3D pancreatic islet formation from endocrine progenitor cells

Abstract

The present disclosure relates to a method of generating pancreatic lineage cells, such as pancreatic islet-like cell aggregates comprising pancreatic beta cells, the method comprises 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 a 3D structure, and culturing the cells under conditions that allow differentiation into pancreatic single hormone beta cells. The disclosure also relates to islet-like cell aggregates obtainable by said method and medical uses thereof.

Description

Technical Field

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

[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 autoimmune destruction of the insulin-producing pancreatic beta cells, while type 2 diabetes is characterized by peripheral insulin resistance and the inability to produce enough insulin to overcome this resistance. Additional, less common forms of diabetes associated with impaired insulin production include gestational diabetes, maturity-onset diabetes of the adolescent, neonatal diabetes, and islet loss in pancreatitis. Patients with type 1 diabetes are treated with injections of exogenous insulin, which can control blood glucose levels to some extent and significantly reduce the incidence of diabetes, but it is not a curative treatment and is associated with short-term and long-term complications. Therefore, while insulin therapy has saved countless diabetic patients from premature death, it represents an ameliorative treatment rather than a cure.

[0003] Pancreatic islets, also known as islets of Langerhans, are regions of the pancreas that contain its endocrine cells. The islets are arranged in dense pathways throughout the human pancreas and are important in the metabolism of glucose. The hormones produced by the islets are secreted directly into the bloodstream by (at least) five types of cells: alpha cells that produce glucagon; beta cells that produce insulin and amylin; delta cells that produce somatostatin; epsilon cells that produce ghrelin and PP cells (gamma cells or F cells) that produce pancreatic polypeptide. The cellular architecture of the islets is essential for cell-cell communication and coordinated hormone secretion.

[0004] Patients with diabetes, especially those with type 1 diabetes, can be cured by transplantation of insulin-producing pancreatic β cells. The pancreatic β cells can be transplanted as islets or islet-like structures. Generating an unlimited supply of human β cells from pluripotent stem cells could provide therapy for millions of patients. Thus, a cure for diabetes could be achieved by replenishing lost β cells for patients in need. Animal models demonstrated this approach decades ago: rats induced to develop diabetes due to the β cell toxin streptozotocin could be cured by injection of syngeneic islets (see Murtaugh 2007 for a review). Transplantation of pancreatic progenitor cells derived from human pluripotent stem cells represents a promising treatment modality for diabetes. However, reliable and safe strategies are needed to obtain the required cell aggregates and rely on efficient differentiation protocols that can be scaled up to meet therapeutic needs.

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

[0006] Many different studies over the past decade have shown that pancreatic cells can be generated from hPSCs, including cells expressing multiple hormones of insulin and cells expressing a single hormone of insulin (US2019 / 0359943; US 10253298; US2011 / 0280842; Nostro et al., (2015); D'Amour et al., (2006)).

[0007] However, prior art methods do not provide pancreatic beta cells with the efficiency and / or reproducibility required for clinical application. Therefore, alternative methods are needed to more efficiently derive desired cell types from pluripotent cells. There are significant challenges to fully utilizing the potential of stem cells, 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 cells generated in vitro; and (iii) eliminating contaminating undesired cell types that may disrupt the function of the desired cell type. The presence of undesired cell types in culture, including, for example, multihormonal pancreatic cells, may, for example, pose safety issues in prospective replacement therapies, or may negatively impact the results of drug screening or disease modeling.

[0008] Therefore, it is apparent from the different parts of this background description that it is desirable to provide differentiation strategies that overcome the disadvantages. Therefore, it is desirable to provide differentiation strategies to effectively obtain high-quality islet-like cell aggregates (or islet-like structures) in vitro in a reproducible manner. Summary of the invention

[0009] It is an object of the present disclosure to provide a differentiation strategy for generating islet-like cell aggregates in vitro that overcomes and / or mitigates the above or other disadvantages of current strategies.

[0010] It is an object of the present disclosure to provide an efficient 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] It is an object of the present disclosure to provide an efficient and reproducible in vitro differentiation protocol that allows for the large-scale generation of islet-like cell aggregates.

[0012] It is also an object of the present disclosure to provide an in vitro differentiation protocol that allows obtaining cultures of islet-like cell aggregates that exhibit a high percentage of pancreatic monohormonal β cells and a low percentage of contaminating cell types. Said islet-like cell aggregates also exhibit monohormonal α cells.

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

[0014] It is also a goal of the present disclosure to provide islet-like cell aggregates that exhibit a high percentage of pancreatic monohormone β cells and a low percentage of contaminating cell types. The islet-like cell aggregates also exhibit monohormone α cells. Specifically, the desired cells must be alive and healthy. Such islet-like cell aggregates or cells obtained therefrom can be used for a variety of applications, including therapeutic applications and scientific applications / biotechnology 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 the skilled person from this disclosure are achieved by the different aspects of the invention as claimed in the accompanying claims and as generally disclosed herein.

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

[0017] The present disclosure is based on the surprising recognition that providing stage 5 endocrine progenitor cells (EP) (also referred to herein as endocrine precursor cells) in a single cell suspension and allowing a population of the EP cells to form a 3D structure, and continuing to culture the population of the EP cells in the form of a 3D structure in a 3D culture condition that allows differentiation into pancreatic monohormone β cells to provide islet-like cell aggregates, results in unexpectedly high quality islet-like cell aggregates. The islet-like cell aggregates contain an unexpectedly high percentage of monohormone β cells, a low percentage of multihormone cells, a low percentage of monohormone α cells (α cells) and / or δ cells, and a low percentage of proliferating cells. The inventors have found 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 (EP) is reached, and dissociating these cells, such as dissociating 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 transfer from 2D culture conditions to 3D culture conditions is very important. Without being bound by theory, it appears that the timing of the cell transfer affects the development / differentiation of EP cells into high quality islet cells. Importantly, according to the methods as disclosed herein, EP cells in single cell suspension allowed to form 3D structures 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) providing a cell population of endocrine progenitor cells (EP), such EPs are, for example, EP cells characterized by expressing NEUROD1; such as EP cells characterized by expressing NKX6.1 and NEUROD1;

[0020] ii) providing a single cell suspension of the population of EP cells;

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

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

[0023] v) thereby generating islet-like cell aggregates comprising a single hormone β cell,

[0024] The islet-like cell aggregates contain at least about 25% of single hormone β cells.

[0025] As used herein, the term "islet-like cell aggregates" or "islet-like aggregates" refers to cell aggregates of pancreatic cells that display the characteristics of islets in vivo. Specifically, the aggregates exhibit the following desired properties: a high number of single hormone β cells, a desired 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. Specifically, 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 the cell types present herein and in terms of their functional properties.

[0026] The skilled person understands that the percentages described herein relate to the average percentages exhibited by islet-like cell aggregates. Thus, for example, 100 aggregates are analyzed and the average number of the cell types is as shown herein. Thus, step v) can be rephrased as "thereby generating a population of islet-like cell aggregates comprising single hormone beta cells, wherein the population comprises islet-like cell aggregates containing at least 25% of single hormone beta cells". In this context, the properties listed below refer to the average percentages in a population of islet-like cell aggregates according to the present disclosure.

[0027] Thus, in one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: 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 60%, such as at least about 65%, such as at least about 70% of single hormone beta cells. In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: about 25% to 70%, such as 30% to 70%, such as 30% to 70%, such as 30% to 70% of single hormone beta cells, such as 35% to 70%, 35% to 70%, such as 40% to 70%, such as 45% to 70%, such as 45% to 65%, such as 45% to 60%, such as 45% to 55%, such as about 50% of single hormone beta cells. In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising about 35% to 65%, such as 40% to 65%, such as 40% to 60%, of single hormonal beta cells.

[0028] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: approximately 7% to 25%, such as 7% to 20%, 10% to 20%, such as 15% to 20%, such as approximately 20% of single hormone alpha cells.

[0029] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: at most about 10%, for example at most about 7%, for example at most about 6%, for example at most about 5%, for example at most about 4%, for example at most about 3%, for example at most about 2%, for example at most about 0.5%, for example at most about 0.3%, for example at most about 0.1% of multi-hormone alpha cells.

[0030] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: at most about 10%, for example at most about 7%, for example at most about 6%, for example at most about 5%, for example at most about 4%, for example at most about 3%, for example at most about 2%, for example at most about 0.5%, for example at most about 0.3%, for example at most about 0.1% of multi-hormone beta cells.

[0031] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising less than 5%, such as less than 4%, such as less than 3%, such as less than 1%, such as less than 1% delta cells.

[0032] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates containing: at most about 5, such as at most about 4%, such as at most about 3%, such as at most about 2%, such as at most about 1%, such as at most about 0.5%, such as at most about 0.1% of proliferating cells, such as proliferating cells expressing Ki-67.

[0033] In one, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: at least 40%, such as at least 50%, of single hormone β cells; about 15% to 20%, such as about 20%, of single hormone α cells and less than about 2%, such as less than about 1%, of proliferating cells.

[0034] In one embodiment, the composition of the islet-like cell aggregates is scored, in other words investigated, at culture day 38-42 or later. For example, at day 38, 39, 40, 41 or 42. In other words, the composition of the islet-like cell aggregates is scored, in other words investigated, at the end of stage 6.

[0035] Pancreatic islets, also known as islets of Langerhans, are regions of the pancreas that contain endocrine (hormone-producing) cells. There are approximately 1 million islets distributed throughout the pancreas of healthy adults, with each islet having an average diameter of approximately 0.2 mm. Each islet is separated from the surrounding pancreatic tissue by a thin fibrous connective tissue capsule that is continuous with the fibrous connective tissue interwoven throughout the rest of the pancreas. The hormones produced by the islets are secreted directly into the bloodstream by (at least) five types of cells. The endocrine cell types in the islets include: alpha cells that produce glucagon, beta cells that produce insulin and amylin, PP cells (gamma cells or F cells) that produce pancreatic polypeptide, delta cells that produce somatostatin, and epsilon cells that produce ghrelin. In humans, beta cells account for approximately 40%-50% of the cells. In addition to endocrine cells, there are stromal cells (fibroblasts), vascular cells (endothelial cells, pericytes), immune cells (granulocytes, lymphocytes, macrophages, dendritic cells), and neural cells. The skilled artisan is familiar with the composition and cellular structure of pancreatic islets.

[0036] Endocrine progenitor (EP) cells 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 Figure 1A ), which NEUROD1 is upregulated. Cells at early developmental stage 4 (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. As explained below, other markers and combinations thereof can be used to identify EP cells. Pancreatic endocrine progenitor cells express at least one, or two, three, or all four of the following markers: PDX1, NKX6.1, NGN3, and NEUROD1.

[0037] Thus, in one embodiment of the method as disclosed herein, the cell population of EP cells provided in step i) is characterized by the expression of NEUROD1 and NKX6.1. In another embodiment, the cell population of EP cells provided in step i) is characterized by the expression of NKX6.1 and NGN3 or by the 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] The skilled person will appreciate that step ii) of providing a single cell suspension of a population of EP cells involves dissociating the EP cells from adherent cultures 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 attachment, allowing the cells to be released from the surface and from each other. Dissociation may be performed by mechanical means. Non-limiting examples of dissociation agents include trypsin, collagenase, displasases, and dissociation agents such as Accumax TM and ACS-3010.

[0039] In an embodiment, step ii) of providing a single cell suspension of a population of EP cells involves dissociating EP cells from an adherent culture on a 2D substrate into single cells. In an embodiment, step ii) of providing a single cell suspension of a population of EP cells involves dissociating EP cells by enzymatic means, such as using a solution comprising enzymes (e.g., proteolytic enzymes and collagenolytic enzymes). For example, such a solution may be The skilled artisan is aware of appropriate methods for dissociating EP cells and providing a single cell suspension of a population of EP cells.

[0040] In one embodiment, step ii) and step iii) are performed prior to subjecting the cells to conditions allowing differentiation into pancreatic monohormone β cells, such as prior to culturing the cells in a medium allowing differentiation into pancreatic monohormone β cells.

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

[0042] Thus, in one embodiment, steps i), ii) and iii) are performed under conditions that allow differentiation into endocrine progenitor cells, whereas step iv) is performed under conditions that allow differentiation into pancreatic monohormonal beta cells.

[0043] In step iii), the EP cells in the single cell suspension are allowed to form a 3D structure. In one embodiment, the 3D culture conditions allow the cells to self-aggregate. The aggregation can be forced or induced aggregation, but 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 analog or agonist thereof. In one embodiment, the concentration of H1152 ranges from >0 μM to 10 μM. As shown in the examples of the present 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] Thus, in one embodiment of the method as disclosed herein, the ROCK inhibitor is present in the culture medium during about 24 hours during phase iii).

[0046] Without being bound by theory, it is envisioned that self-aggregation allows selective enrichment of endocrine progenitor cells. As shown in the attached examples, the formation of 3D structures (also referred to as aggregation) leads to selective enrichment and leads to increased generation of pancreatic monohormone β cells in the culture. As explained above, the aggregates exhibit the following desired properties: a high number of monohormone β cells, a desired number of monohormone α cells, a low number of multihormone cells (including a low number of multihormone β cells and a low number of multihormone α cells) and a low number of proliferating cells. In one embodiment, the pancreatic monohormone β cells are generated as part of a cell aggregate. In one embodiment, the aggregates comprise monohormone β cells. In one embodiment, the aggregates further comprise pancreatic monohormone α cells. In one embodiment, the aggregates further comprise pancreatic monohormone δ cells, such as less than 3%, such as less than 2%, such as less than 1% of δ cells. It is understood by those skilled in the art that the percentages described herein should be interpreted as being related to the average percentages exhibited by islet-like cell aggregates. The percentages can be evaluated for the islet-like cell aggregates themselves or for the populations comprising islet-like cell aggregates. For the sake of clarity, said cell aggregates are referred to herein as islet-like cell aggregates and are obtainable according to the method defined herein.

[0047] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized ("less committed") cell acquires the characteristics of a specialized cell ("more committed"), such as, for example, a pancreatic cell. A differentiated cell is one that occupies a more specialized ("committed") position in a cell lineage.

[0048] As used herein, the term "committed" as applied to a differentiation process refers to a cell that has progressed through a differentiation pathway to a point where it would normally continue to differentiate into a particular cell type or subset of cell types and is normally unable to differentiate into a different cell type or revert to a less differentiated cell type.

[0049] As used herein, the term "lineage" of a cell refers to the heredity of a cell, i.e., which cells it comes from and which cells it can give rise to. A cell's lineage places a cell within a genetic scheme of development and / or differentiation in vivo or in vitro.

[0050] As used herein, the term "lineage-specific marker" refers to a characteristic that is specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of uncommitted cells to a lineage of interest.

[0051] Those skilled in the art are familiar with the different developmental stages of the pancreatic endocrine lineage, such as the pancreatic β-cell lineage.

[0052] As used herein, the term "pancreatic β cell lineage" refers to a genetic program of development and / or differentiation in vivo or in vitro that ultimately results in providing cells that exhibit characteristic properties of pancreatic monohormonal β cells, such as the production of insulin and the expression of at least one of PDX1, NKX6.1, and NEUROD1. The skilled artisan will appreciate that the cells of the pancreatic β cell lineage can be any cell at an early developmental stage of the cell.

[0053] As used herein, the term "precursor thereof" relates to pancreatic lineage cells, such as precursors of pancreatic β cells, and refers to any cell capable of differentiating into pancreatic β cells when cultured under conditions suitable and / or permissive for the differentiation of precursor cells into pancreatic lineage (e.g., precursors of pancreatic β cells), including, for example, pluripotent stem cells, definitive endoderm cells, primitive gut tube cells, posterior foregut cells, pancreatic progenitor cells, or endocrine progenitor cells.

[0054] Differentiation of cells along the pancreatic β cell lineage includes differentiation from less committed to more committed cell types. In short, the development of insulin-producing pancreatic β cells represents the culmination of a complex developmental program and involves the following in vivo steps: posterior foregut cells acquire pancreatic properties, the expanding pancreatic primordium adopts an endocrine fate, and a subset of these precursors become competent to generate β cells. Factors (e.g., transcription factors) that have been shown to be important in the development of cells of the pancreatic β cell lineage include, among others, PDX1 (pancreatic and duodenal homeobox 1), PTF1A (pancreatic-specific transcription factor 1a, NGN3 (neurogenin 3), NEUROD1 (neurogenic differentiation 1), and NKX6.1 (homeobox protein NKX-6.1). The list of factors should be considered non-limiting, and additional factors will be discussed below.

[0055] In addition, the development of cells 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 definitive endoderm in mouse and human embryonic stem (ES) cells, while RA treatment promotes PDX1 expression and pancreatic specialization in ES cell-derived endoderm. The skilled person understands that in vitro differentiation of pancreatic β cell lineage cells requires the addition of extrinsic factors to the cell growth medium at the appropriate stage of the differentiation process and at suitable / permissive concentrations, which in principle mimics the in vivo development of the cells.

[0056] Thus, the skilled artisan recognizes that, for a recited cell type, "differentiation-permissive conditions" refers to conditions that allow the cells to express the characteristics of that cell type, and may include a combination of cell culture medium, the presence and / or absence of extrinsic factors, and their timing.

[0057] The following is a brief summary of the various developmental stages of pancreatic development. The skilled artisan understands that the development can be described in terms of developmental stages. Each developmental stage 0-6 is characterized by the expression of a set of factors (commonly referred to as markers).

[0058] The skilled person understands that the process of differentiating pluripotent stem cells in vitro into functional pancreatic endocrine cells (e.g., monohormonal pancreatic β cells) can in some aspects be viewed as progressing through six sequential stages, as shown in the schematic diagram shown in Figure 1, which correspond to the in vivo developmental stages. The skilled person is very familiar with the in vivo developmental stages and the stages are considered to be encompassed within the common knowledge in the art.

[0059] In this stepwise progression, stage 0 refers to undifferentiated pluripotent cells, such as hES cells or iPS cells; stage 1 refers to cells expressing markers characteristic of definitive endoderm (DE) cells; stage 2 refers to cells expressing markers characteristic of primitive gut tube cells (PGT); stage 3 cells express markers characteristic of posterior foregut cells (PF); stage 4 refers to cells expressing markers characteristic of pancreatic progenitor cells (PP); stage 5 refers to cells expressing markers characteristic of pancreatic endocrine progenitor cells (EP); stage 6 refers to cells expressing markers characteristic of endocrine islet cells, such as pancreatic β cells. Stage 6 cells as defined herein can form in vitro islet-like cell aggregates (in vitro cell aggregates) that mimic islets found in vivo.

[0060] The skilled artisan understands that not all cells in a particular population progress through these stages at the same rate, ie, some cells may progress less or more along the differentiation pathway than the majority of cells present in the population.

[0061] Below is a non-exhaustive description of the features associated with cells at different stages of in vitro culture as described above. The technician will understand that the different stages of in vitro culture correspond to the developmental stages in vivo. The technician should understand that by selectively selecting which protein expression to monitor, people can track the developmental process along the pancreatic endocrine lineage (e.g., pancreatic beta cell lineage). When necessary, the expression of more than one protein feature of the developmental stage can be evaluated. As the cells progress in the developmental process, the expression of certain proteins is upregulated and downregulated, which makes the protein suitable as a marker for different developmental stages.

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

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

[0064] As used herein, the term "posterior foregut cell" refers to an endoderm cell that produces a portion of the stomach, liver, pancreas, gallbladder, and duodenum. Posterior foregut cells express PDX1 or express both PDX1 and HNF6. Thus, posterior foregut cells can be identified by the expression of PDX1, HNF6, or both PDX1 and HNF6.

[0065] As used herein, the term "pancreatic progenitor cells" refers to cells that express at least one, or two, or three, or four, or 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] Thus, pancreatic progenitor cells can be identified by expression of PDX1 and NKX6.1.Pancreatic progenitor cells can be identified by expression of PDX1, NKX6.1, and one or both of PTF1A and SOX9.

[0067] As used herein, the term "endocrine progenitor cell" refers to a pancreatic endoderm cell that is capable of becoming a pancreatic hormone-expressing cell. Pancreatic endocrine progenitor cells express at least one, or 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 be distinguished from pancreatic progenitor cells in that they express NEUROD1 and NGN3, while pancreatic progenitor cells do not express these two. 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 cells" refers to cells 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 also include one or 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 pancreatic beta cells at stage 6. As used herein, the term "islet-like cell aggregates" or "islet-like aggregates" refers to cell aggregates of pancreatic cells that display the characteristics of islets in vivo. Specifically, the aggregates exhibit properties as 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 does not express glucagon or somatostatin. The terms "pancreatic β cell" and "monohormonal pancreatic β cell" are used interchangeably herein. Thus, a pancreatic monohormonal β cell can be identified by the expression of insulin and the lack of expression of glucagon and / or somatostatin.

[0070] Pancreatic beta cells are monohormonal cells, in other words, express only one hormone, namely insulin. In contrast, polyhormonal cells in the context of pancreatic cells express more than one hormone, such as at least two of insulin, glucagon and somatostatin.

[0071] For example, as shown in Figure 1, the progression through the above developmental stages can be followed by marker expression. For example, progression from stage 0 to stage 1 is associated with downregulation of OCT4, NANOG, 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 maintenance of PDX1 and HNF6 expression and upregulation of NKX6.1, PTF1A, and SOX9 expression. Progression from stage 4 to stage 5 is associated with downregulation of PTF1A and SOX9 expression, maintenance of PDX1 and NKX6.1 expression, and upregulation of NGN3 and NEUROD1 expression. Progression from stage 5 to stage 6 pancreatic β cells is associated with downregulation of NGN3 expression, maintenance of PDX1, NKX6.1, and NEUROD1 expression, and upregulation of insulin and C-peptide expression. Thus, for example, upregulation of NKX6.1 in PDX1+ cells marks progression to stage 4.

[0073] It will be appreciated by the skilled artisan that the developmental progression of pancreatic lineage cells can be further divided into additional stages, for example based on the expression levels of markers. For example, in a review article by Verhoeff and colleagues (Stem Cell Reviews and Reports (2022): 18, 2683-2698), seven stages of differentiation into islet-like clusters are summarized, with stages 1-4 and 7 corresponding to stages 1-4 and 6 used in this application. Stage 5 according to Verhoeff is characterized by expression of NKX6.1 and low expression of NGN3, while stage 6 according to Verhoeff is characterized by high expression of NGN3 and expression of endocrine hormones. Stage 5 defined in this disclosure corresponds to stage 5 and stage 6 of Verhoeff.

[0074] The generated cells are identified or characterized by phenotypic characteristics, morphological characteristics and / or expression of cell markers, which are readily understood by those skilled in the art of evaluating such cells. As used herein, the term "marker" refers to a nucleic acid or polypeptide molecule that is differentially expressed in a cell of interest.

[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 described above, the combination of the markers is a feature of different developmental stages along the β cell lineage. As discussed above, it will be appreciated by the skilled artisan that markers can be selected so that the expression or lack of expression of the marker combination allows the cells at different developmental stages along the pancreatic endocrine lineage to be distinguished (see FIG. 1 ). The inventors have used the expression of different markers to distinguish cells at different developmental stages, as illustrated in the accompanying embodiments. As used herein, when it comes to cells of a cell population, the term "characterized by expression ... " should be interpreted as being associated with the expression of a given marker or marker group. On the contrary, when it comes to cells of a cell population, the term "characterized by lack of expression ... " or "lack of expression ... " or "no expression ... " should be interpreted as being associated with the lack of expression of a given marker or marker group. The skilled artisan is well familiar with the use of marker expression as a means of distinguishing cells with different characteristics, such as cells of different developmental stages of the pancreatic β cell lineage.

[0076] The skilled person will appreciate that when analyzing a cell population by a method that is limited in the number of markers that can be scored simultaneously, for example due to limitations of the method itself or due to limitations of the reagents used, a subset of markers that allows the first cell population and the second cell population to be distinguished can be selected. For example, a cell population of pancreatic progenitor cells characterized by the expression of PDX1 and NKX6.1 can be distinguished from a population of endocrine progenitor cells characterized by the expression of PDX1, NKX6.1, NEUROD1, and NGN3. For example, pancreatic progenitor cells are positive for PDX1 or NKX6.1, but negative for one of NGN3 or NEUROD1. EP cells can be scored as double positive for NKX6.1 and at least one marker not expressed by pancreatic progenitor cells, such as double positive for NKX6.1 and NEUROD1; or double positive for NKX6.1 and NGN3. The Examples section applies this principle to distinguish cells with different characteristics. Importantly, if the cells are scored with the other markers listed above, the cells will exhibit a complete marker spectrum for a particular population. The fact that only a subset of markers is used in an experimental setting should never be interpreted as a lack of expression representing the remaining characteristic markers. The technician will understand that marker expression can be evaluated at the nucleotide level, such as the mRNA level or the protein level. Well-known methods for evaluating marker expression include, but are not limited to, immunohistochemistry, in situ hybridization, FACS, RNA sequencing, the use of arrays (e.g., microarrays), and quantitative PCR. The technician knows these and other suitable methods.

[0077] In this context, differential expression means that the level of a positive marker is increased and the level of a negative marker is decreased compared to an undifferentiated cell or a cell at a different stage of development. The detectable level of a marker nucleic acid or polypeptide in a target cell is sufficiently high or low compared to other cells so that any of a variety of methods known in the art can be used to identify the target cell and distinguish it from other cells.

[0078] As used herein, a cell is "positive for," or is "positive" for, a particular marker when the particular marker is sufficiently detected in the cell (in other words, expressed by the cell). Thus, a cell characterized by the expression of a given marker is positive for that marker. Conversely, a cell is "negative" for, or is "negative" for a particular marker when the particular marker is not sufficiently detected in the cell. Thus, a cell characterized by the lack of expression of a given marker is negative for that marker. The use of a "+" or "-" sign associated with a marker is intended to be understood herein as a positive or negative expression for that marker (e.g., NKX6.1 + Cells were positive for the marker NKX6.1).

[0079] Using any in vitro differentiation method, the ability to obtain a colony of cells that exhibit the same or similar characteristics at a given time point is crucial, because cells at different developmental, mature or functional stages may have different responses to external and internal signals. To clarify, factor X may cause the specialization of cell types A and B during development, while treatment with the same factor X may cause selective cell death of mature cell type A. Therefore, it is not only important to understand that the downstream functional results of subjecting cells to a given factor are important, but it is also important to subject the cells to the factor during the time window in which the cells can respond to the factor in a desired manner. Therefore, the technician should understand the importance of obtaining a synchronized cell colony to ensure that the cell colony responds as a whole. For example, it is desirable that most cells in the culture be progenitor cells at a given time point.

[0080] Tissue engineering, stem cells and molecular biology research mainly involve cell culture on flat plastic or glass dishes / cell culture plates. These cultures are considered to be adherent cultures. This technology is called two-dimensional (2D) cell culture. As used herein, the term "2D" in the context of cell culture refers to culture on a flat cell culture plate, wherein these cells adhere directly or indirectly (e.g., via a cell culture substrate) to the culture plate. The plate can be coated with a cell culture substrate. There are two main types of cell culture substrates: 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 the 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 great influence on the way cells grow and attach, and is therefore an important parameter to consider.

[0081] In one embodiment of the method as 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 methods 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, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and Matrigel. TM , for example, may contain one or more components selected from the group consisting of: laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen, gelatin, and matrigel TM , for example, may comprise one or more components selected from the group consisting of: laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and matrigel TM , for example, may comprise one or more components selected from the group consisting of: laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and matrigel TM In particular, the fragment may comprise a functional domain of a protein. Thus, the skilled person will appreciate that a fragment may correspond to a functional domain or comprise a functional domain, for example comprise a functional domain and further comprise additional N-terminal and / or C-terminal amino acids. Matrigel TMDerived from mouse Engelbreth-Holm-Swarm tumors, and contains many unknown components. It is known that laminin is the main component of matrigel. In addition to using a defined culture medium, it may be beneficial to avoid the use of any animal-derived products, especially for cell cultures for therapeutic use, to obtain a defined and reproducible product and avoid any potential patient safety issues. As used herein, functionalized silk refers to a recombinant fusion protein comprising a silk protein and a cell binding motif. In certain embodiments, the functionalized silk comprises a cell binding motif selected from the group consisting of 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: 12). 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 except cysteine; and C1 and C2 are linked by a disulfide bond.

[0083] In a specific embodiment, the functionalized silk comprises a spider silk protein fragment and a cell-binding motif, which cell-binding motif is 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 silk has been described in WO 2016 / 207281 and WO 2017 / 137611, and these disclosures are included herein in their entirety. Specifically, the functionalized silk can be a recombinant polypeptide comprising the following amino acid sequence:

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

[0086] X1 is S or T;

[0087] X2 is G, A or V;

[0088] X3 is 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 a disulfide bond;

[0092] and wherein the spider silk protein fragment comprises protein parts REP and CT, wherein REP is a repeating segment having 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, wherein n is an integer from 2 to 10;

[0093] Each individual A segment is an amino acid sequence of 8 to 18 amino acid residues, wherein 0 to 3 amino acid residues are not Ala, and the remaining amino acid residues are Ala; each individual G segment is an amino acid sequence of 12 to 30 amino acid residues, wherein at least 40% of amino acid 25 residues are Gly; each individual L segment is a linker amino acid sequence of 0 to 30 amino acid residues; and CT is a fragment of 70 to 120 amino acid residues, which has 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 certain embodiments, the spider silk protein 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 to SEQ ID NO: 16 or to amino acid residues 18-277 of SEQ ID NO: 14. In a specific embodiment, the cell binding motif comprises the amino acid sequence CTGRGDSPAC (SEQ ID NO: 11).

[0094] In a specific embodiment, the functionalized silk comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15. In certain embodiments, the functionalized silk has at least 70%, such as at least 80%, such as at least 85%, preferably at least 90%, such as at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15.

[0095] Thus, in one embodiment, the 2D substrate does not contain any animal-derived components. In one embodiment, the 2D substrate is not Matrigel. TM In other words, the components of the 2D substrate may be components produced using recombinant technology.

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

[0097] Laminins are high molecular weight proteins of the extracellular matrix. They are the main component of the basement membrane, which is the protein network foundation of most cells and organs. Laminins are an important and biologically active part of the basement membrane, affecting cell differentiation, migration and adhesion. Therefore, it is important to choose the laminins used as cell culture substrates in order to provide the cells with an appropriate chemical and mechanical sensitive microenvironment, thereby providing optimal culture conditions.

[0098] Each laminin isotype is composed of three mutually spiral chains-α, β and γ chains, and these chains exist with five, four and three genetically different variants respectively. Laminin isotype is named according to its chain composition. For example, the combination of α5 chain, β2 chain and γ1 chain forms laminin 5-2-1 (LN-521). Trimeric protein forms a cross-shaped structure, which can be combined with other extracellular matrix molecules and various cell membrane receptors.

[0099] Therefore, the selection of laminin or its fragment as 2D substrate is very important and will be affected by the cell type of culture. In addition, the difference between cell lines (such as different ES or IPS cell lines or primary cells) may affect the selection of the best 2D substrate. Full-length laminin can be used as a cell culture substrate, but its fragments can also be used for cell culture. Different domains of laminin have been identified, which mediate different activities, such as cell attachment and the influence on cell proliferation, differentiation and movement.

[0100] In one embodiment of the method as disclosed herein, the laminin (LN) and fragments thereof are selected from the group consisting of: LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, LN-121 and fragments thereof, and LN-111 and fragments thereof; for example, the group consisting of: LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof; for example, the group consisting of: LN-521 and fragments thereof, LN-511 and fragments thereof, and LN-332 and fragments thereof; for example, the group consisting of LN-521 and fragments thereof or the group consisting of LN-511 and fragments thereof.

[0101] In one embodiment, the laminin and fragments thereof are selected from the group consisting of: LN-521, LN-511, LN-332, LN-421, LN-121 and LN-111; 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, wherein the laminin and fragments thereof are LN-521 or for example, wherein the laminin and fragments thereof are LN-511.

[0102] Known are laminin E8 fragments, which are truncated proteins consisting 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 glutamic acid residue in the C-terminal tail of the γ chain, but lack other activities, such as the heparin / heparan sulfate binding activity associated with full-length laminin. The E8 fragment represents a functionally minimal form that retains all of the ability to bind to the α6β1 integrin. In one embodiment, the one or more fragments thereof are E8 fragments.

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

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

[0105] In one embodiment as disclosed herein, a method is provided, wherein in step i), 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 EP cells characterized by expression of NEUROD1. For example, the EP cells provided in step i) can be obtained in step c+1) disclosed herein. Therefore, the population of EP cells in step i) can be a part of the total cell population, which contains a part of cells that do not express characteristic markers of EP cells. In one embodiment as disclosed herein, the population of EP cells in step i) is a population comprising EP cells. In one embodiment as disclosed herein, a method is provided, wherein in step i) a population of EP cells is provided, wherein 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 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. In one embodiment as disclosed herein, in step i), the population comprising EP cells comprises more than about 30%, such as more than about 40%, such as more than about 45%, such as 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 exemplified 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 as disclosed herein, step ii) of providing a single cell suspension of a population of EP cells is performed when more than about 15%, such as more than about 20%, such as more than about 25%, such as more than about 30%, such as more than about 35%, such as more than about 40%, such as more than about 45%, such as more than about 50% of the total cell population are EP cells characterized by expression of NEUROD1 or NGN3). In one embodiment, the EP cells are characterized by expression of NEUROD1 and NGN3. In one embodiment, step ii) is performed when more than about 15%, such as more than about 20%, such as more than about 25%, such as more than about 30%, such as more than about 35%, such as more than about 40%, such as more than about 45%, such as more than about 50% of the total cell population are EP cells characterized by expression of NKX6.1 and NEUROD1). In one embodiment, step ii) is performed when the cells do not express hormone expression, such as do not express expression). In one embodiment, step ii) is performed when about the cells do not express hormone expression, such as do not express expression of insulin and / or glucagon. In this context, not showing expression is understood as not showing any detectable expression, such as expression detectable by the methods or means disclosed in this Example section. In one embodiment, step ii) is performed before the cell shows hormone expression (e.g., shows 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 a step iv) of culturing a population of EP cells in the form of a 3D structure under 3D culture conditions that allow differentiation into pancreatic monohormone β cells to provide islet-like cell aggregates comprises culturing in a medium suitable for culturing endocrine progenitor cells under conditions that allow differentiation into monohormone β cells. Non-limiting examples of stage 6 (S6) culture medium are defined in this example. The skilled person understands that other suitable culture media can be used. The culture medium in step iv) can be supplemented with other factors specified herein.

[0108] Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is a water-soluble analog of vitamin E with a strong antioxidant effect. Trolox is also a strong membrane damage inhibitor. In one embodiment, the culture medium in step iv) contains about 5 μM to 15 μM Trolox, such as about 10 μM Trolox. As the skilled person will appreciate, Trolox may be replaced with its derivatives or agonists.

[0109] N-acetyl-L-cysteine ​​is a cell culture component, such as intestinal basal medium for culturing mouse intestinal stem cells, and can also be used as a component of expansion medium. In one embodiment, the medium in step iv) contains about 0.5mM to 3mM N-acetyl-L-cysteine, such as about 1mM N-acetyl-L-cysteine. As the skilled person will appreciate, N-acetyl-L-cysteine ​​can be replaced with its derivatives or agonists.

[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 the skilled person will appreciate, H1152 may be replaced by its derivatives or agonists.

[0111] GC-1 is a thyroid hormone receptor (TR) agonist and is more effective than thyroid hormone T3. Thyroid hormone T3 is important for the development of beta cells and will be discussed in more detail below. In one embodiment, the culture medium in step iv) contains about 0.5 μM to 3 μM GC-1, such as about 1 μM GC-1. As will be appreciated by the skilled artisan, GC-1 may be replaced with its derivatives or agonists.

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

[0113] In one embodiment, step iv) of the method as disclosed herein comprises culturing the EP cell population in a medium comprising about 10 μM H1152, about 1 μM GC-1, about 10 μM Trolox, and about 1 mM N-acetyl-L-cysteine. In one embodiment, step iv) of the method as disclosed herein comprises culturing the EP cell population in a medium comprising about 10 μM H1152, about 1 μM GC-1, about 10 μM Trolox, and about 1 mM N-acetyl-L-cysteine ​​for about 3 weeks, followed by culturing in a medium comprising about 1 μM GC-1, about 10 μM Trolox, and about 1 mM N-acetyl-L-cysteine ​​but not comprising H1152.

[0114] In one embodiment, said culturing in step iv) is on a shaker, such as an orbital shaker.

[0115] As discussed in the context of the present methods, the inventors have discovered that the timing of transferring EP cells from culture on a 2D substrate to culture on a 3D substrate is important for obtaining enriched islet-like cell aggregates in vitro that are enriched for β cells and exhibit a low percentage of other monohormone cells, a low percentage of multihormone cells, and a low percentage of proliferating cells.

[0116] In one embodiment of the method, the cells are not transferred from culture on a 2D substrate to culture on a 3D substrate until they exhibit expression of markers characteristic of endocrine progenitor cells. Endocrine progenitor cells are characterized by expression of PDX1, NKX6.1, and at least one of NEUROD1 and NGN3, as discussed above. As described above, EP cells do not express pancreatic hormones. In one embodiment, step ii) is performed about 24 hours after the EP cells begin to express NEUROD1 and / or NGN3, for example, 24 hours after more than 15%, such as more than about 20%, such as more than about 25%, such as more than about 30%, such as more than about 35%, such as more than about 40%, such as more than about 45%, such as more than about 50% of the total cells in the culture begin to express NEUROD1 and / or NGN3. In one embodiment, step ii) is performed within 6 days, such as within 5 days, such as within 1-4 days, such as within 1-3 days, such as within 1-2 or 2-3 days after the EP cells begin to express NEUROD1 or NGN3. Step ii) is performed prior to expression of insulin and / or glucagon.

[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, this spontaneous formation of the 3D structure can occur by self-aggregation. Alternatively, the formation of the 3D structure in step iii) is a forced (also referred to as 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 to be close together via the shape of a cell culture bottle or flask. In one embodiment, 3D culture conditions allow cells to self-aggregate. Without being bound by theory, this example shows that self-aggregation allows selective enrichment of endocrine progenitor cells, which develop into stage 6 cells. In addition, the selective enrichment results in an increase in the 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 a monohormone β cell. In one embodiment, the pancreatic monohormone β cell is 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 cell α and / or δ cells.

[0118] In one embodiment, the in vitro islet-like cell aggregates are generated by human ES cells and contain at least 40%, such as at least 45%, such as at least 50%, such as at least 55% of β cells, such as at least 60% of β cells, such as at least 65% of single hormone β cells. In one embodiment, the in vitro islet-like cell aggregates are generated by human iPS cells and contain at least 40% β cells, such as at least 45%, such as at least 50%, such as at least 55% of β cells, such as at least 60% of β cells, such as at least 65% of single hormone β cells. In a specific embodiment, the IPS cells are C7 cells, and the islet-like cell aggregates contain at least 60% of β cells, such as at least 65% of single hormone β cells. In one embodiment, the single hormone β cells are characterized by insulin expression.

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

[0120] In one embodiment, the in vitro islet-like cell aggregates contain up to about 5%, such as up to about 4%, such as up to about 3%, such as up to about 2%, such as up to about 1% of proliferating cells, such as proliferating cells expressing Ki-67. In one embodiment, the in vitro islet-like cell aggregates contain up to about 5%, such as up to about 4%, such as up to about 3%, such as up to about 2%, such as up to about 1% of proliferating cells, such as proliferating cells expressing Ki-67. In one embodiment, the in vitro islet-like cell aggregates contain up to about 5%, such as up to about 4%, such as up to about 3%, such as up to about 2%, such as up to about 1% of proliferating cells, such as proliferating cells expressing Ki-67. In one embodiment, the Ki-67 is scored one day after the aggregates are formed. In one embodiment, the Ki-67 is scored on the 15th day of culture. Therefore, the evaluation can be performed after step iv) lasts for about 4 weeks.

[0121] In one embodiment, the in vitro islet-like cell aggregates contain at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75% of cells expressing NEUROD1 and NKX6.1. In one embodiment, the in vitro islet-like cell aggregates contain about 60% to 90%, such as about 60% to 80%, such as about 65% to 80% of cells expressing NEUROD1 and NKX6.1. In one embodiment, the in vitro islet-like cell aggregates contain at least 50%, such as at least 80%, such as at least 85%, such as at least 87%, such as at least 90% of cells expressing NEUROD1. In a specific embodiment, the in vitro islet-like cell aggregates are produced by human ES cells. In one embodiment, the expression of NEUROD1 and / or NKX6.1 is scored on the 15th day of culture. Therefore, the evaluation can be performed in step iv) after the formation of the aggregates. In one embodiment, step iv) comprises culturing the cell population for about 2 weeks or 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 cells that express only one hormone. For example, a monohormone β cell expresses only insulin and does not express other hormones expressed by pancreatic islet cells, such as glucagon or somatostatin. As used herein, the term "multihormone" refers to cells that express at least two different hormones.

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

[0124] Thus, in one embodiment of the methods of the invention disclosed herein, the islet-like cell aggregates comprising the beta cells generated in step v) comprise single hormone beta cells. In one embodiment of the methods of the invention, the single hormone beta cells in step v) express insulin. In one embodiment, the single hormone beta cells in step v) express C-peptide after glucose stimulation.

[0125] Particularly, the single hormone beta cell in step v) does not express glucagon or somatostatin. Particularly, the single hormone beta cell in step v) does not express glucagon and somatostatin.

[0126] In one embodiment, the single hormone β cell in step v) is characterized by expressing insulin. The single hormone β cell may also express at least one of NKX6.1, PDX1 and NEUROD1.

[0127] In one embodiment, the single hormone beta cell is characterized by the expression of insulin and PDX1. In one embodiment, the single hormone beta cell is characterized by the expression of insulin and NKX6.1. In one embodiment, the single hormone beta cell is characterized by the expression of insulin and NEUROD1. In one embodiment, the single hormone beta cell is characterized by the expression of: 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 single hormone beta cell is characterized by the expression of insulin, PDX1, NKX.1, and NEUROD1.

[0128] In one embodiment, a method for generating in vitro islet-like cell aggregates as defined herein is provided, wherein the islet-like cell aggregates in v) 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% of beta cells, such as at least about 60% of monohormonal beta cells, such as at least about 65% of beta cells. In one embodiment, the in vitro islet-like cell aggregates in v) contain about 25% to 70%, such as about 30% to 70%, such as about 40% to 70%, such as about 40% to 60% of monohormonal beta cells.

[0129] In one embodiment, the in vitro islet-like cell aggregates in v) comprise at least 40%, such as at least 45%, such as at least 50%, such as at least 55% of β cells, such as at least 60% of β cells, such as at least 65%, such as at least 70% of β cells 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 aggregates in v) contain at most about 20%, such as at most about 18%, such as at most about 16%, such as at most about 13%, such as at most about 10% of single hormone alpha cells. In one embodiment, the single hormone alpha cells are characterized by expressing glucagon.

[0131] In one embodiment, a method for generating in vitro islet-like cell aggregates as defined herein is provided, wherein the islet-like cell aggregates in v) contain single hormone β cells, and up to 5% α cells, and any one or more of cells selected from the group consisting of: δ cells, acinar cells, ductal cells, and activated stellate cells. In one embodiment of the method, the islet-like cell aggregates in v) contain up to about 5%, such as up to about 4%, 3%, 2% or 1% of multi-hormonal cells. In one embodiment of the method, the islet-like cell aggregates in v) contain up to about 5%, such as up to about 4%, 3%, 2% or 1% of non-endocrine cells.

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

[0133] In addition, the inventors have demonstrated that a short culture time of posterior foregut cells (PF) under conditions that allow differentiation into pancreatic progenitor cells (PP) produces a greater number of endocrine progenitor cells (EP) (also referred to herein as endocrine precursor cells), even though the number of PP cells obtained is lower than in a corresponding method with a longer culture time. As used herein, the terms "endocrine progenitor cells (EP)" and "endocrine precursor (EP) cells" are used interchangeably. Without being bound by theory, it appears that the PP cells obtained by the method of the present invention comprising the short culture time have the ability to develop / differentiate into EP cells. Importantly, the EP cells obtained by the method as disclosed herein have the potential to develop into mature and functional pancreatic β cells, such as monohormonal β cells, which have the ability to respond 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 the steps specifically indicated. Therefore, a corresponding method should be interpreted as the same method, but with a difference in the indicated step, for example, the step may be related to the culture time, such as step b-1); or b); or the culture time during both b-1) and b.

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

[0135] a) providing a cell population of posterior foregut cells, the posterior foregut cells being, for example, posterior foregut cells characterized by expression of PDX1;

[0136] b) culturing the cell population of posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for no longer than about 78 hours, such as no longer than about 72 hours; and

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

[0138] For clarification, the cells of the cell population of posterior foregut cells (PF) characterized by the expression of PDX1 provided in step a) do not express NKX6.1, in other words, lack the expression of NKX6.1.

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

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

[0141] In one embodiment of the method as disclosed herein, the cell population of pancreatic progenitor cells in step c), such as a cell population of pancreatic progenitor cells characterized by expression of PDX1 and NKX6.1, is further characterized by expression of at least one marker selected from the group consisting of: PTF1A, SOX9, HNF6 and CPA, such as a marker selected from the group consisting of: SOX9 and PTF1A. In one embodiment of the method as disclosed herein, the cell population of pancreatic progenitor cells 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 the characteristics of the cell type, and can 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 a culture medium suitable for culturing posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells. Non-limiting examples of stage 4 (S4) culture medium are defined in the accompanying examples. The culture medium in step b) can be supplemented with other factors as specified herein. The technician understands that other suitable culture media can be used. Specifically, culturing under conditions that allow differentiation into pancreatic progenitor cells in step b) as disclosed herein can 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 its derivatives or agonists. Thus, in one embodiment of the method as disclosed herein, the conditions 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 as 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 3 intramolecular disulfide bonds. Binding to the receptor stimulates ligand-induced dimerization, activates intrinsic protein tyrosine kinase activity, and initiates a signal transduction cascade that leads to various biochemical changes in the cell—increased intracellular calcium levels, increased glycolysis and protein synthesis, and increased expression of certain genes including the EGFR gene—ultimately leading to DNA synthesis and cell proliferation. EGF is a member of the EGF protein family. Members of this protein family have highly similar structural and functional characteristics. In addition to EGF itself, other family members include heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), epiregulin (EPR), Epigen, betacellulin (BTC), neuregulin-1 (NRG1), neuregulin-2 (NRG2), neuregulin-3 (NRG3), and neuregulin-4 (NRG4).

[0144] It will be appreciated by the skilled artisan that, as used herein, the term "epidermal growth factor (EGF) or a derivative or agonist thereof" is intended to include factors that enhance or replace EGF signaling. These factors may be involved in signaling downstream of EGF or may be small molecule agonists. A non-limiting list of EGF derivatives or agonists includes high-affinity EGFR ligands such as TGF-α, BTC, and HB-EGF, and low-affinity ligands such as AR, EPR, and Epigen. Therefore, in one embodiment of this aspect, the EGF or its derivative or agonist is selected from the group consisting of: EGF, TGF-α, BTC, HB-EGF, AR, EPR, and Epigen, for example, EGF. In a specific 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 at the meta position, and the primary amide group is the amide of nicotinic acid. Nicotinamide is a well-known cell culture supplement for use in the differentiation of embryonic stem cells and induced pluripotent stem cells, and has been shown to regulate stem cell differentiation in various applications, including the differentiation of pancreatic cells. The skilled person will understand that, as used herein, the term "nicotinamide (NIC) or a derivative or agonist thereof" is intended to include factors that enhance or replace NIC signaling. Non-limiting examples of such derivatives or agonists include NIC, niacin (nicotinic acid), nicotinamide riboside, NAD / NADP, and tryptophan as a precursor of NIC. Therefore, in one embodiment of the present aspect, the NIC or its derivative or agonist is selected from the group consisting of NIC, niacin, nicotinamide riboside, NAD / NADP and tryptophan, for example, wherein the NIC or its derivative or agonist is NIC.

[0146] In one embodiment of the method, in step b), the effective amount of EGF or its derivative or agonist is about 50 to 200 ng / mL, such as about 50 to 150 ng / mL, such as about 75 to 125 ng / mL, such as about 100 ng / mL

[0147] In one embodiment of the method, in step b), the effective amount of NIC or its derivative or agonist is about 5 nM-20 nM, such as about 5 to 15 mM, such as about 8 to 12 mM, such as about 10 mM.

[0148] Step b) of the method as disclosed herein may include culturing the cell population in a culture medium comprising additional factors, such as one or more factors selected from the group consisting of 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 plurality of factors selected from KGF, ActA, retinoic acid, SANT-1, PDBu and LDN. In one embodiment, the step b) comprises culturing the cell population in a culture medium further comprising 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, such as a culture medium further comprising KGF, ActA, retinoic acid, SANT-1, PDBu and LDN. It should be understood that the culture medium may comprise a mixture of factors and their derivatives and / or agonists.

[0149] The skilled artisan will appreciate that the cell culture medium and the factors contained therein may be adjusted by replacing the factors with their derivatives and / or agonists (eg with 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 biologically active 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 does not induce the proliferation of fibroblasts and endothelial cells. It is the main growth factor for skin keratinocytes and is also used in the culture and differentiation of pluripotent cells. It will be appreciated by the technician that KGF can be substituted by its derivatives or agonists in the cell culture described herein. Non-limiting examples of such agonists include other factors that bind to FGFR2b and send signals through the receptor (e.g., FGF10).

[0151] In one embodiment, the KGF or a derivative or agonist thereof is selected from the group consisting of KGF and FGF10, such as FGF10.

[0152] In one embodiment, the culture medium in step b) comprises 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) comprises about 25 ng / ml to 75 ng / ml KGF, such as about 50 ng / mL KGF.

[0153] ActA, or activin A, is a member of the TGF-β superfamily. Both activin and Nodal ligands can signal through the same receptors and effectors to regulate transcription. 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 the Smad transcription factors Smad2 and Smad3.

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

[0155] It has been reported that Activin / Nodal is involved in maintaining the pluripotency of stem cells, whereas Activin / Nodal signaling is also required for endoderm differentiation.

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

[0157] In one embodiment, the ActA or its derivative or agonist is 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) comprises about 1 to 10 ng / mL, such as 2.5 to 7.5 ng / mL of ActA, such as about 5 ng / mL of ActA.

[0158] Retinoic acid (RA) is a metabolite of vitamin A and mediates the functions of vitamin A required for growth and development. Retinoic acid is known to be involved in specifying position along the embryonic anterior-posterior axis, also known as patterning, and is also known to play a role in later stages of pancreatic development to promote the generation of pancreatic endocrine progenitor cells and their differentiation into islets and beta cells.

[0159] Retinoic acid plays a role by combining with retinoic acid receptor (RAR), and the retinoic acid receptor and retinoid X receptor (RXR) are combined with DNA as heterodimers in the region called retinoic acid response element (RARE). The combination of retinoic acid ligand and RAR changes the conformation of RAR, thereby affecting the combination of other proteins of induction or inhibition of nearby gene transcription. Technicians will understand that the term "retinoic acid or its derivatives or agonists" is intended to include factors that enhance or replace retinoic acid signal transduction in this article. These factors may participate in the signal transduction of retinoic acid downstream or are small molecule agonists. The non-limiting list of retinoic acid derivatives or agonists includes all-trans retinoic acid, synthetic retinoid ec23, Ch55, TTNPB, fenretinide, RAR agonists (such as RARA agonists and RARB agonists AC261066), adapalene, AC55649, AM80, AM580, BMS753, tazarotene and Ro 41-5253. Therefore, in one embodiment of the present invention, the retinoic acid or its derivative or agonist is selected from the group consisting of retinoic acid, all-trans retinoic acid, synthetic retinoids ec23, Ch55, TTNPB, fenretinide, RAR agonists (e.g., RARA agonists and RARB agonists AC261066), adapalene, AC55649, AM80, AM580, BMS 753, tazarotene, 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 retinoid 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) comprises about 50nM to 150nM RA or its derivative or agonist, for example about 100nM RA or its derivative or agonist.

[0161] In one embodiment, the culture medium in step b) comprises about 50 nM to 150 nM RA, such as about 100 nM RA.

[0162] Hedgehog (HH or Hh) signaling is known to play a key role in regulating vertebrate organogenesis, such as in the growth of digits and brain organization. The vertebrate hedgehog protein family consists of Sonic Hedgehog (SHH), Indian Hedgehog (IHH) and Desert Hedgehog (DHH), which signal through similar pathways and share many functional features. The key negative regulator of pancreatic development is Sonic Hedgehog (SHH), so it is very important to inhibit SHH at the beginning of pancreatic development, and hedgehog inhibition must be maintained to ensure normal pancreatic development.

[0163] Briefly, Hh signals by interacting with the Hh receptor complex, which consists of two components: Patched (Ptc) and Smoothened (Smo), which transduce the Hh signal into the cell. Ptc is thought to inhibit Hh signaling by binding to Smo in the cell membrane. When Hh ligands are present, this inhibition is relieved and Smo is able to signal. In vertebrates, the zinc finger proteins Gli1, Gli2, and Gli3 are downstream mediators of Hh signaling and are involved in controlling the transcriptional responses of target genes in an Hh-dependent manner.

[0164] SANT-1 is an inhibitor of Hedgehog (Hh) signaling and acts by antagonizing smoothened activity. Non-limiting examples of Hedgehog signaling inhibitors include cyclopamine, IHR 1, IHR-Cy3, itraconazole, allitridine, M 25, MRT 10, PF 04449913 maleate, PF 5274857 hydrochloride, SANT-1 and SANT-2.

[0165] In one embodiment, the SANT-1 or derivative or agonist thereof is selected from the group consisting of cyclopamine, IHR 1, IHR-Cy3, itraconazole, allitridine, 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) comprises about 0.10 μM to 0.50 μM SANT-1, such as about 0.25 μM SANT-1.

[0167] PDBu (phorbol-12,13-dibutyrate) is a strong promoter of nitric oxide (NO) synthesis and an effective activator of protein kinase C. PDBu is reported to be a tumor promoter that activates a variety of cellular responses, including proliferation. Other activators of protein kinase C, such as but not limited to phorbol 12-myristate 13-acetate (PMA) or TPPB, may be substituted for PBDu. In one embodiment, the PDBu or its derivative or agonist is selected from the group consisting of PDBu, PMA and TPPB. In one embodiment, the culture medium in step b) contains approximately 0.25 μM to 0.75 μM PDBu, such as approximately 0.5 μM PDBu.

[0168] LDN193189 (referred to herein as LDN) is a bone morphogenesis (BMP) inhibitor and exerts its effects by inhibiting ALK2 and ALK3. LDN exerts its effects primarily by preventing the phosphorylation of Smad1, Smad5, and Smad8. LDN is an analog of dorsomorphin and noggin, and dorsomorphin may replace LDN. In one embodiment, the LDN or its derivative or agonist is selected from the group consisting of LDN, Noggin, and dorsomorphin. In one embodiment, the culture medium in step b) comprises approximately 100nM to 300nM LDN, such as 100nM-250nM LDN, such as 150nM-250nM LDN, such as approximately 200nM LDN.

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

[0170] As explained above, the present disclosure also describes that a short culture time of posterior foregut cells (PF) under conditions allowing differentiation into pancreatic progenitor cells (PP) produces a greater number of endocrine progenitor cells (EP) 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] Thus, in one embodiment of the present aspect, a method is provided, wherein at least about 70%, such as at least about 75%, such as at least about 80% of the posterior foregut cells in a) are differentiated 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%, such as about 80% to 85%, such as 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%, such as at most about 8%, such as at most about 7%, such as at most about 5%, such as 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%, such as at most about 8%, such as at most about 7%, such as at most about 5%, such as 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%, such as about 25% to 48%, such as 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), about 30% to 50%, such as about 35% to 45%, such as about 40% to 45% of the total cell population expresses NKX6.1. In one embodiment, in step c), about at least about 40%, such as at least about 50%, such as at least about 60% of the cells do not express NEUROD1 and NKX6.1.

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

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

[0175] a-1) providing a cell population of primitive intestinal tube cells, wherein the primitive intestinal tube cells are, for example, primitive intestinal tube cells characterized by expressing HNF1β and / or HNF4α;

[0176] b-1) culturing the cell population of primitive gut tube cells under conditions that allow differentiation into posterior foregut cells for no longer than about 54 hours; and

[0177] c-1) thereby generating a population of posterior foregut cells, which are, for example, posterior foregut cells characterized by expression of PDX1. It will be appreciated that posterior foregut cells may also be characterized by expression of HNF6 or co-expression of PDX1 and HNF6.

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

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

[0180] a-1) providing a cell population of primitive intestinal tube cells, wherein the primitive intestinal tube cells are, for example, primitive intestinal tube cells characterized by expressing HNF1β and / or HNF4α;

[0181] b-1) culturing the cell population of primitive gut tube cells under conditions that allow differentiation into posterior foregut cells for no longer than about 54 hours;

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

[0183] a) providing a cell population of posterior foregut cells generated in step c-1), wherein the posterior foregut cells are, for example, posterior foregut cells characterized by expressing PDX1;

[0184] b) culturing the cell population of posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for no longer than about 78 hours, such as no longer than about 72 hours; and

[0185] c) thereby generating a cell population of pancreatic progenitor cells, which are pancreatic progenitor cells characterized, for example, 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. The complete method set of cultivating a cell colony at a certain developmental stage under appropriate conditions to differentiate it into a colony at the next developmental stage includes steps a), b), c), etc. Each step can also be represented by a letter code with an associated digital indicator. The digital indicator indicates that each step in the complete method set is before (negative digital indicator) or after (positive digital indicator) cultivating the complete method set of the rear foregut cells (PF) to generate the pancreatic progenitor cells (PP). In order to illustrate the method steps associated with the complete method set of cultivating a cell colony at a certain developmental stage under appropriate conditions to allow it to differentiate into a colony at the next developmental stage, it is shown in Table 1 below:

[0187]

[0188]

[0189] Table 1. Summary of method groups and steps. In this document, 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 as disclosed herein, the cell population of primitive gut tube cells provided in step a-1) is characterized by the expression of HNF1β, HNF4α, or both HNF1β and HNF4α.

[0191] In one embodiment, the population of posterior foregut cells is characterized by expression of PDX1 or HNF6. In one embodiment, the population of posterior foregut cells is characterized by expression of PDX1 and HNF6. In one embodiment of the methods disclosed herein, the cell population of posterior foregut cells in step c-1) is characterized by expression of PDX1 and lacks expression of NKX6.1.

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

[0193] Without being bound by theory, it is envisioned that the step b-1) of culturing the cell colony of the primitive gut tube cells under conditions that allow differentiation into posterior foregut cells is beneficial for no more than 54 hours, so as to obtain a colony of posterior foregut cells that are capable of developing into the later stages of the β cell lineage. It is envisioned that primitive gut tube cells that are cultured for a period of time longer than 54 hours under conditions that allow differentiation into posterior foregut cells are less capable of developing into the later stages of the β cell lineage. In one embodiment of the method as disclosed herein, the cell colony in step b-1) is cultured for no more than about 52 hours, such as no more than about 50 hours, such as no more than about 48 hours, such as no more than about 44 hours, such as no more than about 40 hours, such as no more than about 36 hours, such as no more than about 32 hours, such as no more than about 28 hours, such as no more than about 26 hours, such as about 24 hours.

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

[0195] As discussed above, "differentiation-permitting conditions" refer to conditions that allow cells to develop / differentiate to express the characteristics of the cell type, and may include a combination of cell culture medium, the presence and / or absence of extrinsic factors and their timing. The factors and their derivatives and agonists have been discussed in detail with respect to step b) above, and the discussion will not be repeated here for the sake of brevity only.

[0196] In one embodiment, the culture medium in step b-1) is a culture medium suitable for culturing primitive gut tube cells under conditions that allow differentiation into posterior foregut cells. Non-limiting examples of stage 3 (S3) culture medium are defined in this example. The skilled person understands that other suitable culture media can be used. The culture medium in step b-1) can be supplemented with other factors specified herein.

[0197] Thus, in one embodiment, a method is provided, wherein step b-1) comprises culturing the cell population in a culture medium comprising one or more factors selected from the group consisting of 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, the step b-1) comprises culturing the cell population in a culture medium comprising 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, the step b-1) comprises culturing the cell population in a culture medium comprising the following: 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 comprising KGF, retinoic acid, SANT-1, PDBu and LDN. It will be appreciated 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) comprises 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) comprises about 25 ng / ml to 75 ng / ml KGF, such as about 50 ng / mL KGF. In one embodiment, the culture medium in step b-1) comprises about 1 μM to 3 μM RA, such as about 2 μM RA.

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

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

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

[0202] In one embodiment, step b-1) of the method as disclosed herein comprises culturing the cell population in a 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 that allow differentiation into endocrine progenitor cells; and

[0206] c+1) thereby generating a population of endocrine progenitor cells, such as those characterized by expression of NEURO D1, such as those characterized by expression of NKX6.1 and NEURO D1.

[0207] In one embodiment, a method as disclosed herein is provided, wherein before step i), the method comprises the following steps

[0208] a-1) providing a cell population of primitive intestinal tube cells, wherein the primitive intestinal tube cells are, for example, primitive intestinal tube cells characterized by expressing HNF1β and / or HNF4α;

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

[0210] c-1) thereby generating a population of posterior foregut cells, the posterior foregut cells being, for example, posterior foregut cells characterized by expression of PDX1;

[0211] a) providing a cell population of posterior foregut cells generated in step c-1), wherein the posterior foregut cells are, for example, posterior foregut cells characterized by expressing PDX1;

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

[0213] c) thereby generating a cell population of pancreatic progenitor cells, the pancreatic progenitor cells being, for example, pancreatic progenitor cells characterized by expressing both PDX1 and NKX6.1;

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

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

[0216] c+1) thereby generating a population of endocrine progenitor cells, such as endocrine progenitor cells characterized by expression of NEUROD1, such as endocrine progenitor cells characterized by expression of NKX6.1 and NEUROD1.

[0217] In one embodiment, the population of endocrine progenitor cells is further characterized by the expression of at least one of NKX6.1 and NGN3. The skilled person will appreciate that different combinations of the two markers may 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) may be characterized by the expression of: PDX1, NKX6.1, and NEUROD1; PDX1, NKX6.1, and NGN3; or PDX1, NKX6.1, NEUROD1, and NGN3. It will be appreciated 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 the pancreatic progenitor cells have adopted an endocrine progenitor cell fate. The skilled person understands that due to experimental limitations of the staining protocol, cells stained for PDX1 or NKX6.1 may be evaluated for expression of NGN3 or NEUROD1. In one embodiment of the method as disclosed herein, the cell population of endocrine progenitor cells in step c+1) is characterized by expression of: 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 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 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 to be sufficient to produce the population of endocrine progenitor cells of step c+1).

[0220] Similar to the description in steps a)-c), steps a+1)–c+1) are performed 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 for the sake of brevity only. Similar to the description in steps a)-c), steps a-1)–c-1) are performed 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 for the sake of brevity only.

[0221] Thus, in one embodiment of the method as disclosed herein, the cells in step b+1) are cultured on a 2D substrate. Thus, in one embodiment of the 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 fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and matrigel. TM , for example, selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, and matrigel TM, for example, selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen, gelatin, and matrigel TM , for example, selected from the group consisting of laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and matrigel TM .

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

[0224] In one embodiment of the method as disclosed herein, wherein the laminin (LN) and fragments thereof are selected from the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, LN-121 and fragments thereof, and LN-111 and fragments thereof;

[0225] For example, the group consisting of: LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof;

[0226] For example, the group consisting of: LN-521 and fragments thereof, LN-511 and fragments thereof, and LN-332 and fragments thereof;

[0227] For example, the group consisting of LN-521 and fragments thereof or the group consisting of LN-511 and fragments thereof. In one embodiment, the laminin and fragments thereof are selected from the group consisting of LN-521, LN-511, LN-332, LN-421, LN-121 and LN-111; 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 fragments thereof are LN-521 or wherein the laminin and fragments thereof are LN-511.

[0228] In one embodiment, the one or more fragments thereof are E8 fragments. In one embodiment, the laminin and fragments comprise E8 fragments of laminin, such as E8 fragments selected from the group consisting of: E8 fragments of LN-511, E8 fragments of LN-521, E8 fragments of LN-332, E8 fragments of LN-421, E8 fragments of LN-121, and E8 fragments of LN-111; such as the group consisting of: E8 fragments of LN-511, E8 fragments of LN-521, E8 fragments of LN-332, E8 fragments of LN-421, E8 fragments of LN-121, and E8 fragments of LN-111. The E8 fragment of LN-511, the E8 fragment of LN-521 and the E8 fragment of LN-332; for example, the group consisting of the E8 fragment of LN-511 and the E8 fragment of LN-521; for example, the E8 fragment of LN-511 or the E8 fragment of LN-521.

[0229] As discussed above, "differentiation-permitting conditions" refer to conditions that allow cells to express the characteristics of the cell type, and may include a combination of cell culture medium, the presence and / or absence of extrinsic factors and their timing. 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 only. Therefore, in one embodiment, a method is provided, wherein step b+1) comprises culturing the cell population in a culture medium comprising one or more factors selected from the group consisting of BTC and its derivatives and agonists, retinoic acid and its derivatives and agonists, Alk5 inhibitors (e.g., Alk5i II) and its derivatives and agonists, retinoic acid and its derivatives and agonists, γ-secretase inhibitors (e.g., 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 group consisting of BTC, Alk5 inhibitors (e.g., Alk5i II), γ-secretase inhibitors (e.g., GSI-XX), GC-1, LDN, retinoic acid and SANT-1. Therefore, in one embodiment, a method is provided, wherein step b+1) comprises culturing the cell population in a culture medium comprising: BTC or a derivative or agonist thereof; an Alk5 inhibitor (e.g., Alk5i II) or a derivative or agonist thereof; a γ-secretase inhibitor (e.g., GSI-XX) or a derivative or agonist thereof; GC-1 or a derivative or agonist thereof; LDN or a derivative or agonist thereof; retinoic acid or a derivative or agonist thereof: and SANT-1 or a derivative or agonist thereof; for example, BTC, an Alk5 inhibitor (e.g., Alk5i II), a γ-secretase inhibitor (e.g., GSI-XX) GC-1, LDN, retinoic acid and SANT-1. Therefore, in one embodiment, a method is provided, wherein step b+1) includes culturing the cell population in a culture medium comprising: BTC and / or its derivatives and / or agonists; Alk5 inhibitors (e.g., Alk5i II) and / or its derivatives and / or agonists; γ-secretase inhibitors (e.g., 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 culture medium may contain a mixture of factors and their derivatives and / or agonists. In one embodiment of the method as disclosed herein, step b+1) includes culturing the cell population in a culture 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 derivative or agonist and a γ-secretase inhibitor and its derivatives and agonists (e.g., Alk5i II and GSI-XX).

[0230] BTC, also known as betacellulin, is a member of the EGF family of growth factors and induces differentiation of beta cells, and may also induce 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] An example of an ALK5 inhibitor is Alk5i II (ALK5 inhibitor II), which is a cell-permeable selective inhibitor of 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 derivative or agonist is 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 (itself an integral membrane protein) that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. Non-limiting examples of γ-secretase inhibitors include GSI-XX, DAPT, RO4929097, YO-01027, BMS-906024, Aβ42-IN-2, LY-411575, and MK-0752. Therefore, 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 thyroid hormone T3, which is important for the development of beta cells.

[0234] As used herein, the term "GC-1 and its derivatives and agonists" refers to GC-1, T3 and T4. Therefore, 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 culture medium in step b+1) is a culture medium suitable for culturing pancreatic progenitor cells under conditions that allow differentiation into endocrine progenitor cells. Non-limiting examples of stage 5 (S5) culture medium are defined in this example. The skilled person understands that other suitable culture media can be used. The culture medium in step b+1) can be supplemented with other factors specified herein.

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

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

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

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

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

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

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

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

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

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

[0246] During the differentiation protocol, cells may be cultured on a 2D substrate, such as adherent culture on a 2D substrate. It should be understood that the above discussion regarding 2D substrate properties is equally relevant in the context of steps a)-c+1) and will not be repeated here simply for the sake of brevity.

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

[0248] In one embodiment of the method, the cells are not transferred from culture on a 2D substrate to culture on a 3D substrate until they exhibit expression of markers characteristic of endocrine progenitor cells. The endocrine progenitor cells are characterized by 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, the method further comprising steps i)-v) as defined herein after steps a+1)-c+1), comprising transferring the population of endocrine progenitor cells (e.g., endocrine progenitor cells characterized by expression of NKX6.1 and NEUROD1) from culture on a 2D substrate to 3D culture conditions;

[0250] The endocrine progenitor cell population is cultured under conditions that allow differentiation into pancreatic monohormone β cells; and results in the generation of a population of pancreatic monohormone β cells, which are, for example, pancreatic monohormone β cells characterized by expressing insulin. The monohormone β cells may also express at least one of NKX6.1, PDX1, and NEUROD1.

[0251] As discussed in detail above, 3D culture conditions allow cells to self-aggregate.

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

[0253] In one embodiment, step iv) comprises culturing the population of 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 "monohormone" refers to cells that express only one hormone. For example, a monohormone β cell expresses only insulin and does not express other hormones expressed by pancreatic islet cells, such as glucagon or somatostatin. As used herein, the term "multihormone" refers to cells that express at least two different hormones.

[0255] In vivo single hormone beta cells are single hormone cells, and advantageously the population obtained by the method of the invention exhibits the properties of in vivo natural beta cells or in vivo endogenous beta cells, such as the properties of in vivo healthy natural beta cells or in vivo healthy endogenous beta cells.

[0256] Thus, in one embodiment of the method disclosed herein, the population of monohormonal beta cells generated in step v) is monohormonal. Specifically, the population of monohormonal beta cells generated in step v) does not express glucagon or somatostatin. Specifically, the population of monohormonal beta cells generated in step v) does not express glucagon and somatostatin.

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

[0258] In one embodiment as 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 expression of NKX6.1 and at least NEUROD1. As discussed and exemplified above, other combinations of two or more of the markers PDX1, NKX6.1, NEUROD1, and NGN3 can be used as characteristics of 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 expression of 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 to culture the cell population of the posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for about 24 hours or less and / or about 96 hours or longer. Therefore, according to the present invention, the culturing time of step b) leads to an increase in the number of EP cells. As shown in the attached 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). In particular, 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% more endocrine progenitor cells than a corresponding method, 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, in which step b) is to culture the cell population of posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for about 24 hours or less and / or about 96 hours or longer.

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

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

[0263] As explained above, it will be appreciated that the increased proportion and / or number of EP cells obtained by the present method can be converted into a higher proportion of single hormonal beta cells as defined herein, compared to the prior art methods wherein step b) is culturing the cell population of the posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for about 96 hours or longer. It will be appreciated that the increased proportion and / or number of EP cells obtained by the method of the present invention can be converted into a higher proportion of single hormonal beta cells as defined herein, compared to the prior art methods wherein step b-1) is 56 hours or longer and step b) is 96 hours or longer.

[0264] In one embodiment of the method as 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 beta cells, such as single hormone beta cells characterized by expressing 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 beta cells, such as single hormone beta cells characterized by expressing insulin than a corresponding method, in which step b) is culturing the cell population of the posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for about 96 hours. In one embodiment, the method produces at least 30%, such as at least about 35%, such as at least about 40% more single hormone beta cells, such as single hormone beta cells characterized by expressing insulin than a corresponding method, in which step b-1) is 56 hours or longer and step b) is 96 hours or longer. The monohormonal beta cell may be characterized by the expression of insulin and at least one of NKX6.1, PDX1, and NEUROD1, such as insulin and NKX6.1.

[0265] In one embodiment, the monohormonal beta cell expresses insulin and may be further characterized by the expression of NKX6.1, PDX1 and / or NEUROD1. In one embodiment, the monohormonal beta cell is characterized by the expression of: insulin and NKX6.1; insulin and PDX1; or insulin and NEUROD1.

[0266] In one embodiment, the monohormonal beta cell is 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 monohormonal beta cell is characterized by the expression of insulin, PDX1, NKX6.1, and NEUROD1.

[0267] In a specific embodiment, the method produces at least 2 times more pancreatic monohormone β cells than monohormone pancreatic α cells characterized by expression of glucagon, such as pancreatic monohormone β cells characterized by expression of insulin. Thus, the islet-like cell aggregates obtained by the method of the invention comprise at least 2 times more pancreatic monohormone β cells than α cells.

[0268] The technician understands that the monohormone pancreatic β cells not only express insulin and the characteristic markers of mature pancreatic β cells, but also that the monohormone β cells are functional pancreatic β cells and are therefore able to respond to glucose stimulation. The results 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 a C-peptide molecule produced by the β cells. C-peptide itself does not affect blood sugar, but C-peptide is a useful marker for insulin production because C-peptide tends to stay longer in the blood than insulin.

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

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

[0271] The source cell can be a pluripotent cell, such as a cell line of a pluripotent cell, such as an embryonic stem cell or an induced pluripotent stem cell. Therefore, the cell can be from an established cell line, or alternatively, the cell can be a primary cell directly derived from a patient, such as a patient-specific cell. Therefore, in one embodiment of the method as disclosed herein, the cell colony 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 colony in step a) or a-1) or a+1) is a primary cell colony directly derived from a patient. Alternatively, the cell colony in step a) or a-1) or a+1) can be derived from a culture of a specialized cell, such as a cell line that has been limited to an endoderm pedigree. The cell can be a human cell.

[0272] In one embodiment, the cell colony in step a) or a-1) is a mammalian cell colony, such as a human cell colony. Therefore, this also applies to the cell colony in step i). Therefore, the cell colony 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 colony in step i) can be a primary cell colony directly derived from a patient, or a culture derived from a specialized cell, such as a cell line that has been restricted to an endoderm pedigree. The cell can be a human cell.

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

[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 produce daughter cells, including self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm). Stem cells also produce tissues of multiple germ layers after transplantation and contribute significantly to most, if not all, tissues after injection into blastocysts.

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

[0276] As explained above, differentiation is the process by which a non-specialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell (such as, for example, a nerve cell or a muscle cell). A differentiated cell or a differentiation-induced cell is a cell that occupies a more specialized ("committed") position in the lineage of the cell. The term "committed" as applied to the differentiation process refers to a cell that has progressed to a point in the differentiation pathway that would normally continue to differentiate into a specific cell type or subset of cell types, and that would normally not differentiate into a different cell type or revert to a less differentiated cell type. "Dedifferentiation" refers to the process by which a cell reverts to a less specialized (or committed) position within a cell lineage.

[0277] It should be noted that the technology of the present invention can be put into practice using any human pluripotent embryonic stem cell, including people's pluripotent embryonic stem cells derived without destroying human embryo, such as from the oocyte activated by parthenogenesis. In specific embodiments, the cell colony in step i) is derived from a human embryonic stem cell colony. In one embodiment, the cell colony in step i) is derived from a human embryonic stem cell colony, which is obtained without destroying human embryo. In specific embodiments, the cell colony in step a) or step a-1) or step a+1) is derived from a human embryonic stem cell colony. In one embodiment, the cell colony in step a) or step a-1) or step a+1) is derived from a human embryonic stem cell colony, which is obtained without destroying human embryo.

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

[0279] In a specific 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 specific embodiment, the IPS cell is selected from the group consisting of patient-derived IPS cells and IPS cell lines. In a specific embodiment, the IPS cell line, for example, is CTRL-7-II (C7). C7 is described in Kele M et al. 2016.

[0281] As discussed above, the method may also include a step of cryopreservation of cells, and in particular it may be suitable for cryopreservation of endocrine progenitor cells. Therefore, in one embodiment, a method is provided comprising the step of cryopreservation of endocrine progenitor cells. In one embodiment, the cryopreservation is the cryopreservation of endocrine progenitor cells generated in step c+1). In one embodiment, the method as disclosed herein includes cryopreservation of 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 previously cryopreserved. As shown in the accompanying examples, cryopreservation does not negatively affect the generation of the islet-like cell aggregates or single hormone β cells.

[0282] In a second aspect of the disclosure, isolated islet-like cell aggregates or isolated islet-like cell aggregate populations obtainable by a 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 beta 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 the pancreatic islets in vivo, both in terms of the distribution of the cell types present herein and in terms of their functional properties. The skilled person will recognize that

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

[0284] Therefore, in one embodiment, the islet-like cell aggregates wherein said separation 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% of single hormone beta cells. In one embodiment, the islet-like cell aggregates contain about 25% to 70%, such as about 30% to 70%, such as about 40% to 70%, such as about 40% to 60% of single hormone beta cells.

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

[0286] In one embodiment, the islet-like cell aggregates contain up to about 20%, such as up to about 18%, such as up to about 16%, such as up to about 13%, such as up to about 10% of a single hormone alpha cell. In one embodiment, the single hormone alpha cell is characterized by expressing glucagon.

[0287] In one embodiment, the isolated islet-like cell aggregates contain single hormone beta cells and alpha cells, and contain less than about 5%, such as 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) contain up to about 5%, such as up to about 4%, 3%, 2% or 1% of multihormonal cells. In one embodiment of the method, the islet-like cell aggregates in v) contain up to about 5%, such as up to about 4%, 3%, 2% or 1% of non-endocrine cells. Therefore, in one embodiment, the isolated islet-like cell aggregates contain the following, or the isolated population contains islet-like cell aggregates containing the following: 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 60%, such as at least about 65%, such as at least about 70% of single hormone beta cells. In one embodiment, the isolated islet-like cell aggregates comprise, or the isolated population comprises, islet-like cell aggregates comprising: about 25% to 70%, such as 30% to 70%, such as 30% to 70%, of single hormone beta cells, such as 35% to 70%, 35% to 70%, such as 40% to 70%, such as 45% to 70%, such as 45% to 65%, such as 45% to 60%, such as 45% to 55%, such as about 50% of single hormone beta cells. In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: about 35% to 65%, such as 40% to 65%, such as 40% to 60%, of single hormone beta cells.

[0288] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: approximately 7% to 25%, such as 7% to 20%, 10% to 20%, such as 15% to 20%, such as approximately 20% of single hormone alpha cells.

[0289] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: at most about 10%, for example at most about 7%, for example at most about 6%, for example at most about 5%, for example at most about 4%, for example at most about 3%, for example at most about 2%, for example at most about 0.5%, for example at most about 0.3%, for example at most about 0.1% of multi-hormone alpha cells.

[0290] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising: at most about 10%, for example at most about 7%, for example at most about 6%, for example at most about 5%, for example at most about 4%, for example at most about 3%, for example at most about 2%, for example at most about 0.5%, for example at most about 0.3%, for example at most about 0.1% of multi-hormone beta cells.

[0291] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates comprising less than 5%, such as less than 4%, such as less than 3%, such as less than 1% delta cells.

[0292] In one embodiment, the islet-like cell aggregates comprise, or the population comprises, islet-like cell aggregates containing: at most about 5%, such as at most about 4%, such as at most about 3%, such as at most about 2%, such as at most about 1%, such as at most about 0.5%, such as at most about 0.1% of proliferating cells, such as proliferating cells expressing Ki-67.

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

[0294] It should be understood that the islet-like cell aggregates of the separation, its colony or the cell therefrom can be used in therapy, such as cell replacement therapy, and drug development or other research applications. Specifically, it should be understood that, without the need for any additional cell selection or sorting, it is advantageous to provide a colony showing a high percentage or high score of the desired cell type. For clarification, the term "separated" related to the islet-like cell aggregates of separation, its colony or the cell from the islet-like cell aggregates refers to removing (in other words, separating) the cells from their natural environment (such as in vivo environment). It should be understood that the islet-like cell aggregate colony of separation, its colony or the cell from the islet-like cell aggregates as disclosed herein can be a part or all of the cell aggregates formed during cell culture. The aggregate (also referred to as islet-like cell aggregates) may also include, for example, pancreatic α cells and / or δ cells or one or more pancreatic α and / or δ cell lineages in addition to pancreatic β cells.

[0295] Therefore, in one embodiment, the islet-like cell aggregates or islet-like cell aggregates of the separation are provided, wherein the cells constituting the islet-like cell aggregates have not been subjected to enrichment for the desired phenotype, for example, have not been subjected to enrichment by manual intervention or by the mode of machine (in other words, automatic sorting). In one embodiment, the cells are not subjected to enrichment before forming the 3D structure in step iii). Therefore, in one embodiment of the islet-like cell aggregates of separation or the islet-like cell aggregates of separation, the cells constituting the islet-like cell aggregates have not been subjected to enrichment for the desired phenotype, for example, have not been subjected to sorting for the desired phenotype, for example, based on sorting of desired marker expression or for example based on sorting of desired phenotype by FACS. Technicians understand that selection / sorting can be based on the presence of the desired phenotype (for example, based on marker expression) or based on the absence of an undesirable phenotype, in which case, undesirable cells are removed from the colony, and therefore the colony is enriched to show cells of the desired phenotype. In one embodiment, the cells are not subjected to sorting before forming a 3D structure in step iii). For the sake of clarity, as used herein, the term "enrichment" refers to enrichment of cells by (manually or by machine) intervention, such as sorting cells based on the characteristics of cells, rather than referring to a process occurring 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 sorted based on desired marker expression. In one embodiment, the cells constituting one or more of the islets have not been subjected to sorting for a desired phenotype based on FACS. In one embodiment, the cells comprising one or more of the islets have not been subjected to enrichment by removing an undesirable phenotype, such as based on marker expression, such as by means of FACS.

[0296] The islet-like cell aggregates of the present invention are characterized by a high percentage of cells with desired characteristics obtained by the differentiation method itself. Therefore, in one embodiment of the present aspect, isolated islet-like cell aggregates are provided, wherein more than about 40%, such as about 40 to 60%, such as about 40 to 50% of the total cell population of one or more of the islets are single hormone β cells, such as single hormone β cells characterized by expressing insulin. In one embodiment, the colony contains at least 2 times (e.g., 3, 4, 5 times) more single hormone pancreatic β cells characterized by expressing insulin than single hormone pancreatic α cells characterized by expressing glucagon.

[0297] The single hormone beta cell may be characterized as described above, for example by expressing insulin and at least one or more, for example two, of the markers NKX6.1, PDX1, and NEUROD1. The single hormone beta cell 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 single hormone beta cell may be characterized by expressing insulin, NKX6.1, PDX1, and NEUROD1. In one embodiment, the single hormone beta cell may comprise a portion of a cell aggregate, for example a portion of a cell aggregate that also comprises pancreatic single hormone alpha and / or delta cells.

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

[0299] As mentioned above, it is envisioned that the isolated islets or isolated islet-like cell aggregates as disclosed herein, or cells obtained therefrom, will be useful in treating and / or preventing diabetes.

[0300] Thus, in a third aspect of the present disclosure, there is provided an isolated islet-like cell aggregate, an isolated islet-like cell aggregate population or cells obtained therefrom as disclosed herein for use in therapy (in other words, as a medicament).

[0301] It should be understood that the islet-like cell aggregates or cells therefrom may be transplanted into a patient in need thereof for the purpose of cell replacement therapy, which may be for the purpose of providing pancreatic β cells to a patient who has no endogenous β cells or only non-functional β cells, or to a patient who needs more pancreatic β cells because his or her endogenous population is reduced or less functional than that required for a healthy patient state.

[0302] The islet-like cell aggregates or cells can be derived from donors, and are therefore allogeneic. For example, the islet-like cell aggregates or cells can be derived from relatives or unrelated donors or derived from established cell lines, such as stem cell lines with the ability to develop along the pancreatic β cell lineage. Non-limiting examples include hES cell lines, non-embryonic stem cell lines (such as special, oligopotent or unipotent cell lines) with the potential to develop along the endocrine lineage, iPS cell lines, and cell lines derived from iPS cells with the ability to develop along the pancreatic β cell lineage. The cell can also be endogenous to the patient, such as derived from iPS cells obtained from the patient or cells derived from other patients with the ability to develop along the pancreatic β cell lineage.

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

[0304] In a fourth aspect, there is provided an islet-like cell aggregate, a colony of islet-like cell aggregates or a cell therefrom as disclosed herein for use in treating, preventing and / or improving 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 aggregate populations, or cells obtained therefrom as disclosed herein are provided for use in treatment in therapy, wherein the isolated islets, isolated islet-like cell aggregate populations, or cells obtained therefrom as disclosed herein have been generated by a method as defined herein.

[0306] In one embodiment, there is provided an isolated islet-like cell aggregate, an isolated islet-like cell aggregate population or a cell therefrom for use in therapy (in other words, for use as a medicament), wherein the use comprises the step of generating an isolated islet-like cell aggregate cell, an isolated islet-like cell aggregate population or a cell therefrom according to a method as defined herein; and

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

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

[0309] In one embodiment, isolated islet-like cell aggregates or cells obtained therefrom are provided for use in treating, preventing and / or ameliorating diabetes (e.g., type 1 or type 2 diabetes), wherein the islet-like cell aggregates or cells have been generated by a method as defined herein.

[0310] In another embodiment, isolated islet-like cell aggregates or cells therefrom are provided for use in treating, preventing and / or ameliorating diabetes (e.g., type 1 or type 2 diabetes), wherein the use comprises the following steps:

[0311] Generating the islet-like cell aggregates or cells according to the method defined herein; and

[0312] The cells of therapeutically effective amount are administered to the patient. In one embodiment, the islet-like cell aggregates can be administered in the form of islet-like cell aggregates, in other words, administered in the form of cell aggregates, as defined above. Alternatively, the islet-like cell aggregates can be dissociated and these cells can be administered in the form of cell suspensions (e.g., single cell suspensions). In one embodiment, the use also includes separating cells from the patient, such as cells for generating IPS cells, or separating stem cells from the patient, and using the cells to generate the islet-like cell aggregates or cells produced therefrom according to the method defined herein. Similar to that discussed about the third aspect, other cell types are considered to be useful in this regard, including allogeneic cells and endogenous cells.

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

[0314] A therapeutically effective amount of the islet-like cell aggregates is administered to a patient.

[0315] Thus, in one embodiment, the use comprises the steps 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 will be appreciated that the cell replacement therapy may involve the transplantation of islet-like cell aggregates (cell aggregates) or cells obtained 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 comprises transplanting the islet-like cell aggregates of the cells into a patient in need. In one embodiment, the use comprises transplanting all cells or a subset of the cells (e.g., single hormone pancreatic β cells) obtained from the dissociated islet-like cell aggregates into a patient in need.

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

[0320] It is also envisioned that the islet-like cell aggregates separated as disclosed herein or cells therefrom or pharmaceutical compositions as disclosed herein may be useful together with the load substrate promoting the growth, proliferation and / or optional differentiation of the cell colony.Therefore, in the fourth and sixth aspects of the present disclosure, a complete set of kits is provided, which comprises the islet-like cell aggregates separated as disclosed herein or cells therefrom or pharmaceutical compositions as disclosed herein and suitable load substrates.The suitable load substrate can be a 3D scaffold.

[0321] In one embodiment, a kit is provided, wherein the loaded substrate is a 3D substrate and wherein the cells are monohormone beta cells. In other embodiments, the kit comprises a 2D substrate. Non-limiting examples of such 2D substrates are the substrates discussed herein.

[0322] It should be understood that one or more separated islets as disclosed herein or cells therefrom can have many uses in biological research, such as for drug screening, such as in vitro drug screening.It should be understood that one or more separated islets as disclosed herein or cells therefrom will be beneficial, because the percentage of total cells of the desired cell type will be higher than the percentage in the islet-like cell aggregates of the cell colony obtained by the culture method described previously.Therefore, compared with the islet-like cell aggregates or cells generated according to the prior art, the islet-like cell aggregates of the present invention or cells therefrom, if even if necessary, will also be at least to a lesser extent required to undergo cell sorting or similar stress and potential destructive techniques, in order to obtain a homogeneous population of the desired cell type with a high percentage.The homogeneous population of the desired cell type with a high percentage may generate data in drug screening assays, which are less or not hindered by one or more potential responses from other irrelevant or contaminated cell types.Therefore, in the seventh aspect of the present disclosure, there is provided the use of the islet-like cell aggregates as disclosed herein or cells therefrom in drug screening (such as in vitro drug screening).

[0323] In this article, a method for in vitro drug screening is provided, the method comprising the following steps: generating isolated islet-like cell aggregates or cells therefrom according to the method defined herein; and exposing the islet-like cell aggregates or cells to at least one candidate drug compound. The method may also include a step of evaluating the response of the isolated islet-like cell aggregates or cells therefrom to the candidate drug compound.

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

[0325] In one embodiment, a method for in vitro drug screening is provided, comprising the steps of generating isolated islet-like cell aggregates according to the 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 of treating a patient in need thereof is provided, the method comprising administering to the patient a therapeutically effective amount of an isolated islet-like cell aggregate as disclosed herein or a cell therefrom. In addition, a method of treating diabetes in a patient in need thereof, the method comprising the steps of: generating an isolated islet-like cell aggregate or a cell therefrom according to a method as defined herein; and

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

[0328] In one embodiment, the method is used to treat diabetes, such as type 1 or type 2 diabetes. Thus, the patient may suffer from type 1 or type 2 diabetes.

[0329] Thus, a method of treating diabetes in a patient in need thereof is provided, the method comprising administering to the patient a therapeutically effective amount of an isolated islet-like cell aggregate as disclosed herein or a cell therefrom. In addition, a method of treating a patient in need thereof is provided, the method comprising the steps of: generating an isolated islet-like cell aggregate or a cell therefrom according to a method as defined herein; and

[0330] The patient is administered the isolated islet-like cell aggregates or cells therefrom of a therapeutically effective amount. The method may also include separating cells from the patient, such as cells or stem cells for generating IPS cells, and using the cells to generate isolated islet-like cell aggregates or cells therefrom according to the method as defined herein. Islet-like cell aggregates or cells can be administered by transplantation.

[0331] Thus, in one embodiment, said administering comprises transplanting said islet-like cell aggregates or cells into said patient.

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

[0333] It should be understood that any discussion related 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 simply for the sake of brevity. In one embodiment, a method for treating diabetes in a patient in need thereof as disclosed herein is provided, wherein the patient suffers from type 1 or type 2 diabetes. In one embodiment, the administration comprises transplanting the colony 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 steps of:

[0335] a-1) providing a cell population of primitive intestinal tube cells, wherein the primitive intestinal tube cells are, for example, primitive intestinal tube cells characterized by expressing HNF1β and / or HNF4α;

[0336] b-1) culturing the cell population of primitive gut tube cells under conditions that allow differentiation into posterior foregut cells for no longer than about 54 hours;

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

[0338] a) providing a cell population of posterior foregut cells generated in step c-1), wherein the posterior foregut cells are, for example, posterior foregut cells characterized by expressing PDX1;

[0339] b) culturing the cell population of posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for no longer than about 78 hours, such as no longer than about 72 hours;

[0340] c) thereby generating a cell population of pancreatic progenitor cells, the pancreatic progenitor cells being, for example, pancreatic progenitor cells characterized by expressing both PDX1 and NKX6.1;

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

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

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

[0344] i) providing a population of endocrine progenitor cells (EP) generated in step c+1), wherein the endocrine progenitor cells are, for example, EP cells characterized by expressing NEUROD1; for example, EP cells characterized by expressing NKX6.1 and NEUROD1;

[0345] ii) providing a single cell suspension of the population of EP cells;

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

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

[0348] v) thereby generating islet-like cell aggregates comprising a single hormone β cell,

[0349] The islet-like cell aggregates contain at least 25% of single hormone β cells.

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

[0351] As used herein, the phrase "a population of..." when referring to a certain type of cell is intended to be interpreted as a population comprising said cells. For example, the phrase "a population of EP cells" should be understood as a population comprising EP cells. The population may also additionally comprise other cells, such as, but not limited to, cells at an early developmental stage, such as, for example, PP cells.

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

[0353] The skilled person knows that numerical values ​​related to measurements are subject to measurement errors, which limit 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 the rounding convention to the last decimal place, e.g., for a measurement of 3.5 cm, the error tolerance is 3.45-3.54. When interpreting numerical ranges in patent specifications, the skilled person proceeds on the same basis.

[0354] Although the present invention has been described with reference to various exemplary aspects and embodiments, it will be appreciated by those skilled in the art that, without departing from the scope of the present invention, various changes may be made and its elements may be replaced with equivalents. In addition, without departing from the essential scope of the present invention, many modifications may be made to adapt specific situations, culture conditions or cell colonies to the teachings of the present invention. Therefore, it is intended that the present invention is not limited to any particular embodiment contemplated, but that the present invention will include all embodiments falling within the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0355] Figure 1 is a schematic diagram of the developmental stages along the pancreatic β-cell lineage, including the expression of markers characteristic of each developmental stage. (A) Pancreatic endocrine cell types can be generated from human pluripotent embryonic stem cells (hES) or IPS cells by recapitulating embryonic pancreatic development in a petri dish. Pancreatic differentiation can be divided into multiple stages, including definitive endoderm (DE), primitive gut tube (PGT), posterior foregut (PF), multipotent pancreatic progenitors (PP), endocrine progenitors (EP), and pancreatic islets (ISL). Key markers expressed at each stage are indicated in the figure. (B) Figure 1B It is a schematic diagram according to the long differentiation scheme of the prior art and the short differentiation scheme as disclosed herein. The long duration and short duration of stage 3 and stage 4 (S3+S4) were analyzed on the impact of subsequent endocrine differentiation. When stage 2 (S2) ends, when stage 3 (S3) ends, when stage 4 (S4) ends, when entering stage 5 (S5) four days and when stage 6 (S6) ends, the expression of stage-specific markers is checked. 2D LN-521 is an example of 2D substrate and can be replaced by other 2D substrates as disclosed herein.

[0356] Fig. 2 shows the 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 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) stage 5 are shown. Column charts representing the results of HS980 and H1 cells are shown. The results are calculated based on three independent samples. The percentages of (A and B) PDX1+ / NKX6.1+ and (C) NKX6.1+ / NEUROD1+ cells in the three substrates were compared, and unpaired 2-tail t tests were performed 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 gut tube cells (PGT) and post-foregut cells (PF). HS980 and H1 cells were differentiated toward 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 1 or 2 days into S3. Representative confocal microscopy images and dot plots of HS980 cells are shown. (A) HS980 cells. (B) H1 cells.

[0358] Fig. 4 shows the evaluation results of the effect of S4 duration on endocrine differentiation. HS980, H1 and H9 cells were differentiated on LN-521 coated plates. The duration of S4 was 1 day, 2 days, 3 days, 4 days or 5 days. The expression of progenitor cell markers NKX6.1 and NEUROD1 was examined by flow cytometry at the end of S4 or four days into S5. The expression of endocrine markers insulin (INS) and glucagon (GCG) was examined at the end of S6. The results were calculated based on multiple independent experiments. In order to compare the percentages of different cell populations in different S4 durations, an unpaired 2-tailed t-test was performed in Microsoft Excel. (A) Schematic diagram of different durations of S4. (B) Representative dot plots of progenitor cell markers NKX6.1 and NEUROD1 expression analysis in HS980 cells at S4 and S5. (C) Representative dot plots of progenitor cell markers NKX6.1 and NEUROD1 expression analysis in H1 and H9 cells at S5. (D) Bar graph showing the calculated percentage of NKX6.1+ / NEUROD1+ and NKX6.1+ / NEUROD1- cells. The results show the percentage of H980, H1, and H9 cells. (E) Bar graph showing the calculated percentage of NEUROD1+ and NKX6.1+ cells at S5. The results show the percentage of H980, H1, and H9 cells. (F) Representative dot plots of INS and GCC expression analysis in HS980, H1, and H9 cells at S6. (G) Bar graph showing the calculated percentage 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 evaluation of pancreatic differentiation on different coated substrates. Using the short protocol, HS980, H1 and H9 cells were differentiated towards S5EP cells on plates coated with different recombinant human laminin isotypes (LN) or matrigel. H1 cells were also differentiated on plates coated with fibronectin (FN) or vitronectin (VTN). At four days into S5, the expression of NKX6.1 and NEUROD1 was measured by flow cytometry. HS980 cells on LN-332, LN-511, LN-521 and matrigel were further differentiated towards S6 islet cells in 3D suspensions. The expression of INS and GCG was measured by flow cytometry at the end of S6. Representative dot plots of S5 EP cells are shown in Figure 5. Figure 5A-1 and 5A-2 Representative dot plots of S6 islet cells are shown in Figure 5CTo compare the percentage of NKX6.1+ / NEUROD1+ cells between laminin isotypes and Matrigel, 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 showing the timeline of HS980 cell differentiation on LN-521 using the short protocol. Cells were dissociated into single cells at the end of S4 (upper timeline) or at 4 or 6 days into S5 (lower timeline). 4x10 6 Individual S4 and S5 cells were maintained in 3D suspension and further differentiated into S6 islet cells for analysis. (B) At the end of S6, islet-like aggregates were counted under a microscope and then dissociated into single cells for cell counting. The expression of INS and GCG was measured by flow cytometry. Representative dot plots are shown. The percentage of cell populations and the number of aggregates / cells per well are shown in the bar graph and are calculated based on three independent samples. 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.

[0361] Figure 7 shows the enrichment of S5 EP cells during aggregate formation in suspension. HS980 and H1 cells were differentiated towards S5 on LN-521-coated 2D surfaces. At 4 days into 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 the single cell suspension as indicated. The next 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 from three independent experiments, as shown in the bar graph in (A). 6 The number of islet cells generated from a single cell was calculated based on three independent samples and is shown in the bar graph in (D). Unpaired 2-tailed t-test was performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001.

[0362] Fig. 8 shows the comparative results of short pancreatic differentiation scheme and long pancreatic differentiation scheme.HS980 and H1 cells were differentiated into S5 EP cells on LN-521 coated plates using short schemes and comparative long schemes as disclosed herein.When entering S5 for four days, cells were dissociated into single cells and further matured into islet-like cell aggregates in 3D suspensions.As shown in the figure (S3-S6), the expression of key markers was examined by flow cytometry at each stage.Insulin c-peptide secretion stimulated by glucose in vitro was examined at the end of S6.Islet-like aggregates were counted under a microscope and then dissociated into single cells for cell counting.The results were calculated based on multiple independent samples.Unpaired 2-tail t tests were performed in Microsoft Excel.(A) Representative dot plots of NKX6.1 and NEUROD1 expression in HS980 cells during S3-5.(B) Quantitative bar graphs showing NKX6.1+ / NEUROD1+ and NKX6.1+ / NEUROD1- obtained using short schemes and long schemes. (C) Representative dot plots of INS and GCG expression in HS980 and H1 cells at S6 obtained using the short and long protocols and a bar graph showing their quantification. (D) Bar graph showing a comparison of the results of in vitro glucose-stimulated insulin secretion in cells obtained using the short and long protocols, and a bar graph 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] Fig. 9 shows immunocytochemical analysis of stage-specific marker expression in cell culture. HS980 cells differentiated using short and long protocols were analyzed using specific antibodies for 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 to visualize cells.

[0364] Figure 10 shows the comparison results between spontaneous aggregation and forced aggregation using microplates in 3D suspensions.H1 cells differentiate towards S5 on LN-521 coated 2D surfaces.When entering S5 for four days, cells were dissociated into single cells and then maintained in 3D suspensions (free), 96-well plates (96 wells) or AggreWell plates (AggreWell).The expression of NKX6.1, NEUROD1 and Ki-67 was checked by flow cytometry before and after aggregation formation at S5.Representative dot plots are shown in Figure (A).The percentage of S5NKX6.1+ / NEUROD1+EP cells and Ki-67+ proliferating cells is calculated based on multiple independent samples and as shown in the bar graph in (B).Differentiation towards S6 islet cells was carried out in suspensions and AggreWell plates.In S6, the expression of INS, GCG and Ki-67 was checked by flow cytometry.Representative dot plots are shown in Figure (C). Unpaired 2-tailed t-test was performed in Microsoft Excel. *p<0.05, **p<0.01.

[0365] Fig.11 Schematic diagram of the in vitro differentiation protocol disclosed herein. Factors and duration of each stage are as indicated. Cells on LN-521 coated surfaces were dissociated into single cells at day 14 and then maintained in 3D suspension.

[0366] Figure 12 shows pancreatic differentiation in human ESC and iPSC lines. Cells were differentiated using a short protocol. The expression of key markers was examined by flow cytometry at the end of S5 for four days and S6. Insulin c-peptide secretion stimulated by glucose in vitro was examined at the end of S6. The results were calculated based on multiple independent experiments. Unpaired 2-tail t tests were performed in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. (A) and (B) show representative dot plots of INS and GCG expression during S6 and NKX6.1 and NEUROD1 expression during S5. (A) shows a bar graph (left) showing INS+ / GCG-, INS- / GCG+ and INS+ / GCG+ cell population quantification and a bar graph (right) showing the results of insulin secretion stimulated by glucose in vitro. (A) Differentiation of hESC lines. (B) Differentiation of human iPSC line C7.

[0367] Figure 13 shows the comparison results 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 two published protocols. The duration, factors, and 2D / 3D culture system of each stage are shown. (B) UMAP and cell type prediction (top) and expression levels of marker genes INS and GCG in predicted cell types (bottom).

[0368] Fig.14 It is shown that frozen S5 EP cells are able to generate S6 islet-like aggregates. HS980 and H1 cells differentiate towards S5 on LN-521 coated 2D surfaces. After entering S5 for four days, the cells are dissociated into single cells and then maintained in 3D suspensions to generate S6 aggregates. Alternatively, single cells are frozen and stored in liquid nitrogen. In order to generate 3D aggregates, frozen S5 cells are thawed and cultured in 3D suspensions. The expression of markers INS and GCG and insulin c-peptide release stimulated by glucose in vitro are examined at the end of S6. A bar graph (left) showing quantification of INS+ / GCG-, INS- / GCG+ and INS+ / GCG+ cell populations and a bar graph (right) showing the results of insulin secretion stimulated by glucose in vitro are shown. The results are calculated based on multiple independent experiments. Unpaired 2-tailed t-tests are performed in Microsoft Excel. *p<0.05, **p<0.01. Example

[0369] Overview: This example demonstrates the method of the invention disclosed herein, which includes the step of culturing a cell population of posterior foregut cells under conditions permissive for differentiation into pancreatic progenitor cells for no more than about 78 hours, such as 72 hours or 48 hours, 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] The hESC line HS980 was derived under xeno-free and defined conditions as previously described (Rodin, S., et al. 2014). The hESC lines WA01 / H1 and WA09 / H9 were obtained from the WiCell Research Institute (Madison, WI). The human iPSC line CTRL-7-II (C7) is described in Kele M et al., 2016). The hESC lines were maintained in NutriStem hPSC XF medium (Biological Industries, Israel; 05-100-1A) on Sarstedt multiwell cell culture plates coated with 10 μg / mL human recombinant laminin (LN)-521 (BioLamina, Sweden; LN-521) in a 37°C incubator with 5% CO2, 5% O2 and 100% humidity. Every 3-5 days, 12,000-24,000 cells / cm 2 Enzymatic passaging of hESCs was performed.

[0373] For routine passaging, hESC cultures on LN-521 were maintained in a Ca-free medium. 2+ and Mg 2+ The cells were briefly washed in D-PBS (Thermo Fisher Scientific; 14190169) and incubated with Gibco TrypLE Select (Thermo Fisher Scientific; A1285901) for 5 minutes at 37° C. The cells were collected into fresh NutriStem hPSC XF medium by gently pipetting 5-10 times using a P1000 pipette, centrifuged at 300 g for 4-5 minutes, resuspended in fresh NutriStem hPSC XF medium, and seeded onto newly coated cell culture plates.

[0374] In vitro pancreatic differentiation of hESCs

[0375] The stepwise pancreatic differentiation protocol described here was modified from 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 24,000 cells / cm 2 Plated on LN-521 coated cell culture plates in NutriStem hPSC XF medium. Pancreatic differentiation was initiated four days later, reaching 95%-100% confluency. 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) + 25 mM NaHCO3 (Sigma; S6297) + 1X GlutaMAX (Thermo Fisher Scientific; 35050038) + 50 U / mL penicillin-streptomycin (Thermo Fisher Scientific; 15140122) + 2.5 mM D-glucose (8 mM final concentration, Sigma; G8769) + 0.2% or 0.5% fatty acid-free bovine serum albumin (FAF-BSA, Sigma; A8806).

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

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

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

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

[0383] Short differentiation protocol

[0384] Stage 1 Definitive endoderm (3 days): Undifferentiated H1, H9 and HS980 cells were cultured with Ca 2+ and Mg 2+ The cells were rinsed once with 5 μM 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 (R&D; 338-AC). For the next two days, the 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] Stage 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. FAF-BSA concentration was 0.2% for HS980 cells and 0.5% for H1 and H9 cells.

[0386] Stage 3 posterior foregut (1 day): cells were induced for 24 hours in S3 medium with 50 ng / ml KGF, 2 μM retinoic acid (Sigma; R2625), 0.25 μM SANT-1 (Sigma; S4572), 0.5 μM PDBu (Tocris; 4153) and 200 nM LDN193189 (Tocris; 6053).

[0387] Stage 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; N0636), 100 nM Retinoic Acid, 0.25 μM SANT-1, 0.5 μM PDBu and 200 nM LDN193189. To analyze the effect of the duration of stage 4, cells were also differentiated for 1-5 days in this step.

[0388] Stage 5 endocrine progenitor cells (5 days): Cells were induced in S5 medium with 20 ng / ml betacellulin (Research and Development Corporation; 261-CE), 100 nM retinoic acid, 0.25 μM SANT-1, 100 nM GSI-XX (Sigma; 565789), 10 μM ALK5 inhibitor II (Cayman Chemical; 14794), 1 μM GC-1 (Tocris; 4554) and 100 nM LDN193189.

[0389] Four days into stage 5, cells were incubated with Ca-free 2+ and Mg 2+ The cells were rinsed once with DPBS, treated with Accutase for 10 min at 37 °C, and then dissociated into single cells in S5 medium by pipetting several times using a P1000 pipette. Single cells were pelleted by centrifugation at 300 g for 5 min and then centrifuged at 1.0-1.5 x 10 6 10 cells / ml were resuspended in S5 medium supplemented with 10 μM H1152 (Tocris; 2414) and other factors. To generate islet-like aggregates, cells were transferred to ultra-low attachment 6-well plates (Corning; 3471) with a total of 4-6 x 10 cells / ml per well in 4 ml. 4 cells and incubated overnight in an incubator at 95 rpm on an orbital shaker (Infors HT Celltron).

[0390] To investigate the effect 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] Stage 6 (4 weeks): Cell aggregates were maintained in S6 medium further 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. Aggregates were maintained in an incubator on an orbital shaker at 95 rpm.

[0392] The medium was changed daily from stage 1 to stage 5 and every 2-3 days during stage 6.

[0393] Long Differentiation Program:

[0394] hESCs were differentiated towards endocrine progenitors using the same factors and media as the short protocol above, but the duration of Stage 3 and Stage 4 were 2 and 5 days, respectively.

[0395] Freezing of Stage 5 Endocrine Progenitor Cells

[0396] Four days into S5, cells were dissociated into single cells as described above. These single cells were pelleted by centrifugation at 300 g for 5 min and then centrifuged at 1 × 10 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 freezing containers (Thermo Fisher Scientific; 5100-0001). For controlled cooling, a programmable cooling device can be used to cool cells at 1°C per minute. For long-term cryopreservation, cryogenic tubes were transferred to liquid nitrogen for storage.

[0397] Thawing of stage 5 endocrine progenitor cells

[0398] Frozen S5 cells in cryovials were removed from liquid nitrogen storage and rapidly thawed at 37°C. Each 1 ml of cell suspension was diluted and gently mixed into 5 ml of pre-warmed complete S5 medium. The cells were pelleted by centrifugation at 300 g for 5 min and then plated at 1.0-1.5 x 10 6 The cells were resuspended in complete S5 medium at 100 cells / ml. Further differentiation procedures were performed as described above.

[0399] Aggregate formation in micropores

[0400] To generate islet-like aggregates in microwells, S5 cells were dissociated into single cells as described above. These single cells were plated at 1.5 x 10 6 The cells were resuspended at 10 cells / ml and then seeded into AggreWell 400 6-well plates (StemCell Technologies; 34421), with a total of 6 x 10 cells / ml per well in 4 ml. 4 Alternatively, 10 cells in 50 μl 4 Single cells were inoculated into each well of a 96-well Microtest plate (Sydno, 82.1583.001), and the cells were cultured in an incubator overnight.

[0401] Differentiate into S6 islet-like cell aggregates as described above. Maintain aggregates in AggreWell 400 6-well plates without shaking.

[0402] Aggregate and cell counting

[0403] S6 islet-like aggregates were counted under a bright field microscope. The aggregates were then placed in a Ca-free 2+ and Mg 2+ Rinse once in D-PBS and incubate with Accutase for 15 min at 37 °C. Dissociate aggregates into single cells by pipetting several times using a P1000 pipette, centrifuge at 300 g for 4-5 min, and resuspend in Ca-free PBS. 2+and Mg 2+ The cells were counted using an ORFLOMOXI Z mini automatic cell counter (ORFLO Corporation; MXZ001).

[0404] Flow cytometry

[0405] Cells were dissociated into single cells by treatment with Accutase at 37°C for 15 min. The cells were then washed twice and cultured at 10 6 Cells were resuspended at 100 cells / ml in D-PBS without Mg2+ and Ca2+. The cells were incubated on ice for 30 min using the Live / Dead Fixable Dead Cell Stain 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). The cells were then washed twice in 1X BD Bioscience permeabilization / washing buffer (554723) and incubated on ice for 30 minutes with conjugated antibodies diluted in 1X BD Bioscience permeabilization / washing buffer. After washing twice in 1X BD Bioscience permeabilization / washing buffer, cells were resuspended in FACS buffer (D-PBS without Ca2+ or Mg2+, containing 2% fetal bovine serum (Thermo Fisher Scientific; 10082147) and 1mM EDTA (Thermo Fisher Scientific; 15575020)) and analyzed using Beckman Coulter CytoFLEX S flow cytometer. Flow cytometry data were analyzed using BD Bioscience FlowJo v10.8 software. The conjugated antibodies were all 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 min and then washed with Mg-free2+ and Ca 2+ For immunostaining, the cells were washed three times with D-PBS containing 0.3% (vol / vol) Triton X-100 (Sigma; T9284). 2+ and Ca 2+ The cells were blocked with 5% normal donkey serum (Merck Millipore; S30-100 mL) at RT for 1 h and then incubated with D-PBS (without Mg) containing 0.1% (vol / vol) Triton X-100 or Tween 20 (Sigma; P9416) and 5% normal donkey serum. 2+ and Ca 2+ ) were incubated overnight at 4°C. The next day, cells were incubated with secondary antibodies for 1 hour at room temperature. After each incubation step, cells were incubated with Mg-free 2+ and Ca 2+ The cells were washed three times with D-PBS. The primary antibodies used were as follows: goat anti-human PDX-1 (1 / 300, Research and Development; AF2419), mouse anti-Nkx6.1 (1 / 100, DSHB; F55A12-s), sheep anti-human neurogenin 3 (NGN3) (1 / 100, Research and Development; AF3444), goat anti-human / mouse NeuroD1 (1 / 100, Research and Development; AF2746), guinea pig anti-C-peptide (1 / 100, abcam; ab30477), rat anti-C-peptide (1 / 50, DSHB; GN-ID4-s), mouse anti-glucagon (1 / 1000, Sigma; G2654), and rabbit anti-somatostatin (1 / 500, Sigma; 332A-1).

[0408] In vitro glucose stimulation

[0409] At the end of stage 6, twenty to thirty hESC-derived aggregates were incubated overnight in S6 medium without ITS-X and additional glucose (finally 5mM). The next day, the aggregates were transferred to 24-well ultra-low attachment plates (Corning; 3473) and washed twice with 2ml Krebs buffer (129mM NaCl, 4.8mM KCl, 2.5mM CaCl2, 1.2mM MgSO2, 1mMNa2HPO4, 1.2mM KH2PO4, 5mM NaHCO3, 10mM HEPES and 0.1% FAF-BSA). The aggregates were then pre-incubated for 2 hours in 2ml Krebs buffer containing 2mM glucose to remove residual insulin. Afterwards, the aggregates were washed twice with 2ml Krebs buffer and then incubated for 30min in 2ml Krebs buffer containing 2mM glucose. 500μl supernatant samples (low glucose samples) were collected after incubation. Aggregate is washed once with 2ml Krebs buffer, then incubated 30min in 2ml Krebs buffer containing 20mM glucose.500μl supernatant samples (high glucose samples) are collected after incubation. Aggregate is washed twice in 2mL Krebs buffer, then incubated 30min in 2mLKrebs buffer containing 2mM glucose again.500μL supernatant samples (low glucose samples) are collected. Aggregate is washed once in 2mL Krebs buffer, then incubated 30min in 2mL Krebs buffer containing 2mM glucose and 30mM KCl (polarization stimulation).500μl supernatant samples (KCl stimulated samples) are collected.After KCl stimulates, aggregate is dispersed into single cell by processing with Accutase for 15 minutes, and ORFLO MOXI Z cell counter is used to count total number of cells.Use human c peptide ELISA kit (research and development company; DICP00) to process the supernatant samples containing secreted insulin collected. Human insulin C-peptide measurements were normalized by total cell number and expressed as 10 3 If ELISA was not performed on the same day, the samples were stored at -80°C.

[0410] scRNA sequencing sample preparation

[0411] hESC-derived islets were collected for scRNA sequencing at the end of S6. Islet-like aggregates were cultured in a Ca-free 2+ and Mg 2+The cells were rinsed twice in D-PBS and incubated with TrypLE on an orbital shaker at 37°C for 15-20 min. Aggregates were dissociated into single cells by multiple pipetting using a P1000 pipette, centrifuged at 300 g for 4-5 min, and resuspended in D-PBS containing 0.04% FAF-BSA (without Ca2+). 2+ and Mg 2+ ) and then filtered using a 40 μm cell strainer (VWR, 732-2760). To determine the total cell number and live cell ratio, 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 Nextseq2000 machine using the 10x Genomics Chromium Next GEM Single Cell 3′ Kit v3.1 (10x Genomics, CG000315 or CG000388), sometimes with the Cell Multiplex Oligonucleotide Labeling Protocol (10x Genomics, CG000391). Analysis steps were performed in Cell Ranger 3.1.0 to generate FastQ files and feature barcode matrices. 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 example, the ability of cell culture substrates to support pancreatic differentiation was compared using different hESC lines.Pancreatic differentiation was assessed based on the expression of key markers by pancreatic progenitor cells (PP) at stage 4 (S4) and endocrine progenitor cells (EP) at stage 5 (S5).

[0416] Protocols for in vitro pancreatic differentiation of hPSCs cultured on 2D surfaces coated with feeder cells or Matrigel have been developed (Pagliuca et al. 2014; D'Amour et al., 2006; Kroon et al., 2008). However, for clinical applications of hPSC-derived islets, a xeno-free, chemically defined coating substrate is highly preferred. Therefore, we compared the ability of human recombinant LN-511 and LN-521 to support pancreatic differentiation in the presence of Matrigel. The hESC lines HS980 and H1 (see Figure 2) were differentiated on these three substrates using a long differentiation protocol. Figure 1B), and the expression of key progenitor markers PDX1, NKX6.1, and NEUROD1 were 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 on all substrates in HS980 and H1 cell lines ( Figure 2A However, the levels of PDX1+ / NKX6.1+ cells decreased significantly on Matrigel and to a lesser extent on LN-511, but remained relatively stable on LN-521 when S4 cells were further differentiated to S5 ( Figure 2B ). The percentage of S5 NKX6.1+ / NEUROD1+ endocrine progenitor cells (EP) was much higher on LN-521 and, to a lesser extent, on LN-511 compared with that on Matrigel ( Figure 2C ).

[0418] Taken together, the data suggest that Matrigel, LN-511, and LN-521 are able to support differentiation into S4 PP cells, and in particular, LN-521 also provides a permissive surface environment for endocrine differentiation of S4 PP cells. Therefore, we decided to continue all our experiments on LN-521 instead of Matrigel. Since the percentage of S5EP cells on LN-521 was only 20%-30%, modifications were required to optimize the differentiation protocol.

[0419] Example 2

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

[0421] Phase 3 Evaluations of different lengths

[0422] It has been previously reported that short-duration S3 promotes S4 PDX1 + / NKX6.1 + The PP cell population is activated and inhibits the immature plurihormone cell population (Nostro et al., 2015). However, it was unclear whether the duration of S4 would also affect later differentiation stages in a similar manner. Therefore, we decided to analyze / optimize the duration of S3 and S4. To identify the shortest duration of S3, HS980 and H1 cells were differentiated into post-S3 foregut cells (PF) 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 one day after entering S3, and PDX1 was expressed in most cells on days 6, 7, and 8 of culture as shown by immunocytological analysis. + As shown by representative dot blots of cell numbers, expression increased only slightly after 2 days (Figure 3).

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

[0425] 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 at S3 for 1 day and then at S4 for 1, 2, 3, 4, or 5 days (as 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 dot plots are shown in Figure 4B 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. + / NEUROD1 + The percentage of EP cells decreased as the duration of S4 increased ( Figure 4B ). Similar inhibitory effects were observed for H1 and H9 cells. When the duration exceeded 2-3 days, NKX6.1 + / NEUROD1 + The percentage of EP cells began to decrease ( Figure 4C The experiment was repeated several times and the results showed that 2-3 days under S4 was necessary for subsequent differentiation into S5 NKX6.1 + / NEUROD1 + EP cells are optimal ( Figure 4D ). In contrast, an increase in duration at S4 generated a significantly higher percentage of NKX6.1 at S5. + / NEUROD1 - cell( Figure 4D ).NKX6.1 + / NEUROD1 -The cells are likely actually S4 NKX6.1 + PP cells, due to long-term inhibition, failed to further differentiate into S5 NKX6.1 + / NEUROD1 + EP cells ( Figure 4D The inhibitory effect of long S4 duration was particularly evident in H9 cells and was reduced from 5 days to 2 days, which reduced the total NEUROD1 + The percentage of endocrine cells increased to similar levels in HS980 and H1 cells ( Figure 4E The results also showed that 1 day under S4 was not sufficient to induce NKX6.1 ( Figure 4E ).

[0428] Therefore, it was concluded that a 2-3 day S4 duration allowed for a higher percentage of NKX6.1 + / NEUROD1 + EP cells, and thus benefit from differentiation into S5 EP cells. Optimal S4 duration can also minimize cell line variation during pancreatic differentiation.

[0429] Effect of Stage 4 Duration on 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) were analyzed at the end of S6.

[0431] result : The results show that when the duration of S4 becomes longer than 3 days, INS + The percentage of monohormone β cells decreased, and GCG + The percentage of monohormetic α cells increased significantly, as shown in the representative dot plots ( Figure 4E ) and column charts ( Figure 4F ). Notably, a 2-day duration of S4 resulted in a high percentage of EP cells in H1 cell cultures and H9 cell cultures, whereas a 3-day duration of S4 resulted in a high percentage of EP cells in H980 cultures ( Figure 4D ). However, a duration of 3 days resulted in the highest percentage of S6INS+β cells ( Figure 4F ).

[0432] In summary: Taken together, these results suggest that long S4 duration promotes differentiation into S4NKX6.1 + PP cells, but inhibited further differentiation into S5 NKX6.1 + / NEUROD1 + On the other hand, short S4 durations generate less S4NKX6.1 + PP cells, but with increased S5NKX6.1 + / NEUROD1 + EP cells. The results also showed that S4 duration had opposite effects on α and β cell populations. 3 days of S4 promoted INS + β cells, 5 days duration strongly promotes GCG + Importantly, this result was confirmed in three different hESC lines (H1, H9 and HS980).

[0433] Example 3

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

[0435] Chemically defined and xeno-free cell culture substrates, 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 on 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 Matrigel as indicated in the table. Cell adhesion and survival were examined under a microscope after 3-4 days. + indicates that cells attached to the bottom and formed a monolayer, - indicates that cells failed to attach to the coated surface.

[0439] To determine whether hESC can also be differentiated into pancreatic endocrine cells on other matrix proteins, we passaged HS980, H1 and H9 cells onto cell culture plates coated with matrigel (1 / 100, Corning; 354277) or human recombinant LN-111, LN-121, LN-211, LN-221, LN-332, LN-411, LN-421, LN-511 or LN-521 (all obtained from Bio-Lemina). HS980, H1 and H9 cells were attached to LN-111, LN-121, LN-332, LN-421, LN-511, LN-521 and matrigel (Table 2). Short protocols were used to differentiate cells into S5 EP cells, and the expression of NKX6.1 and NEUROD1 was analyzed at four days into stage 5 (Fig. 5A). The results showed that LN-332, LN-521, and LN-511 supported differentiation into S5 NKX6.1+ / NEUROD1+EP cells more effectively than Matrigel ( Figure 5A , Figure 5B ). Notably, when the short protocol was used instead of the long protocol, LN-511 was as effective as LN-521 in enhancing S5 EP differentiation of HS980 and H1 cells (Figure 5A, Figure 5B ). H1 cells were also differentiated on plates coated with 10 μg / mL fibronectin (FN, Sigma; F0895) or vitronectin (VTN, Sigma; 5051). The results showed that FN and VTN were able to support S5 EP differentiation ( Figure 5B ).

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

[0441] In summary, the results demonstrate that the short protocol is highly effective on a variety of xenobiotic-free, defined substrates.

[0442] Example 4

[0443] In this example, it was investigated whether S4 or S5 cells are better suited for forming islets in vitro.

[0444] Purified S4 PP cells have been used to generate islets in previous reports (Cogger et al., 2017; Ameri et al., 2017; Kelly et al., 2011). To determine which cell population is more suitable for islet formation, HS980 cells were dissociated into single cells at the end of S4 or at 4 days into S5. The cells were then kept in suspension to generate islet aggregates ( Fig. 6A ). The expression of key endocrine markers INS and GCG, the number of islet-like aggregates, and the number of islet cells were analyzed at the end of S6.

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

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

[0447] Taken together, these data indicate that S5 EP cells, but not S4 PP cells, efficiently form islet-like aggregates in 3D suspension and that a duration of 4 days under S5 is sufficient for aggregate formation in suspension.

[0448] Example 5

[0449] In this example, the effect of cell culture in 3D single cell suspension was investigated.

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

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

[0452] To determine whether ROCK inhibitors are 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 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 ( Fig.7D ).

[0454] Taken together, these results suggest that S5 EP cells are selectively enriched due to the formation of 3D aggregates from single cells, which is consistent with previous studies on fetal pancreatic development (Gouzi et al., 2017).

[0455] Example 6

[0456] In this example, a short pancreatic differentiation protocol was compared to a long pancreatic differentiation protocol by comparing the percentages of pancreatic progenitor cells (PP) and endocrine progenitor cells (EP) obtained.

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

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

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

[0460] result : The results showed that the short regimen induced INS + The percentage of monohormonal β cells was higher than that of the long regimen, but GCG + The percentage of monohormone α cells was lower than that of the long protocol ( Figure 8C A similar effect of S3+S4 duration was observed in H1 cells ( Figure 8C The short protocol also generated greater numbers of islet-like aggregates and islet cells than the long protocol ( Fig.8D Results from in vitro glucose stimulation experiments confirmed that aggregates at the end of S6 were fully functional and could increase insulin secretion 10-fold in response to high glucose concentrations, but aggregates generated using the short protocol secreted more insulin at high glucose levels ( Fig.8D Altogether, these results suggest that a short S3+S4 duration has a strong positive effect on islet maturation during S6.

[0461] The expression of stage-specific markers was also analyzed using immunocytochemistry (ICC) (Figure 9). The long protocol induced a very dense layer at S4, which mainly contained PDX1 + / NKX6.1 + PP cells ( Fig. 9A ). In contrast, the short regimen induced less S4PDX1 + / NKX6.1 +PP cells, some cells still maintain NKX6.1 - ( Fig. 9A ). NKX6.1 was observed at S4 in both protocols. - Premature induction of NEUROD1 in cells ( Fig. 9A ). Further differentiation into S5 EP cells showed the opposite effect. The short protocol generated more S5 NKX6.1 than the long protocol + / NEUROD + EP cells ( Fig. 9B The results also showed that aggregates generated using the short and long protocols had different ratios between β cells and α cells ( Fig. 9C The short regimen induced more insulin c-peptide (CPEP)-positive β cells, and the long regimen induced more GCG + α cells ( Fig. 9C Some somatostatin (SST)-positive δ cells were also observed ( Fig. 9C ). In summary, the results of ICC support the conclusions of the above flow cytometry analysis.

[0462] Example 7

[0463] In this example, the effects of spontaneous aggregation of S5 cells into 3D cultures compared to forced aggregation using AggreWell 400 plates and 96-well plates on the production of single hormonal pancreatic β cells were investigated.

[0464] For spontaneous aggregation, H1 cells were dissociated into single cells at S5 and maintained in 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 investigated by flow cytometry one day after 3D aggregation and again at the end of S6.

[0465] result: The results showed that the aggregates generated in the microwells mainly contained NKX6.1 + / NEUROD1 + / Ki-67 - EP cells, because aggregates spontaneously form in 3D suspension culture ( Fig. 10A , Fig. 10B ). Proliferative Ki-67 + Cells are removed from aggregates in microwells as in 3D suspensions ( Fig. 10A , Fig. 10B ). At the end of S6, the aggregates generated in the microwells contained INS similar to the spontaneously generated aggregates. + β cells and GCG + The percentage of α cells ( Fig. 10C). Only a very small amount of Ki-67 remains in these aggregates. + Proliferating cells ( Fig. 10C ).

[0466] Taken together, these results suggest that S5 EP cells can segregate from other non-endocrine cell types and then undergo spontaneous self-aggregation.

[0467] Example 8

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

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

[0470] Results: The results showed that in all three hESC lines, single hormone INS + β cells and GCG + The percentage of α cells was comparable and the aggregates were functional and could increase insulin secretion in a similar manner in response to high glucose concentrations ( Fig. 12A The results also showed that the human iPSC line could be successfully differentiated into S5NKX6.1 + / NEUROD1 + EP cells, and subsequently differentiated into S6 monohormone INs + β cells and GCG + α cells ( Fig. 12B ).

[0471] Altogether, these results show relatively low cell line variability between multiple human ESC and iPSC lines differentiated using the short pancreatic differentiation protocol.

[0472] Example 9

[0473] In this example, a 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. Fig.13A ).

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

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

[0476] Altogether, these results indicate that islets generated by the short pancreatic differentiation protocol contain primarily mature monohormonal β and α cells, whereas islets generated by two recently published protocols contain immature multihormonal or non-endocrine cells.

[0477] Example 10

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

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

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

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

[0482] References:

[0483] 1. Rodin, S., et al. Clonal culturing of human embryonic stem cells on laminin-521 / E-cadherin matrix in defined and xeno-free environment. Nat Commun, 2014. 5: 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. Nat Commun, 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. Nat Commun, 2017. 8(1): 331.

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

[0493] 11. Gouzi, M., et al., Neurogenin3 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 Rep, 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. 2016 Nov;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) providing a population comprising endocrine progenitor cells (EP), such as a population of EP cells, such as EP cells characterized by expression of NEUROD1; such as EP cells characterized by expression of NKX6.1 and NEUROD1;

[0500] ii) providing a single cell suspension of the population of EP cells;

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

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

[0503] v) thereby generating islet-like cell aggregates comprising a single hormone β cell,

[0504] The islet-like cell aggregates comprise at least about 25%, such as at least about 30%, such as at least about 35%, such as at least about 40%, such as at least about 45% of single hormone beta cells.

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

[0506] 3. A method for generating in vitro islet-like cell aggregates as described in item 2, wherein the 2D substrate comprises one or more components selected from the group consisting of: laminin (LN) and its fragments, vitronectin and its fragments, fibronectin and its fragments, collagen and its fragments, gelatin and its fragments, functionalized silk (FN silk), and matrix glue TM, such as one or more components selected from the group consisting of: laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and matrigel TM .

[0507] 4. A method for generating in vitro islet-like cell aggregates as described in item 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. A 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, a group consisting of: LN-521, LN-511, LN-332, LN-421 and LN-121, for example, a group consisting of: LN-521, LN-511 and LN-332; for example, a 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. A method for generating in vitro islet-like cell aggregates as described in item 3 or 4, wherein the laminin (LN) and fragments include 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 -511 E8 fragment, LN-521 E8 fragment, LN-332 E8 fragment, as well as LN-421 E8 fragment and 121 E8 fragment; for example, the group consisting of: LN-511 E8 fragment, LN-521 E8 fragment and LN-332 E8 fragment; for example, the group consisting of: LN-511 E8 fragment and LN-521 E8 fragment; for example, LN-511 E8 fragment or LN-521 E8 fragment.

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

[0511] or

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

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

[0514] or

[0515] wherein step ii) of providing a single cell suspension of a population of EP cells is performed when more than about 15%, such as more than about 20%, such as more than about 25%, such as more than about 30%, such as more than about 35%, such as more than about 40%, such as more than about 45%, such as more than about 50% of the total cell population are EP cells characterized by expression of NKX6.1 and NEUROD1.

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

[0517] 10. A method for generating in vitro islet-like cell aggregates as described in any of items 1-9, wherein the islet-like cell aggregates generated in step v) contain at most about 5%, for example at most about 4%, for example at most about 3%, for example at most about 2%, for example at most about 1%, for example at most about 0.5%, for example at most about 0.1% of proliferating cells, for example proliferating cells expressing Ki-67.

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

[0519] 12. The method for producing in vitro islet-like cell aggregates according to any one of items 1 to 11, wherein step ii) is performed before culturing these cells in a culture medium allowing differentiation into pancreatic monohormonal β cells.

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

[0521] 14. The method for producing in vitro islet-like cell aggregates according to any one of items 1 to 13, wherein the step ii) is performed within 1 to 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 to 14, wherein the formation of the 3D structure in step iii) is 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 to 14, wherein the forming of the 3D structure in step iii) is forced or assisted forming of the 3D structure.

[0524] 17. The method for producing islet-like cell aggregates in vitro as described in any one of items 1-16, wherein step iv) comprises culturing the EPs 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 single hormone β cell generated in step v) has the ability to express insulin, for example, wherein the single hormone β cell generated in step v) expresses insulin.

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

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

[0528] 21. A 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) contain at least 40% of monohormone β cells; 7%-25% of monohormone α cells and less than 2% of proliferating cells.

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

[0530] a) providing a cell population of posterior foregut cells, the posterior foregut cells being, for example, posterior foregut cells characterized by expression of PDX1;

[0531] b) culturing the cell population of posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for no longer than about 78 hours, such as no longer than about 72 hours; and

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

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

[0534] a-1) providing a cell population of primitive intestinal tube cells, wherein the primitive intestinal tube cells are, for example, primitive intestinal tube cells characterized by expressing HNF1β and / or HNF4α;

[0535] b-1) culturing the cell population of primitive gut tube cells under conditions that allow differentiation into posterior foregut cells for no longer than about 54 hours; and

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

[0537] 24. A 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 before steps a)-c).

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

[0539] a-1) providing a cell population of primitive intestinal tube cells, wherein the primitive intestinal tube cells are, for example, primitive intestinal tube cells characterized by expressing HNF1β and / or HNF4α;

[0540] b-1) culturing the cell population of primitive gut tube cells under conditions that allow differentiation into posterior foregut cells for no longer than about 54 hours;

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

[0542] a) providing a cell population of posterior foregut cells generated in step c-1), wherein the posterior foregut cells are, for example, posterior foregut cells characterized by expressing PDX1;

[0543] b) culturing the cell population of posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for no longer than about 78 hours, such as no longer than about 72 hours; and

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

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

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

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

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

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

[0550] a-1) providing a cell population of primitive intestinal tube cells, wherein the primitive intestinal tube cells are, for example, primitive intestinal tube cells characterized by expressing HNF1β and / or HNF4α;

[0551] b-1) culturing the cell population of primitive gut tube cells under conditions that allow differentiation into posterior foregut cells for no longer than about 54 hours;

[0552] c-1) thereby generating a population of posterior foregut cells, the posterior foregut cells being, for example, posterior foregut cells characterized by expression of PDX1;

[0553] a) providing a cell population of posterior foregut cells generated in step c-1), wherein the posterior foregut cells are, for example, posterior foregut cells characterized by expressing PDX1;

[0554] b) culturing the cell population of posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for no longer than about 78 hours, such as no longer than about 72 hours;

[0555] c) thereby generating a cell population of pancreatic progenitor cells, the pancreatic progenitor cells being, for example, pancreatic progenitor cells characterized by expressing both PDX1 and NKX6.1;

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

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

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

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

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

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

[0562] 31. A method for generating in vitro islet-like cell aggregates as described in any of items 22-30, wherein in step c), the cell population of pancreatic progenitor cells, such as 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, such as a marker 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. A method for generating in vitro islet-like cell aggregates as described in any of items 32-33, wherein the effective amount of EGF or its derivative or agonist 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 of items 32-34, wherein the effective amount of NIC or its derivative or agonist is about 5mM to 20mM, for example, about 5mM to 15mM, for example, about 8mM to 12mM, for example, about 10mM.

[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 adherently on a 2D substrate.

[0569] 38. A 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, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and matrix glue TM , such as one or more components selected from the group consisting of: laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and matrigel TM .

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

[0571] LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, LN-121 and fragments thereof, and LN-111 and fragments thereof; for example, the group consisting of LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof;

[0572] For example, the group consisting of: LN-521 and fragments thereof, LN-511 and fragments thereof, and LN-332 and fragments thereof;

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

[0574] 40. A method for generating in vitro islet-like cell aggregates as described in any 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, a group consisting of: LN-521, LN-511, LN-332, LN-421 and LN-121, for example, a group consisting of: LN-521, LN-511 and LN-332; for example, a 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 include 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-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. Group: E8 fragment of LN-511, E8 fragment of LN-521, E8 fragment of LN-332, and E8 fragment of LN-421 and E8 fragment of 121; for example, the group consisting of: E8 fragment of LN-511, E8 fragment of LN-521 and E8 fragment of LN-332; for example, the group consisting of: E8 fragment of LN-511 and E8 fragment of LN-521; for example, E8 fragment of LN-511 or E8 fragment of LN-521.

[0576] 42. A method for generating in vitro islet-like cell aggregates as described in any of items 22-41, wherein at least about 60%, such as at least about 55%, such as at least about 70%, such as at least about 75%, such as at least about 80% of the posterior foregut cells in a), such as the posterior foregut 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. A method for generating in vitro islet-like cell aggregates as described in any of items 22-42, wherein in step c), at least about 75%, such as at least about 80%, such as about 80 to 85%, such as about 80 to 90% of the total cell population express 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), no more than about 10% of the total cell population expresses NEUROD1.

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

[0580] 46. ​​A 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, such as no more than about 50 hours, such as no more than about 48 hours.

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

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

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

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

[0585] 51. A method for generating in vitro islet-like cell aggregates as described in any of items 26-50, wherein the population of endocrine progenitor cells, such as 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 of items 26-51, wherein in step b+1), the cell population of pancreatic progenitor cells is cultured for about 3 to 5 days, for example, about 3 to 4 days or about 4 to 5 days, for example, about 4 days or for example, about 5 days.

[0587] 53. A 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 culture medium comprising 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, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk), and matrix glue TM , such as one or more components selected from the group consisting of: laminin (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, and matrigel TM 56. The method for generating in vitro islet-like cell aggregates according to any one of items 26-55, wherein the laminin (LN) and fragments thereof are selected from the group consisting of: LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, LN-121 and fragments thereof, and LN-111 and fragments thereof;

[0590] For example, the group consisting of: LN-521 and fragments thereof, LN-511 and fragments thereof, LN-332 and fragments thereof, LN-421 and fragments thereof, and LN-121 and fragments thereof;

[0591] For example, the group consisting of: LN-521 and fragments thereof, LN-511 and fragments thereof, and LN-332 and fragments thereof;

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

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

[0594] 57. A method for generating in vitro islet-like cell aggregates as described in any 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, a group consisting of: LN-521, LN-511, LN-332, LN-421 and LN-121, for example, a group consisting of: LN-521, LN-511 and LN-332; for example, a 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 include E8 fragments of laminin, such as E8 fragments of laminin selected from the group consisting of: E8 fragments of LN-511, E8 fragments of LN-521, E8 fragments of LN-332, E8 fragments of LN-421, E8 fragments of LN-121, and E8 fragments of LN-111; for example, composed of Group: E8 fragment of LN-511, E8 fragment of LN-521, E8 fragment of LN-332, and E8 fragment of LN-421 and E8 fragment of 121; for example, the group consisting of: E8 fragment of LN-511, E8 fragment of LN-521 and E8 fragment of LN-332; for example, the group consisting of: E8 fragment of LN-511 and E8 fragment of LN-521; for example, E8 fragment of LN-511 or E8 fragment of LN-521.

[0596] 59. A method for generating in vitro islet-like cell aggregates as described in any of items 26-58, wherein in step c+1), more than about 30%, for example more than about 40%, for example more than about 45%, for example more than about 50% of the total cell population are endocrine progenitor cells, for example, 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 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 a corresponding method, in which step b) is to culture the cell population of the 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 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, in which step b) is to culture the cell population of the posterior 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, further comprising culturing the endocrine progenitor cells under 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, adherently cultured 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 of items 26-63, wherein the cells are not transferred from culture on a 2D substrate to culture on a 3D substrate before expressing characteristic markers of endocrine progenitor cells, for example, wherein the cells are not transferred from 2D substrate adherent culture to culture on a 3D substrate before expressing characteristic markers of endocrine progenitor cells.

[0602] 65. A 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. A method for generating in vitro islet-like cell aggregates as described in any of items 22-65, wherein the cell population provided in step a-1), a) or a+1) is derived from a culture of pluripotent stem cells, such as a culture of induced pluripotent stem cells or a culture of embryonic stem cells, such as a culture of human induced pluripotent stem cells or a culture of human embryonic stem cells.

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

[0605] 68. A 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 of items 65-68, 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, 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. 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 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, further comprising cryopreserving EP cells before step i).

[0609] 72. An isolated islet-like cell aggregate, which can be obtained by the method as described in any one of the preceding items.

[0610] 73. An isolated population of islet-like cell aggregates, which can be obtained by the method described in any one of the preceding items.

[0611] 74. An isolated islet cell aggregate as described in item 72 or an isolated islet cell aggregate population as described in item 73, wherein the islet cell aggregates contain 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 are single hormone beta cells, for example, single hormone beta cells characterized by expressing insulin.

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

[0613] 76. An isolated islet-like cell aggregate as described in any one of items 72-75 or an isolated islet-like cell aggregate population as described in any one of items 73-75, wherein the cells constituting the islet-like aggregates have not been subjected to sorting for the desired phenotype based on marker expression, wherein the cells have not been subjected to sorting before forming the 3D structure in step iii).

[0614] 77. An isolated islet-like cell aggregate as described in any one of items 72-76 or an isolated islet-like cell aggregate population as described in any one of items 73-76, wherein prior to formation, the cells constituting the islet-like cell aggregates have not been subjected to FACS-based sorting for the desired phenotype, wherein the cells have not been subjected to sorting before forming the 3D structure in step iii).

[0615] 78. An isolated islet cell aggregate as described in any one of items 72-77 or an isolated islet cell aggregate population as described in any one of items 73-77, wherein the islet cell aggregates contain 7% to 25%, for example 7% to 20%, 10% to 20%, for example 15% to 20%, for example approximately 20% of monohormonal alpha cells.

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

[0617] 80. A cell obtained from at least one isolated islet as defined in any one of items 72 to 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 an isolated islet-like cell aggregate population as described in any one of items 73-79 or a cell as described in item 80, for use in therapy.

[0619] 82. An isolated islet cell aggregate as described in any one of items 72-79 and 81, or an isolated islet cell aggregate population as described in any one of items 73-79 and 81, or a cell as described in item 80 or 81, for use in treating, preventing and / or ameliorating diabetes, such as type 1 or type 2 diabetes.

[0620] 83. An isolated islet-like cell aggregate as described in any of items 72-79 or a population of isolated islet-like cell aggregates as described in any of items 73-79, for use in treatment in therapy, wherein the islet-like cell aggregates or cells have been generated by a method as described in any of items 1-71.

[0621] 84. An isolated islet cell aggregate as described in any one of items 72-79 and 81, or an isolated islet cell aggregate population as described in any one of items 73-79 and 81, or a cell as described in item 80 or 81, for use in treating, preventing and / or ameliorating diabetes, such as type 1 or type 2 diabetes, wherein the islet cell aggregates or cells have been generated by a method as 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 an isolated islet-like cell aggregate population as described in any one of items 73-79 and 81 or a cell as described in item 80 or 81 for use in treatment in therapy,

[0623] The use comprises the following steps:

[0624] Producing isolated islet-like cell aggregates according to the method of any one of items 1-71; and

[0625] Administering a therapeutically effective amount of the islet-like cell aggregates to a patient

[0626] or

[0627] The use comprises the following steps:

[0628] Producing isolated islet-like cell aggregates according to the method of any one of items 1-71;

[0629] Dissociation of these islet-like cell aggregates, and

[0630] A therapeutically effective amount of the dissociated pancreatic islet cells is administered to a patient.

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

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

[0633] or

[0634] The use comprises the following steps:

[0635] Producing isolated islet-like cell aggregates according to the method of any one of items 1-71;

[0636] Dissociation of these islet-like cell aggregates, and

[0637] A therapeutically effective amount of the dissociated pancreatic islet cells is administered to a patient.

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

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

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

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

[0642] 91. Use of the isolated islet-like cell aggregates as described in any one of items 72-79 and 81, or the isolated islet-like cell aggregate populations as described in any one of items 73-79 and 81, or the cells as described in item 80 or 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 the method of 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 method for in vitro drug screening according to item 92, comprising the following steps: generating isolated islet-like aggregates according to the method according to any one of items 1-71;

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

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

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

[0649] 95. A method of treating a patient in need thereof, such as a method of treating diabetes in a patient in need thereof, 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] administering a therapeutically effective amount of the islet-like cell aggregates to the patient

[0651] or

[0652] Producing isolated islet-like cell aggregates according to the method of any one of items 1-71;

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

[0654] A therapeutically effective amount of the dissociated pancreatic islet cells is administered to the patient.

[0655] 96. A method for treating diabetes in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of isolated islet cell aggregates as described in any one of items 72-79 and 81 or a population of isolated islet 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 a patient in need thereof as described in item 95 or 96, wherein the patient suffers from type 1 or type 2 diabetes.

[0657] 98. A method for treating diabetes in a patient in need thereof 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 as described in any one of items 72-79 and 81, or the isolated islet-like cell aggregate populations as described in any one of items 73-79 and 81, or the cells as described in item 80 or 81 for the manufacture of a medicament for treating diabetes in a patient in need thereof.

[0659] 100. The use as described in item 99, wherein the manufacture of the medicament comprises generating islet-like aggregates by a method as described in 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) providing a cell population of primitive intestinal tube cells, wherein the primitive intestinal tube cells are, for example, primitive intestinal tube cells characterized by expressing HNF1β and / or HNF4α;

[0662] b-1) culturing the cell population of primitive gut tube cells under conditions that allow differentiation into posterior foregut cells for no longer than about 54 hours;

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

[0664] a) providing a cell population of posterior foregut cells generated in step c-1), wherein the posterior foregut cells are, for example, posterior foregut cells characterized by expressing PDX1;

[0665] b) culturing the cell population of posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for no longer than about 78 hours, such as no longer than about 72 hours;

[0666] c) thereby generating a cell population of pancreatic progenitor cells, the pancreatic progenitor cells being, for example, pancreatic progenitor cells characterized by expressing both PDX1 and NKX6.1;

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

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

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

[0670] i) providing a population of endocrine progenitor cells (EP) generated in step c+1), wherein the endocrine progenitor cells are, for example, EP cells characterized by expressing NEUROD1; for example, EP cells characterized by expressing NKX6.1 and NEUROD1;

[0671] ii) providing a single cell suspension of the population of EP cells;

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

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

[0674] v) thereby generating islet-like cell aggregates,

[0675] wherein the islet-like cell aggregates comprise at least about 25% of single hormone β cells; or

[0676] v) thereby generating a population of islet-like cell aggregates comprising a single hormone β cell,

[0677] wherein the population comprises islet-like cell aggregates comprising at least about 25% single hormonal beta cells.

Claims

1. A method for generating in vitro islet-like cell aggregates, the method comprising the following steps: i) providing a population comprising endocrine progenitor cells (EP), such as a population of EP cells, such as EP cells characterized by expression of NEUROD1; such as EP cells characterized by expression of NKX6.1 and NEUROD1; ii) providing a single cell suspension of the population of EP cells; iii) allowing a population of said EP cells in a single cell suspension to form a 3D structure; iv) culturing the population of EP cells in the form of a 3D structure under 3D culture conditions that allow differentiation into pancreatic monohormone β cells to provide islet-like cell aggregates; and v) thereby generating islet-like cell aggregates comprising a single hormone β cell, The islet-like cell aggregates contain at least about 25% of single hormone β cells.

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

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

4. A 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; 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.

5. A method for generating in vitro islet-like cell aggregates as described in claim 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, a group consisting of: LN-521, LN-511, LN-332, LN-421 and LN-121, for example, a group consisting of: LN-521, LN-511 and LN-332; for example, a 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.

6. The method for generating in vitro islet-like cell aggregates as described in any one of claims 1 to 5, wherein step ii) of providing a single cell suspension of the EP cell population is performed when more than about 15%, such as more than about 20%, such as more than about 25%, such as more than about 30%, such as more than about 35%, such as more than about 40%, such as more than about 45%, such as more than about 50% of the total cell population are EP cells characterized by expressing NEUROD1.

7. The method for producing in vitro islet-like cell aggregates according to any one of claims 1 to 6, wherein the islet-like cell aggregates contain, for example, at least about 40% of single hormone β cells.

8. A method for generating in vitro islet-like cell aggregates as described in any one of claims 1-7, wherein the islet-like cell aggregates generated in step v) contain approximately 7% to 25%, for example 7% to 20%, 10% to 20%, for example 15% to 20%, for example approximately 20% of monohormonal α cells.

9. A method for generating in vitro islet-like cell aggregates as described in any one of claims 1 to 8, wherein the islet-like cell aggregates generated in step v) contain at most about 5%, such as at most about 4%, such as at most about 3%, such as at most about 2%, such as at most about 1%, such as at most about 0.5%, such as at most about 0.1% of proliferating cells, such as proliferating cells expressing Ki-67.

10. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 9, wherein the islet-like cell aggregates generated in step v) are scored after 38 to 42 days of culture.

11. The method for producing in vitro islet-like cell aggregates according to any one of claims 1 to 10, wherein step ii) is performed before culturing these cells in a culture medium allowing differentiation into pancreatic monohormonal β cells. 12 . The method for generating in vitro islet-like cell aggregates according to claim 1 , wherein the formation of the 3D structure in step iii) is spontaneous formation of the 3D structure.

13. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 11, wherein the forming of the 3D structure in step iii) is forced or assisted forming of the 3D structure.

14. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 13, wherein step iv) comprises culturing the EPs 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.

15. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 14, wherein the single hormone β cell generated in step v) has the ability to express insulin, for example, wherein the single hormone β cell generated in step v) expresses insulin.

16. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 15, wherein the single hormone β cells generated in step v) have the ability to express C-peptide after glucose stimulation, for example, wherein the single hormone β cells generated in step v) express C-peptide after glucose stimulation.

17. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 16, wherein the monohormonal β cells generated in step v) do not express glucagon and / or somatostatin.

18. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 17, wherein the islet-like cell aggregates generated in step v) comprise at least 40% of monohormone β cells; 7%-25% of monohormone α cells and less than 2% of proliferating cells.

19. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 18, wherein before step i), the method comprises steps a-1) to c-1) and steps a) and c) a-1) providing a cell population of primitive intestinal tube cells, wherein the primitive intestinal tube cells are, for example, primitive intestinal tube cells characterized by expressing HNF1β and / or HNF4α; b-1) culturing the cell population of primitive gut tube cells under conditions that allow differentiation into posterior foregut cells for no longer than about 54 hours; c-1) thereby generating a population of posterior foregut cells, the posterior foregut cells being, for example, posterior foregut cells characterized by expression of PDX1; a) providing a cell population of posterior foregut cells generated in step c-1), wherein the posterior foregut cells are, for example, posterior foregut cells characterized by expressing PDX1; b) culturing the cell population of posterior foregut cells under conditions that allow differentiation into pancreatic progenitor cells for no longer than about 78 hours, such as no longer than about 72 hours; and c) thereby generating a cell population of pancreatic progenitor cells, which are pancreatic progenitor cells characterized, for example, by the expression of both PDX1 and NKX6.

1.

20. The method for generating in vitro islet-like cell aggregates according to claim 19, further comprising steps a+1)-c+1) after steps a)-c): a+1) providing a cell population of pancreatic progenitor cells generated in step c; b+1) culturing the cell population of pancreatic progenitor cells under conditions that allow differentiation into endocrine progenitor cells; and c+1) thereby generating a population of endocrine progenitor cells, such as endocrine progenitor cells characterized by expression of NEUROD1, such as endocrine progenitor cells characterized by expression of NKX6.1 and NEUROD1.

21. A method for generating in vitro islet-like cell aggregates as described in any one of claims 19-20, wherein in step b), the cell population is cultured for a period of about 42 to 78 hours, such as a period of about 44 to 76 hours, such as a period of about 46 to 74 hours, such as a period of about 48 to 72 hours.

22. The method for generating in vitro islet-like cell aggregates according to any one of claims 19 to 21, wherein the cells are cultured on a 2D substrate, for example wherein the cells are cultured adherently on a 2D substrate.

23. The method for generating in vitro islet-like cell aggregates according to claim 22, wherein the 2D substrate is as defined in any one of claims 3-5.

24. A method for generating in vitro islet-like cell aggregates as described in any one of claims 19-23, wherein in step b-1), the cell population is cultured for a period of about 18 to 54 hours, for example, a period of about 20 to 52 hours, for example, a period of about 22 to 50 hours, for example, a period of about 24 to 48 hours.

25. A method for generating in vitro islet-like cell aggregates as described in any one of claims 20-24, wherein in step b+1), the cell population of pancreatic progenitor cells 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 or for example about 5 days.

26. The method for generating in vitro islet-like cell aggregates according to any one of claims 20 to 25, wherein the cells are cultured on a 2D substrate, such as adherently cultured on a 2D substrate during steps a-1) to c+1).

27. A method for generating in vitro islet-like cell aggregates as described in any one of claims 1-26, wherein the cells are not transferred from culture on a 2D substrate, such as from adherent culture on a 2D substrate, to culture on a 3D substrate before expressing characteristic markers of endocrine progenitor cells.

28. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 27, 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.

29. The method for generating in vitro islet-like cell aggregates as claimed in claim 28, wherein the cell population is a mammalian cell population, such as a human cell population.

30. The method for generating in vitro islet-like cell aggregates as claimed in any one of claims 28-29, wherein the cell population is derived from a human embryonic stem cell line selected from the group of embryonic stem cell lines consisting of: HS980 cells, H1 cells and H9 cells.

31. The method for generating in vitro islet-like cell aggregates as described in any one of claims 28-29, wherein the cell population is derived from a human induced pluripotent stem cell population.

32. The method for generating in vitro islet-like cell aggregates according to any one of claims 1 to 31, further comprising cryopreserving EP cells prior to step i).

33. An isolated islet-like cell aggregate obtainable by the method of any one of the preceding claims.

34. An isolated population of islet-like cell aggregates obtainable by the method of any one of the preceding claims.

35. An isolated islet cell aggregate as described in claim 33 or an isolated islet cell aggregate population as described in claim 34, wherein the islet cell aggregates contain more than about 40%, such as about 40% to 70%, such as about 40% to 60%, such as about 40% to 50% of the total cell population are single hormone beta cells, such as single hormone beta cells characterized by expressing insulin.

36. An isolated islet-like cell aggregate as described in any one of claims 33-35 or an isolated islet-like cell aggregate population as described in any one of claims 34-35, wherein the cells constituting the islet-like cell aggregates have not been subjected to enrichment for a desired phenotype, for example, have not been subjected to enrichment by manual or automatic intervention, wherein the cells have not been subjected to enrichment before forming these 3D structures in step iii).

37. An isolated islet-like cell aggregate as described in any one of claims 33-36 or an isolated islet-like cell aggregate population as described in any one of claims 34-35, wherein the cells constituting the islet-like aggregates have not been subjected to sorting for the desired phenotype based on marker expression, wherein the cells have not been subjected to sorting before forming these 3D structures in step iii).

38. An isolated islet-like cell aggregate as described in any one of claims 33-37 or an isolated islet-like cell aggregate population as described in any one of claims 34-37, wherein prior to formation, the cells constituting the islet-like cell aggregates have not been subjected to FACS-based sorting for a desired phenotype, wherein the cells have not been subjected to sorting before forming these 3D structures in step iii).

39. An isolated islet-like cell aggregate as described in any one of claims 33-38 or an isolated islet-like cell aggregate population as described in any one of claims 34-38, wherein the islet-like cell aggregates contain 7% to 25%, for example 7% to 20%, 10% to 20%, for example 15% to 20%, for example approximately 20% of single hormone alpha cells.

40. The isolated islet-like cell aggregates of any one of claims 33-39 or the isolated islet-like cell aggregate populations of any one of claims 34-39, wherein the islet-like cell aggregates comprise at least 40%, for example at least 50%, of single hormone β cells; approximately 15% to 20%, for example approximately 20%, of single hormone α cells, and less than approximately 2%, for example less than approximately 1% of proliferating cells.

41. A cell obtained from at least one isolated islet as defined in any one of claims 33 to 40, for example by dissociating said islet-like cell aggregates.

42. An isolated islet-like cell aggregate as described in any one of claims 33-40 or an isolated islet-like cell aggregate population as described in any one of claims 34-40 or a cell as described in claim 41 for use in therapy.

43. The isolated islet-like cell aggregates as described in any one of claims 33-40 or the isolated islet-like cell aggregate populations as described in any one of claims 34-40 or the cells as described in claim 41, for use in treating, preventing and / or ameliorating diabetes, such as type 1 or type 2 diabetes.

44. An isolated islet-like cell aggregate as described in any one of claims 33-40 or an isolated islet-like cell aggregate population as described in any one of claims 34-40 or a cell as described in claim 41 for use according to any one of claims 42-43, The use of The following steps are involved: Producing isolated islet-like cell aggregates according to the method of any one of claims 1-32; and A therapeutically effective amount of the islet-like cells or cell aggregates is administered to a patient.

45. The isolated islet-like cell aggregates as described in any one of claims 33-40 or the isolated islet-like cell aggregate populations as described in any one of claims 34-40 or the cells as described in claim 41, for use according to any one of claims 42-44, wherein the use comprises transplanting the islet-like cell aggregates or cells into a patient in need.

46. ​​A pharmaceutical composition comprising 33-40 or the isolated islet-like cell aggregate population described in any one of claims 34-40 or the cells described in claim 41, and at least one pharmaceutically acceptable excipient or carrier.

47. A kit of parts comprising the isolated islet-like cell aggregates as described in any one of claims 33-40 or the isolated islet-like cell aggregate populations as described in any one of claims 34-40 or the cells as described in claim 41 or the pharmaceutical composition as described in claim 46, and a suitable loading substrate.

48. The kit of parts according to claim 47, wherein the suitable support substrate is a 3D substrate.

49. Use of the isolated islet-like cell aggregates as described in any one of claims 33 to 40, or the isolated islet-like cell aggregate populations as described in any one of claims 34 to 40, or the cells as described in claim 41 in drug screening, such as in vitro drug screening.

50. A method for in vitro drug screening, the method comprising the following steps: Producing isolated islet-like aggregates according to the method of any one of claims 1-32; as well as The islet-like cell aggregates are exposed to at least one candidate drug compound.

51. A method of treating a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the isolated islet-like cell aggregates described in any one of claims 33-40 or the isolated islet-like cell aggregate population described in any one of claims 34-40 or the cells described in claim 41.

52. A method of treating a patient in need thereof, such as a method of treating diabetes in a patient in need thereof, the method comprising the steps of: Producing isolated islet-like cell aggregates according to the method of any one of claims 1-32; as well as A therapeutically effective amount of the islet-like cell aggregates is administered to the patient.

53. Use of the isolated islet-like cell aggregates as described in any one of claims 33-40, or the isolated islet-like cell aggregate populations as described in any one of claims 34-40, or the cells as described in claim 41 for the manufacture of a medicament for treating diabetes in a patient in need thereof.

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