro method for generating in vitro islet-like cell aggregates as described in any one of items 1-8, wherein the islet-like cell aggregates generated in step v) comprise approximately 7 to 25%, for example 7 to 20%, 10 to 20%, for example 15 to 20%, or for example approximately 20% monohormone α cells.

[0517] 10. A method for generating in vitro islet-like cell aggregates as described in any one of items 1-9, wherein the islet-like cell aggregates generated in step v) contain 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% of proliferating cells, such as proliferating cells expressing Ki-67.

[0518] 11. A method for generating in vitro islet-like cell aggregates as described in any one of items 1-10, wherein the islet-like cell aggregates generated in step v) are scored after 38-42 days of culture.

[0519] 12. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-11, wherein step ii) is performed prior to culturing these cells in a medium that allows differentiation into pancreatic monohormone β cells.

[0520] 13. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-12, wherein step ii is performed approximately 6 days after the EP cells begin to express NEUROD1.

[0521] 14. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-13, wherein step ii is performed within 1-5 days after the EP cells begin to express NEUROD1.

[0522] 15. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-14, wherein the formation of the 3D structure in step iii) is a spontaneous formation of the 3D structure.

[0523] 16. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-14, wherein the formation of the 3D structure in step iii) is forced or assisted in the formation of the 3D structure.

[0524] 17. A method for producing islet-like cell aggregates in vitro as described in any one of items 1-16, wherein step iv) comprises culturing EP for about 2 weeks or longer, such as about 3 weeks or longer, such as about 3 to 5 weeks, such as about 4 weeks.

[0525] 18. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-17, wherein the mono-hormone β cells generated in step v) have the ability to express insulin, for example, wherein the mono-hormone β cells generated in step v) express insulin.

[0526] 19. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-18, wherein the mono-hormone β cells generated in step v) have the ability to express insulin, for example, wherein the mono-hormone β cells generated in step v) express C-peptide upon glucose stimulation.

[0527] 20. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-19, wherein the mono-hormone β cells generated in step v) do not express glucagon and / or somatostatin.

[0528] 21. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-20, wherein the islet-like cell aggregates generated in step v) comprise at least 40% monohormone β cells; 7%-25% monohormone α cells and less than 2% proliferating cells.

[0529] 22. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-21, wherein prior to step i), the method comprises steps a)-c):

[0530] a) Provide a population of hindbrain cells, which are, for example, hindbrain cells characterized by expression of PDX1;

[0531] b) The cell population of the posterior foregut cells is cultured under conditions that allow differentiation into pancreatic progenitor cells for no more than approximately 78 hours, for example, no more than approximately 72 hours; and

[0532] c) This generates a population of pancreatic progenitor cells, which are, for example, pancreatic progenitor cells characterized by the expression of both PDX1 and NKX6.1.

[0533] 23. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-22, wherein prior to step i), the method comprises steps a-1)–c-1):

[0534] a-1) Provide a population of primitive intestinal cells, which are, for example, primitive intestinal cells characterized by the expression of HNF1β and / or HNF4α;

[0535] b-1) The cell population of the primitive intestinal cells shall be cultured for no more than approximately 54 hours under conditions that allow differentiation into hindforegut cells; and

[0536] c-1) thereby generating a population of hindbrain cells, which are, for example, hindbrain cells characterized by expression of PDX1.

[0537] 24. The method for generating in vitro islet-like cell aggregates as described in any one of items 22-23, wherein steps a-1)–c-1) are performed prior to steps a)–c).

[0538] 25. The method for generating in vitro islet-like cell aggregates as described in any one of items 22-24, wherein prior to step i), the method comprises steps a-1)–c-1) and steps a) and c):

[0539] a-1) Provide a population of primitive intestinal cells, which are, for example, primitive intestinal cells characterized by the expression of HNF1β and / or HNF4α;

[0540] b-1) The cell population of the primitive intestinal cells was cultured for no more than approximately 54 hours under conditions that allowed differentiation into hindforegut cells;

[0541] c-1) thereby generating a population of hindbrain cells, which are, for example, hindbrain cells characterized by expression of PDX1.

[0542] a) Provide a cell population of hindbrain cells generated in step c-1), which are, for example, hindbrain cells characterized by expressing PDX1;

[0543] b) The cell population of the posterior foregut cells is cultured under conditions that allow differentiation into pancreatic progenitor cells for no more than approximately 78 hours, for example, no more than approximately 72 hours; and

[0544] c) This generates a population of pancreatic progenitor cells, which are, for example, pancreatic progenitor cells characterized by the expression of both PDX1 and NKX6.1.

[0545] 26. The method for generating in vitro islet-like cell aggregates as described in any one of items 22-25, wherein after steps a)-c), the method further comprises steps a+1)–c+1):

[0546] a+1) provides the cell population of pancreatic progenitor cells generated in step c;

[0547] b+1) Culture the pancreatic progenitor cell population under conditions that allow differentiation into endocrine progenitor cells; and

[0548] c+1) thereby generating a population of endocrine progenitor cells, which are endocrine progenitor cells characterized, for example, by expressing NEUROD1, such as by expressing NKX6.1 and NEUROD1.

[0549] 27. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-26, wherein prior to step i), the method comprises the following steps:

[0550] a-1) Provide a population of primitive intestinal cells, which are, for example, primitive intestinal cells characterized by the expression of HNF1β and / or HNF4α;

[0551] b-1) The cell population of the primitive intestinal cells was cultured for no more than approximately 54 hours under conditions that allowed differentiation into hindforegut cells;

[0552] c-1) thereby generating a population of hindbrain cells, which are, for example, hindbrain cells characterized by expression of PDX1;

[0553] a) Provide a cell population of hindbrain cells generated in step c-1), which are, for example, hindbrain cells characterized by expressing PDX1;

[0554] b) The cell population of the posterior foregut cells is cultured under conditions that allow differentiation into pancreatic progenitor cells for no more than about 78 hours, for example, no more than about 72 hours.

[0555] c) This generates a population of pancreatic progenitor cells, which are, for example, pancreatic progenitor cells characterized by the expression of both PDX1 and NKX6.1;

[0556] a+1) provides the cell population of pancreatic progenitor cells generated in step c;

[0557] b+1) Culture the pancreatic progenitor cell population under conditions that allow differentiation into endocrine progenitor cells; and

[0558] c+1) thereby generating a population of endocrine progenitor cells, which are endocrine progenitor cells characterized, for example, by expressing NEUROD1, such as by expressing NKX6.1 and NEUROD1.

[0559] 28. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-27, wherein in step b), the cell population is cultured for a period of approximately 42 to 78 hours, for example, a period of approximately 44 to 76 hours, for example, a period of approximately 46 to 74 hours, for example, a period of approximately 48 to 72 hours.

[0560] 29. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-28, wherein in step b), the cell population is cultured for a period of approximately 66 to 78 hours, for example, approximately 68 to 76 hours, for example, approximately 70 to 74 hours, for example, approximately 72 hours.

[0561] 30. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-29, wherein in step b), the cell population is cultured for a period of approximately 42 to 54 hours, for example, a period of approximately 44 to 52 hours, for example, a period of approximately 46 to 50 hours, for example, a period of approximately 48 hours.

[0562] 31. The method for generating in vitro islet-like cell aggregates as described in any one of items 22-30, wherein in step c), the cell population of pancreatic progenitor cells, for example, a cell population of pancreatic progenitor cells characterized by expressing PDX1 and NKX6.1, is further characterized by expressing at least one marker selected from the group consisting of: PTF1A, SOX9, HNF6 and CPA, for example, markers selected from the group consisting of: PTF1A and SOX9.

[0563] 32. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-31, 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.

[0564] 33. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-32, 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.

[0565] 34. The method for generating in vitro islet-like cell aggregates as described in any one of items 32-33, wherein the effective amount of said EGF or its derivatives or agonists is about 50 ng / mL to 200 ng / mL, for example about 50 ng / mL to 150 ng / mL, for example about 75 ng / mL to 125 ng / mL, for example about 100 ng / mL.

[0566] 35. A method for generating in vitro islet-like cell aggregates as described in any one of items 32-34, wherein the effective amount of said NIC or its derivative or agonist is about 5 mM to 20 mM, for example about 5 mM to 15 mM, for example about 8 mM to 12 mM, for example about 10 mM.

[0567] 36. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-35, wherein step b) comprises culturing the cell population in a culture medium further comprising KGF, activin A, retinoic acid, SANT-1, PDBu and LDN.

[0568] 37. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-36, wherein the cells are cultured on a 2D substrate, for example, wherein the cells are cultured in an adherent manner on a 2D substrate.

[0569] 38. The method for generating in vitro islet-like cell aggregates as described in item 37, wherein the 2D substrate comprises one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized filaments (FN filaments), and matrix gel. TM For example, one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, and matrix gel. TM .

[0570] 39. The method for generating in vitro islet-like cell aggregates as described in item 38, wherein the laminin (LN) and its fragments are selected from the group consisting of:

[0571] 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;

[0572] For example, the following groups: LN-521 and its fragments, LN-511 and its fragments, and LN-332 and its fragments;

[0573] For example, a group consisting of LN-521 and its fragments or a group consisting of LN-511 and its fragments.

[0574] 40. A method for generating in vitro islet-like cell aggregates as described in any one of items 38-39, 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, wherein the laminin and its fragments are LN-521.

[0575] 41. A method for generating in vitro islet-like cell aggregates as described in any one of items 38-39, wherein the laminin (LN) and fragments comprise the E8 fragment of laminin; for example, the E8 fragment of laminin selected from the group consisting of: the E8 fragment of LN-511, the E8 fragment of LN-521, the E8 fragment of LN-332, the E8 fragment of LN-421, the E8 fragment of LN-121, and the E8 fragment of LN-111; for example, the E8 fragment of LN-111. Groups: E8 fragments of LN-511, E8 fragments of LN-521, E8 fragments of LN-332, and E8 fragments of LN-421 and 121; for example, groups consisting of: E8 fragments of LN-511, E8 fragments of LN-521 and E8 fragments of LN-332; for example, groups consisting of: E8 fragments of LN-511 and E8 fragments of LN-521; for example, E8 fragments of LN-511 or E8 fragments of LN-521.

[0576] 42. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-41, wherein at least about 60%, for example at least about 55%, for example at least about 70%, for example at least about 75%, for example at least about 80% of the hindbrain cells, such as hindbrain cells characterized by expressing PDX1, differentiate into pancreatic progenitor cells, such as pancreatic progenitor cells characterized by expressing PDX1 and NKX6.1, in c).

[0577] 43. The method for generating in vitro islet-like cell aggregates as described in any one of items 22-42, wherein in step c), at least about 75%, for example at least about 80%, for example about 80 to 85%, for example about 80 to 90%, of the total cell population expresses PDX1.

[0578] 44. The method for generating in vitro islet-like cell aggregates as described in any one of items 22-43, wherein in step c), up to about 10% of the total cell population expresses NEUROD1.

[0579] 45. A method for generating in vitro islet-like cell aggregates as described in any one of items 22-44, wherein in step c), approximately 40% to 70%, for example approximately 30% to 50%, of the total cell population express NKX6.1.

[0580] 46. ​​The method for generating in vitro islet-like cell aggregates as described in any one of items 23-45, wherein in step b-1), the cell population is cultured for no more than about 52 hours, for example no more than about 50 hours, for example no more than about 48 hours.

[0581] 47. A method for generating in vitro islet-like cell aggregates as described in any one of items 23-46, wherein in step b-1), the cell population is cultured for a period of approximately 18 to 54 hours, for example, a period of approximately 20 to 52 hours, for example, a period of approximately 22 to 50 hours, for example, a period of approximately 24 to 48 hours.

[0582] 48. A method for generating in vitro islet-like cell aggregates as described in any one of items 23-47, wherein in step b-1), the cell population is cultured for a period of approximately 42 to 54 hours, for example, a period of approximately 44 to 52 hours, for example, a period of approximately 46 to 50 hours, for example, approximately 48 hours.

[0583] 49. A method for generating in vitro islet-like cell aggregates as described in any one of items 23-47, wherein in step b-1), the cell population is cultured for a period of approximately 18 to 30 hours, for example, a period of approximately 20 to 28 hours, for example, a period of approximately 22 to 26 hours, for example, approximately 24 hours.

[0584] 50. The method of any one of items 23-49, wherein step b-1) comprises culturing the cell population in a medium containing KGF, retinoic acid, SANT-1, PDBu and LDN.

[0585] 51. The method for generating in vitro islet-like cell aggregates as described in any one of items 26-50, wherein the population of endocrine progenitor cells, for example, a population of endocrine progenitor 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.

[0586] 52. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-51, wherein in step b+1), the cell population of said 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.

[0587] 53. The method for generating in vitro islet-like cell aggregates as described in any one of items 26-52, wherein step b+1) comprises culturing the cell population in a medium containing BTC, Alk5i II, GSI-XX, GC-1, LDN, retinoic acid and SANT-1.

[0588] 54. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-53, wherein the cells are cultured on a 2D substrate at least until endocrine progenitor cells are generated in step c+1).

[0589] 55. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-54, wherein the 2D substrate comprises one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized filaments (FN filaments), and matrix gel. TM For example, one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, and matrix gel. TM 56. The method for generating in vitro islet-like cell aggregates as described in any one of items 26-55, 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;

[0590] 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;

[0591] For example, the following groups: LN-521 and its fragments, LN-511 and its fragments, and LN-332 and its fragments;

[0592] For example, the group consisting of: LN-521 and its fragments, and LN-511 and its fragments;

[0593] For example, a group consisting of LN-521 and its fragments or a group consisting of LN-511 and its fragments.

[0594] 57. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-56, 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, wherein the laminin and its fragments are LN-521 or wherein the laminin and its fragments are LN-511.

[0595] 58. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-56, wherein the laminin (LN) and fragments comprise the E8 fragment of laminin, for example, the E8 fragment of laminin selected from the group consisting of: the E8 fragment of LN-511, the E8 fragment of LN-521, the E8 fragment of LN-332, the E8 fragment of LN-421, the E8 fragment of LN-121, and the E8 fragment of LN-111; for example, the group consisting of: Groups: E8 fragments of LN-511, E8 fragments of LN-521, E8 fragments of LN-332, and E8 fragments of LN-421 and 121; for example, groups consisting of: E8 fragments of LN-511, E8 fragments of LN-521 and E8 fragments of LN-332; for example, groups consisting of: E8 fragments of LN-511 and E8 fragments of LN-521; for example, E8 fragments of LN-511 or E8 fragments of LN-521.

[0596] 59. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-58, wherein in step c+1), more than 30%, for example more than 40%, for example more than 45%, for example more than 50% of the total cell population are endocrine progenitor cells, such as endocrine progenitor cells characterized by expressing NKX6.1 and NEUROD1.

[0597] 60. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-58, wherein the number of endocrine progenitor cells in step c+1) is higher than the number of endocrine cells obtained using the corresponding method, wherein step b) is culturing the cell population of said posterior foregut cells under conditions allowing differentiation into pancreatic progenitor cells for about 24 hours or less and / or about 96 hours or longer.

[0598] 61. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-60, wherein the method produces at least about 10%, for example at least about 15%, for example at least about 20%, for example at least about 30%, for example at least about 40%, for example at least 50%, for example at least 60% more endocrine progenitor cells than a corresponding method, wherein in the corresponding method, step b) is culturing the cell population of said foregut cells under conditions that allow differentiation into pancreatic progenitor cells for about 24 hours or less and / or about 96 hours or longer.

[0599] 62. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-61, the method further comprising culturing the endocrine progenitor cells in conditions that allow differentiation into monohormonal pancreatic β cells.

[0600] 63. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-62, wherein the cells are cultured on a 2D substrate, for example, adherent culture on a 2D substrate during steps a-1) to c+1).

[0601] 64. A method for generating in vitro islet-like cell aggregates as described in any one of items 26-63, wherein the cells are not transferred from a 2D substrate culture to a 3D substrate culture before the expression of characteristic markers of endocrine progenitor cells is observed, for example, wherein the cells are not transferred from a 2D substrate adherent culture to a 3D substrate culture before the expression of characteristic markers of endocrine progenitor cells is observed.

[0602] 65. The method for generating in vitro islet-like cell aggregates as described in any one of items 1-64, wherein the EP cell population 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.

[0603] 66. The method for generating in vitro islet-like cell aggregates as described in any one of items 22-65, wherein the cell population provided in steps 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.

[0604] 67. The method for generating in vitro islet-like cell aggregates as described in any one of items 65-66, wherein the provided cell population is derived from a culture of human embryonic stem cells.

[0605] 68. The method for generating in vitro islet-like cell aggregates as described in any one of items 65-67, wherein the cell population is a mammalian cell population, such as a human cell population.

[0606] 69. A method for generating in vitro islet-like cell aggregates as described in any one of items 65-68, wherein the cell population is derived from a human embryonic stem cell line selected from: a group of embryonic stem cell lines consisting of HS980 cells, H1 cells and H9 cells, for example, a group of embryonic stem cell lines consisting of HS980 cells and H1 cells, or a group of embryonic stem cell lines consisting of H1 and H9 cells, or a group of embryonic stem cell lines consisting of HS980 cells and H9 cells.

[0607] 70. The method for generating in vitro islet-like cell aggregates as described in any one of items 65-68, wherein the cell population is derived from a human induced pluripotent stem cell population.

[0608] 71. A method for generating in vitro islet-like cell aggregates as described in any one of items 1-70, the method further comprising cryopreserving EP cells prior to step i).

[0609] 72. An isolated aggregate of islet-like cells, which can be obtained by any of the preceding items.

[0610] 73. A population of isolated islet-like cells that can be obtained by any of the methods described in any of the preceding items.

[0611] 74. An isolated islet-like cell aggregate as described in item 72 or a population of isolated islet-like cell aggregates as described in item 73, wherein the islet-like cell aggregates comprise more than about 40%, for example about 40% to 70%, for example about 40% to 60%, for example about 40% to 50% of the total cell population, and are monohormone β cells, for example monohormone β cells characterized by expressing insulin.

[0612] 75. An isolated islet-like cell aggregate as described in any one of items 72-74 or a population of isolated islet-like cell aggregates as described in any one of items 73-74, wherein the cells constituting the islet-like cell aggregates have not been enriched for a desired phenotype, for example, not enriched by manual or automated intervention, such that the cells have not been enriched before forming a 3D structure in step iii).

[0613] 76. An isolated islet-like cell aggregate or a population of isolated islet-like cell aggregates as described in any one of items 72-75, wherein the cells constituting the islet-like aggregates have not been sorted for a desired phenotype based on marker expression, such that the cells have not been sorted prior to forming a 3D structure in step iii).

[0614] 77. An isolated islet-like cell aggregate or a population of isolated islet-like cell aggregates as described in any one of items 72-76, wherein the cells constituting the islet-like cell aggregates have not been subjected to FACS-based sorting for a desired phenotype prior to their formation, such that the cells have not been sorted prior to the formation of the 3D structure in step iii).

[0615] 78. An isolated islet-like cell aggregate or a population of isolated islet-like cell aggregates as described in any one of items 72-77, wherein the islet-like cell aggregates comprise 7% to 25%, for example 7% to 20%, 10% to 20%, for example 15% to 20%, for example approximately 20% monohormone α cells.

[0616] 79. An isolated islet-like cell aggregate as described in any one of items 72-78 or a population of isolated islet-like cell aggregates as described in any one of items 73-78, wherein the islet-like cell aggregate comprises at least 40%, for example, at least 50%, monohormone β cells; about 15% to 20%, for example, about 20%, monohormone α cells; and less than about 2%, for example, less than about 1%, proliferating cells.

[0617] 80. A cell obtained from at least one isolated islet as defined in any one of items 72-79, for example by dissociating said islet-like cell aggregates.

[0618] 81. An isolated islet-like cell aggregate as described in any one of items 72-79, or a population of isolated islet-like cell aggregates as described in any one of items 73-79, or cells as described in item 80, for use in a therapy.

[0619] 82. An isolated islet-like cell aggregate as described in any one of items 72-79 and 81, or a population of isolated islet-like cell aggregates as described in any one of items 73-79 and 81, or cells as described in item 80 or 81, for use in the treatment, prevention and / or improvement of diabetes, such as type 1 or type 2 diabetes.

[0620] 83. An isolated islet-like cell aggregate or a population of isolated islet-like cell aggregates as described in any one of items 72-79, for use in a therapeutic treatment, wherein said islet-like cell aggregates or cells have been generated by the method described in any one of items 1-71.

[0621] 84. An isolated islet-like cell aggregate or a population of isolated islet-like cell aggregates or cells as described in any one of items 72-79 and 81, for use in the treatment, prevention and / or improvement of diabetes, such as type 1 or type 2 diabetes, wherein said islet-like cell aggregates or cells have been generated by the method described in any one of items 1-71.

[0622] 85. An isolated islet-like cell aggregate as described in any one of items 72-79 and 81, or a population of isolated islet-like cell aggregates as described in any one of items 73-79 and 81, or cells as described in item 80 or 81, for use in a therapeutic treatment.

[0623] The use of the above includes the following steps:

[0624] The isolated islet-like cell aggregates are generated according to the method described in any one of items 1-71; and

[0625] Administer a therapeutically effective amount of the islet-like cell aggregate to the patient

[0626] or

[0627] The use of the above includes the following steps:

[0628] The isolated islet-like cell aggregates are generated according to the method described in any one of items 1-71;

[0629] Dissociate these islet-like cell aggregates, and

[0630] The patient is given a therapeutically effective amount of the dissociated islet cells.

[0631] 86. An isolated islet-like cell aggregate as described in any one of items 72-79 and 81, or a population of isolated islet-like cell aggregates as described in any one of items 73-79 and 81, or cells as described in item 80 or 81, for use in the treatment, prevention, and / or improvement of diabetes, such as type 1 or type 2 diabetes, wherein said use comprises the steps of: generating isolated islet-like cell aggregates according to the method as described in any one of items 1-71; and

[0632] Administer a therapeutically effective amount of the cells to the patient

[0633] or

[0634] The use of the above includes the following steps:

[0635] The isolated islet-like cell aggregates are generated according to the method described in any one of items 1-71;

[0636] Dissociate these islet-like cell aggregates, and

[0637] The patient is given a therapeutically effective amount of the dissociated islet cells.

[0638] 87. An isolated islet-like cell aggregate as described in any one of items 72-79 and 81, or a population of isolated islet-like cell aggregates as described in any one of items 73-79 and 81, or cells as described in items 80 or 81 to 87, wherein the use comprises transplanting the islet-like cell aggregates or cells into a patient in need.

[0639] 88. A pharmaceutical composition comprising an isolated islet-like cell aggregate as described in any one of items 72-79 and 81, or a population of isolated islet-like cell aggregates as described in any one of items 73-79 and 81, or cells as described in item 80 or 81, and at least one pharmaceutically acceptable excipient or carrier.

[0640] 89. A kit comprising isolated islet-like cell aggregates as described in any one of items 72-79 and 81, or isolated islet-like cell aggregate populations as described in any one of items 73-79 and 81, or cells as described in item 80 or 81, or a pharmaceutical composition as described in item 88, and a suitable loading substrate.

[0641] 90. The kit as described in item 89, wherein the suitable loading substrate is a 3D substrate.

[0642] 91. Use of isolated islet-like cell aggregates or populations of isolated islet-like cell aggregates or cells as described in any one of items 72-79 and 81 in drug screening, such as in vitro drug screening.

[0643] 92. A method for in vitro drug screening, the method comprising the steps of: generating isolated islet-like aggregates according to any one of items 1-71; and

[0644] The islet-like cell aggregates are exposed to at least one candidate drug compound.

[0645] 93. The in vitro drug screening method as described in item 92, the method comprising the step of: generating isolated islet-like aggregates according to the method described in any one of items 1-71;

[0646] Dissociate these islet-like cell aggregates; and

[0647] At least a portion of the dissociated pancreatic islet cells are exposed to at least one candidate drug compound.

[0648] 94. A method of treating a patient in need, the method comprising administering to the patient a therapeutically effective amount of an isolated islet-like cell aggregate as described in any one of items 72-79 and 81, or a population of isolated islet-like cell aggregates as described in any one of items 73-79 and 81, or cells as described in item 80 or 81.

[0649] 95. A method for treating patients in need, such as a method for treating diabetes in patients in need, the method comprising the steps of: generating isolated islet-like cell aggregates according to the method of any one of items 1-71; and

[0650] Administer a therapeutically effective amount of the islet-like cell aggregate to the patient.

[0651] or

[0652] The isolated islet-like cell aggregates are generated according to the method described in any one of items 1-71;

[0653] Dissociate these islet-like cell aggregates; and

[0654] The patient is given a therapeutically effective amount of the dissociated islet cells.

[0655] 96. A method of treating diabetes in a patient in need, the method comprising administering to the patient a therapeutically effective amount of an isolated islet-like cell aggregate as described in any one of items 72-79 and 81, or a population of isolated islet-like cell aggregates as described in any one of items 73-79 and 81, or cells as described in item 80 or 81.

[0656] 97. A method for treating diabetes in patients in need, as described in item 95 or 96, wherein the patients have type 1 or type 2 diabetes.

[0657] 98. A method of treating diabetes in a patient in need, as described in any one of items 94 to 97, wherein the administration comprises transplanting the islet-like cell aggregates or cells into the patient.

[0658] 99. Use of the isolated islet-like cell aggregates or populations of isolated islet-like cell aggregates or as described in any one of items 72-79 and 81, or the cells described in item 80 or 81, for the manufacture of a medicament for the treatment of diabetes in patients in need.

[0659] 100. The use as described in item 99, wherein the manufacture of said drug comprises generating islet-like aggregates by any one of items 1-71, and optionally dissociating them.

[0660] 101. A method for generating in vitro islet-like cell aggregates, the method comprising the following steps:

[0661] a-1) Provide a population of primitive intestinal cells, which are, for example, primitive intestinal cells characterized by the expression of HNF1β and / or HNF4α;

[0662] b-1) The cell population of the primitive intestinal cells was cultured for no more than approximately 54 hours under conditions that allowed differentiation into hindforegut cells;

[0663] c-1) thereby generating a population of hindbrain cells, which are, for example, hindbrain cells characterized by expression of PDX1.

[0664] a) Provide a cell population of hindbrain cells generated in step c-1), which are, for example, hindbrain cells characterized by expressing PDX1;

[0665] b) The cell population of the posterior foregut cells is cultured under conditions that allow differentiation into pancreatic progenitor cells for no more than about 78 hours, for example, no more than about 72 hours.

[0666] c) This generates a population of pancreatic progenitor cells, which are, for example, pancreatic progenitor cells characterized by the expression of both PDX1 and NKX6.1;

[0667] a+1) provides the cell population of pancreatic progenitor cells generated in step c);

[0668] b+1) Culture the pancreatic progenitor cell population under conditions that allow differentiation into endocrine progenitor cells;

[0669] c+1) thereby generating a population of endocrine progenitor cells, which are, for example, endocrine progenitor cells characterized by the expression of NEUROD1, such as those characterized by the expression of NKX6.1 and NEUROD1;

[0670] i) Provide a population of endocrine progenitor cells (EPs) generated in step c+1), which are, for example, EP cells characterized by expression of NEUROD1; for example, EP cells characterized by expression of NKX6.1 and NEUROD1;

[0671] ii) Provide a single-cell suspension of the population of said EP cells;

[0672] iii) Allows the population of said EP cells in a single-cell suspension to form a 3D structure;

[0673] iv) Culture the EP cells in a 3D structure under 3D culture conditions that allow differentiation into pancreatic monohormonal β cells to provide islet-like cell aggregates; and

[0674] v) This leads to the formation of islet-like cell aggregates.

[0675] The islet-like cell aggregates described therein comprise at least approximately 25% monohormonal β cells; or

[0676] v) This results in a population of islet-like cells containing mono-hormone β cells.

[0677] The population comprises islet-like cell aggregates, which contain at least approximately 25% monohormone β cells.

Claims

1. A method for generating in vitro islet-like cell aggregates, the method comprising the following steps: i) Provide a population of endocrine progenitor cells (EPs) characterized by the expression of NKX6.1 and NEUROD1, wherein the population is EP cells cultured adherently on a 2D substrate; ii) Provide a single-cell suspension of the population of said EP cells; iii) Allows the population of said EP cells in a single-cell suspension to form a 3D structure; iv) Culture the EP cells in a 3D structure under 3D culture conditions that allow differentiation into pancreatic monohormonal β cells to provide islet-like cell aggregates; and v) This results in the formation of islet-like cell aggregates containing mono-hormone β cells. The islet-like cell aggregates contain up to 2% proliferating cells, and the islet-like cell aggregates contain at least 40% monohormone β cells.

2. The method for generating in vitro islet-like cell aggregates as described in claim 1, wherein, The population containing EP cells is characterized by the expression of NKX6.1, NEUROD1, and PDX1.

3. The method for generating in vitro islet-like cell aggregates as described in claim 1 or 2, wherein the 2D substrate comprises one or more components selected from the group consisting of: laminin (LN) and fragments thereof, fibronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized filaments (FN filaments), and matrix gel. TM .

4. The method for generating in vitro islet-like cell aggregates as described in claim 3, 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.

5. The method for generating in vitro islet-like cell aggregates as described in claim 4, wherein the laminin (LN) and its fragments are selected from the group consisting of LN-521, LN-511 and LN-332.

6. The method for generating in vitro islet-like cell aggregates as described in claim 1, wherein step ii) is performed when more than 15% of the total cell population consists of EP cells expressing NEUROD1.

7. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein the islet-like cell aggregates generated in step v) comprise 7% to 25% monohormone α cells.

8. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein the islet-like cell aggregates generated in step v) comprise up to 1% proliferating cells expressing Ki-67.

9. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein the islet-like cell aggregates generated in step v) are scored after 38-42 days of culture.

10. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein step ii) is performed prior to culturing these cells in a culture medium that allows differentiation into pancreatic monohormone β cells.

11. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein the formation of the 3D structure in step iii) is a spontaneous formation of the 3D structure.

12. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein the formation of the 3D structure in step iii) is a forced or assisted formation of the 3D structure.

13. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein step iv) comprises culturing EP for 2 weeks or longer.

14. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein step iv) comprises culturing EP for 4 weeks.

15. The method for generating in vitro islet-like cell aggregates as described in claim 1, wherein the mono-hormone β cells generated in step v) have the ability to express insulin.

16. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein the mono-hormone β cells generated in step v) have the ability to express C-peptide upon glucose stimulation.

17. The method for generating in vitro islet-like cell aggregates as described in claim 1, wherein the mono-hormone β cells generated in step v) do not express glucagon and / or somatostatin.

18. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein the islet-like cell aggregates generated in step v) comprise at least 40% monohormone β cells; 7%-25% monohormone α cells and less than 2% proliferating cells.

19. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein prior to step i), the method comprises steps a-1)–c-1) and steps a)–c). a-1) Provide a population of primitive intestinal cells characterized by the expression of HNF1β and / or HNF4α; b-1) The cell population of the primitive intestinal cells shall be cultured for no more than 54 hours under conditions that allow differentiation into hindforegut cells; c-1) This generates a population of hindgut cells characterized by PDX1 expression; a) Provide a cell population of hindgut cells characterized by PDX1 expression generated in step c-1); b) The cell population of the posterior foregut cells shall be cultured for no more than 78 hours under conditions that allow differentiation into pancreatic progenitor cells; and c) This generates a cell population of pancreatic progenitor cells characterized by the expression of both PDX1 and NKX6.

1.

20. The method for generating in vitro islet-like cell aggregates as described in claim 19, wherein after steps a)-c), it further comprises steps a+1)-c+1): a+1) Provide the cell population of pancreatic progenitor cells generated in step c; b+1) Culture the pancreatic progenitor cells into a cell population under conditions that allow them to differentiate into endocrine progenitor cells; and c+1) thus generates a population of endocrine progenitor cells.

21. The method for generating in vitro islet-like cell aggregates as described in claim 20, wherein the endocrine progenitor cells are characterized by expressing NKX6.1 and NEUROD1.

22. The method for generating in vitro islet-like cell aggregates as described in claim 19, wherein in step b), the cell population is cultured for a period of 42 to 78 hours.

23. The method for generating in vitro islet-like cell aggregates as described in claim 19, wherein the cells are cultured on a 2D substrate.

24. The method for generating in vitro islet-like cell aggregates as described in claim 23, wherein the 2D substrate is defined as in any one of claims 3-4.

25. The method for generating in vitro islet-like cell aggregates as described in claim 19, wherein in step b-1), the cell population is cultured for a period of 18 to 54 hours.

26. The method for generating in vitro islet-like cell aggregates as described in claim 25, wherein the cell population is cultured for a period of 24 to 48 hours.

27. The method for generating in vitro islet-like cell aggregates as described in claim 20, wherein in step b+1), the cell population of said pancreatic progenitor cells is cultured for 3 to 5 days.

28. The method for generating in vitro islet-like cell aggregates as described in claim 20, wherein in step b+1), the cell population of said pancreatic progenitor cells is cultured for 5 days.

29. The method for generating in vitro islet-like cell aggregates as described in claim 20, wherein the cells are cultured on a 2D substrate during steps a-1) to c+1).

30. The method for generating in vitro islet-like cell aggregates as claimed in claim 1, wherein the cells are not transferred from a 2D substrate culture to a 3D substrate culture before they exhibit expression of characteristic markers of endocrine progenitor cells.

31. The method for generating in vitro islet-like cell aggregates as described in claim 1, wherein the EP cell population in step i) is derived from a culture of pluripotent stem cells or a culture of embryonic stem cells.

32. The method for generating in vitro islet-like cell aggregates as described in claim 31, wherein the cell population is a mammalian cell population.

33. The method for generating in vitro islet-like cell aggregates as described in claim 31, wherein the cell population is derived from a human embryonic stem cell line selected from the group consisting of HS980 cells, H1 cells, and H9 cells.

34. The method for generating in vitro islet-like cell aggregates as described in claim 31, wherein the cell population is derived from a human induced pluripotent stem cell population.

35. The method for generating in vitro islet-like cell aggregates as described in claim 1, further comprising cryopreserving EP cells prior to step i).

36. An isolated islet-like cell aggregate, which can be obtained by the method of any one of the preceding claims, wherein the islet-like cell aggregate comprises at least 40% monohormone β cells; 15% to 20% monohormone α cells and less than 2% proliferating cells.

37. A population of isolated islet-like cell aggregates, which can be obtained by the method of any one of claims 1-35, wherein the islet-like cell aggregates comprise at least 40% monohormone β cells; 15% to 20% monohormone α cells and less than 2% proliferating cells.

38. The isolated islet-like cell aggregates of claim 36 or the isolated islet-like cell aggregate populations of claim 37, wherein the islet-like cell aggregates comprise 40% to 70% monohormone β cells.

39. An isolated islet-like cell aggregate as described in any one of claims 36 or 38, or a population of isolated islet-like cell aggregates as described in any one of claims 37-38, wherein the monohormonal β cells are characterized by expressing insulin.

40. The isolated islet-like cell aggregates of claim 36 or the isolated islet-like cell aggregate populations of claim 37, wherein the cells constituting the islet-like cell aggregates have not been enriched for the desired phenotype.

41. The isolated islet-like cell aggregates of claim 36 or the isolated islet-like cell aggregate populations of claim 37, wherein the cells constituting the islet-like aggregates have not been subjected to sorting for a desired phenotype based on marker expression.

42. The isolated islet-like cell aggregates of claim 36 or the isolated islet-like cell aggregate populations of claim 37, wherein the cells constituting the islet-like cell aggregates have not undergone FACS-based sorting for the desired phenotype prior to their formation.

43. The isolated islet-like cell aggregates of claim 36 or the isolated islet-like cell aggregate populations of claim 37, wherein the cells have not been sorted prior to forming these 3D structures in step iii).

44. The isolated islet-like cell aggregates of claim 36 or the isolated islet-like cell aggregate populations of claim 37, wherein the islet-like cell aggregates comprise 7% to 20% monohormone α cells.

45. The isolated islet-like cell aggregates of claim 36 or the isolated islet-like cell aggregate population of claim 37, wherein the islet-like cell aggregates comprise 15% to 20% monohormone α cells.

46. ​​The isolated islet-like cell aggregate of claim 36 or the isolated islet-like cell aggregate population of claim 37, wherein the islet-like cell aggregate comprises at least 50% monohormone β cells; 15% to 20% monohormone α cells and less than 2% proliferating cells.

47. Use of the isolated islet-like cell aggregates of claim 36 or the isolated islet-like cell aggregate populations of claim 37 in the preparation of a medicament for use in a therapy.

48. Use of the isolated islet-like cell aggregates of claim 36 or the isolated islet-like cell aggregate populations of claim 37 in the preparation of a medicament for use in the treatment, prevention and / or improvement of diabetes.

49. The use according to claim 48, wherein the diabetes is type 1 or type 2 diabetes.

50. The use according to any one of claims 47-48, The use of Includes the following steps: The method according to any one of claims 1-35 generates isolated islet-like cell aggregates; and Administer a therapeutically effective amount of the islet-like cells or cell aggregates to the patient.

51. The use according to any one of claims 47-48, wherein the use comprises transplanting the islet-like cell aggregates or cells into a patient in need.

52. A pharmaceutical composition comprising isolated islet-like cell aggregates or populations of isolated islet-like cell aggregates as claimed in any one of claims 36 or 38-46, and at least one pharmaceutically acceptable excipient or carrier.

53. A kit comprising isolated islet-like cell aggregates as claimed in any one of claims 36 or 38-46, or isolated islet-like cell aggregate populations as claimed in any one of claims 37-46, or a pharmaceutical composition as claimed in claim 52, and a suitable loading substrate.

54. The kit of claim 53, wherein the suitable loading substrate is a 3D substrate.

55. Use of the isolated islet-like cell aggregates as described in any one of claims 36 or 38-46, or the isolated islet-like cell aggregate populations as described in any one of claims 37-46, in in vitro drug screening.

56. A method for in vitro drug screening, the method comprising the following steps: Separated islet-like aggregates are generated according to the method of any one of claims 1-35; as well as The islet-like cell aggregates are exposed to at least one candidate drug compound.

57. Use of the isolated islet-like cell aggregates or populations of isolated islet-like cell aggregates as described in any one of claims 38-46 for the manufacture of a medicament for the treatment of diabetes in patients in need.

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