WO2025119299A1

WO2025119299A1 - Method for differentiating pancreatic beta cells

Info

Publication number: WO2025119299A1
Application number: PCT/CN2024/137256
Authority: WO
Inventors: Xiaofeng Wang; Huijuan HUA; Yong Jiang; Yue PU
Original assignee: Hangzhou Reprogenix Bioscience Inc
Current assignee: Hangzhou Reprogenix Bioscience Inc
Priority date: 2023-12-08
Filing date: 2024-12-06
Publication date: 2025-06-12
Anticipated expiration: 2026-06-08

Abstract

Provided is a method of differentiating human pluripotent stem cells (hPSCs) into mature pancreatic β cells, comprising contacting hPSC-derived, differentiated immature pancreatic β cells with a histone deacetylase (HDAC) inhibitor.

Description

METHOD FOR DIFFERENTIATING PANCREATIC BETA CELLS

FIELD OF THE INVENTION

The disclosure generally relates to a method of inducing cell differentiation as well as uses of the differentiated cells (hPSC-β cells) in cell therapy.

BACKGROUND OF THE INVENTION

How to generate a large amount of functionally mature cells in vitro is one of the most important scientific questions of regenerative medicine. Human pluripotent stem cells constitute a promising source of cells because of their robust differentiation potential and unlimited proliferation capacity. However, most human pluripotent stem cell-derived, terminally differentiated cells display a fetal-like immature phenotype and functionality, limiting their clinical application. Pluripotent stem cell differentiation has long benefited from developmental biology. But the poor accessibility of human donor organs at late stages of development has greatly hindered research on functional maturation after cell fate determination. Previous studies showed that, after transplantation, immature cells gradually acquired mature cell functionality. This maturation process provides an available alternative to study the functional maturation of human cells, which could enhance the overall understanding of late-stage development and facilitate the generation of mature cells from human pluripotent stem cells (hPSCs) .

Human pluripotent stem cell-derived islets can restore endogenous insulin secretion by replenishing pancreatic β cells, which are essential to maintain circulating glucose concentrations in a narrow physiological range by adjusting insulin output. However, a common dilemma of hPSC-derived β cells (hPSC-β cells) is their immature functionality, represented by gene expression level, intracellular Ca²⁺ oscillation, mitochondrial respiration, and insulin secretion ability, which has limited their efficacy in application. Therefore, methodology of generating mature hPSC-β cells and small molecules to improve hPSC-β cell functionality in vitro need to be elucidated.

SUMMARY OF THE INVENTION

The present disclosure provides a method of differentiating human pluripotent stem cells (hPSC) into mature pancreatic β cells, comprising contacting hPSC-derived, differentiated immature pancreatic β cells with a histone deacetylase (HDAC) inhibitor.

In some embodiments, said differentiated immature pancreatic β cells are pancreatic β cells presenting a fetal and neonatal islet state.

In some embodiments, said hPSC is human induced pluripotent stem cells (hiPSCs) and/or human embryonic stem cells (hESCs) .

In some embodiments, said method is an in vitro method.

In some embodiments, said method is capable of promoting the acquisition of functional maturity of said hPSC-derived, differentiated immature pancreatic β cells.

In some embodiments, said acquisition of functional maturity of pancreatic β cells comprises one or more of the functional characteristics described below: a. said functionally mature pancreatic βcells have a glucose-stimulated insulin release (GSIR) index that is increased threefold or more than threefold compared to said differentiated immature pancreatic β cells; b. said functionally mature pancreatic β cells undergo a decrease in ceramide accumulation compared to said differentiated immature pancreatic β cell; c. gene expression levels of MAFA, NFIC, UCN3 and/or IAPP in said functionally mature pancreatic β cells undergo upregulation compared to said differentiated immature pancreatic β-cells; d. calcium ion fluorescence signal of said functionally mature pancreatic β cells is enhanced compared to said differentially immature pancreatic β cells; e. the mitochondrial oxidative respiration function of said functionally mature pancreatic β cells is improved compared to said differentially immature pancreatic β cells; f. insulin intracellular proteins are more normally sheared, folded and processed in said functionally mature pancreatic β cells; g. number of mature insulin granules in said functionally mature pancreatic β cells is improved twice or more than twice compared to said differentiated immature pancreatic β cells; h. after hPSC-islets have been transplanted in vivo (in some certain embodiments, hPSC-islets are transplanted into diabetic mice) , diabetes reversal is more rapid in said functionally mature pancreatic β cells from said hPSC-islets.

In some embodiments, said HDAC inhibitor comprises Scriptaid, Crotonoside, TH34, and/or Sulforaphane.

In some embodiments, said HDAC inhibitor is capable of presenting selectivity for HDAC6, HDAC8, and HDAC10.

In some embodiments, said HDAC inhibitor comprises TH34.

In some embodiments, said method comprises providing an in vitro culture environment comprising said HDAC inhibitor, and culturing said differentiated immature pancreatic β cells in said in vitro culture environment.

In some embodiments, said in vitro culture environment is a culture medium.

In some embodiments, said HDAC inhibitor comprises TH34, and micromolar concentration of said TH34 within said culture medium is from about 2.0μM to about 20.0μM.

In some embodiments, said TH34 has a micromolar concentration of about 10μM.

The present disclosure provides a mature pancreatic β cell obtained by the method described herein.

The present disclosure provides a cell population of mature pancreatic β cells obtained by the method described herein.

The present disclosure provides a culture comprising said mature pancreatic β cells obtained by the method described herein.

The present disclosure provides use of the mature pancreatic β cell described herein, the cell population of mature pancreatic β cells described herein, and/or the culture described herein for promoting insulin secretion and/or achieving diabetes reversal.

The present disclosure provides use of the mature pancreatic β cell described herein, the cell population of mature pancreatic β cells described herein, and/or the culture described herein for alleviating or treating abnormal insulin secretion, abnormal glucose metabolism, or diabetes.

In some embodiments, said diabetes is type I diabetes.

In some embodiments, said use is via one or more of the in vitro achievements described below: a. glucose-stimulated insulin release (GSIR) index is increased threefold or more than threefold; b. ceramide accumulation is decreased; c. gene expression levels of MAFA, NFIC, UCN3 and/or IAPP undergo upregulation; d. calcium ion fluorescence signal is enhanced; e. the mitochondrial oxidative respiration function is improved; f. insulin intracellular proteins are more normally sheared, folded and processed; g. number of mature insulin granules is improved twice or more than twice; h. after hPSC-islets have been transplanted in vivo, diabetes reversal is more rapid.

The present disclosure provides an in vitro method of alleviating or treating abnormal insulin secretion, abnormal glucose metabolism, or diabetes, comprising administering an effective amount of the mature pancreatic β cell described herein, the cell population of mature pancreatic β cells described herein, and/or the culture described herein to a subject in need thereof.

The present disclosure provides a method in vitro for promoting insulin secretion and/or achieving diabetes reversal, comprising transplanting the mature pancreatic β cell described herein, the cell population of mature pancreatic β cells described herein, and/or the culture described herein into a host.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

Figure 1 illustrates Characterization of hPSC-islets at Stage 6 before transplantation. Figure 1A shows schematic diagram of differentiation and cryopreservation of hPSC-islets, differentiated following our previously published protocol and cryopreserved in single cells at Stage 6 Day 2. Figure 1B shows representative images of flow cytometry analyzing the proportions of βcells (NKX6.1⁺C-peptide⁺) , α cells (ARX⁺GCG⁺) , δ cells (HHEX⁺SST⁺) , and EC cells (LMX1A⁺TPH1⁺) in hPSC-islets at Stage 6. The column graph showed the results from 3 independent batches of hPSC-islets. Figure 1C shows qRT-PCR assessment of marker genes in human primary islets (n = 3) and hPSC-islets (n = 3 independent batches of hPSC-islets) at Stage 6. Data are presented as mean ± SEM.

Figure 2 illustrates small molecule screening identified TH34 and assessment of TH34-treated hPSC-islets. Figure 2A shows class of small molecules predicted by drug enrichment analysis. Figure 2B shows schematic diagram of small molecule screening and assessment of TH34-treated hPSC-islets. The cryopreserved hPSC-islets were reaggregated and cultured in the Aggrewell plate for 1 day and then transferred to the ultra-low adherent plate to further culture for another day. They were used for screening or functional assessment 2 days after thawing. The sample “before treatment” was collected 2 days after hPSC-islets recovery. The samples “Null” , “DMSO” and “TH34” were cultured for additional 7 days in different conditions, wherein “Null” represents sample without small molecule treatment, and “DMSO” represents sample with vehicle control, and “TH34” represents sample treated with 10 μM of TH34 molecule.

Figure 3 illustrates TH34 promoted functional maturation of hPSC-β cells in vitro. Figure 3A shows small molecule screening to identify candidate compounds increasing the index of GSIR, wherein primary hits exhibit GSIR index over 3. Figure 3B shows static GSIR test of hPSC-β cells under sequential glucose challenge or continuously increased glucose concentration (n = 3 independent batches of hPSC-islets) . Data are presented as mean ± SEM.

Figure 4 illustrates the characterization of hPSC-β cells after TH34 treatment. Figure 4A shows representative images of flow cytometry analyzing the proportions of β cells (NKX6.1⁺C-peptide⁺) , α cells (ARX⁺GCG⁺) , δ cells (HHEX⁺SST⁺) , EC cells (LMX1A⁺TPH1⁺) and double-positive cells (C-peptide⁺GCG⁺) in hPSC-islets before and after TH34 treatment. The column graph showed the results from 3 independent batches of hPSC-islets (right) . Figure 4B shows qRT-PCR assessment of mature β cells marker genes after TH34 treatment (n = 3 independent batches of hPSC-islets) . Figure 4C shows representative immunofluorescence staining of mature β cells marker after TH34 treatment (Scale bar, 25 μm) . Data are presented as mean ± SEM. Significance was indicated by the number of asterisks.

Figure 5 illustrates representative Ca²⁺ flux in hPSC-islets and human primary islets (n = 10 βcells) . Data are presented as mean ± SEM.

Figure 6 illustrates OCR of hPSC-islets and human primary islets in response to 2.8 mM glucose, 16.7 mM glucose, oligomycin (2 μM) , FCCP (1 μM) , and rotenone/antimycin A (R&A, 1 μM) (n = 3 independent batches of hPSC-islets) . Data are presented as mean ± SEM. Significance was indicated by the number of asterisks.

Figures 7A-7B illustrate hormone contents and proinsulin/insulin ratio of human primary islets and hPSC-islets cultured under different conditions (n = 4 independent batches of hPSC-islets) . Data are presented as mean ± SEM. Significance was indicated by the number of asterisks.

Figure 8 illustrates representative electron micrographs and quantitative assessment of insulin granules in hPSC-islets cultured under different conditions (Scale bars, 1 μm) . The percentage of mature insulin granules was calculated (n = 6 hPSC-β cells) . In detail, 6 hPSC-β cells were from one differentiation batch. For each hPSC-β cell, 90 ± 43 granules in a presentative field were counted. Data are presented as mean ± SEM. Significance was indicated by the number of asterisks.

Figure 9 illustrates TH34 treatment remodeled ceramide metabolism in hPSC-islets. Figure 9A shows measurement of intracellular ceramides (Cer) by LC-MS /MS (n = 3 independent batches of hPSC-islets) . Figure 9B shows qRT-PCR assessment of key genes in ER stress (n = 3 independent batches of hPSC-islets) . Figure 9C shows GSIR of hPSC-islets under different culture conditions (n = 4 independent batches of hPSC-islets) , wherein “ceramide” represents exogenous supplementation of ceramide (5 μM) . Figure 9D shows representative results of relative expression of genes involved in ceramide metabolism detected by qRT-PCR (n = 4 technical replicates) . Data are presented as mean ± SEM. Significance was indicated by the number of asterisks.

Figure 10 illustrates fasting blood glucose and body weight of diabetic mice after hPSC-islet transplantation, wherein n represents the number of mice for the indicated treatment. Normal fasting blood glucose level was indicated by the dotted line of the left plot. Data are presented as mean ± SEM. Significance was indicated by the number of asterisks.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
I. Definition

As used herein, the term “contacting” (i.e., contacting a pluripotent stem cell with a compound) is intended to include incubating the compound and the cell together in vitro (e.g., adding the compound to cells in culture) . In some embodiments, the term “contacting” is not intended to include the in vivo exposure of cells to the compounds as disclosed herein that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process) . The step of contacting a pluripotent stem cell with a compound as in the embodiments related to the production of pancreatic β cells can be conducted in any suitable manner. For example, the cells may be treated in adherent culture, or in suspension culture. It is understood that the cells contacted with a compound can also be simultaneously or subsequently contacted with another agent, such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further. Similarly, a pluripotent stem cell can be contacted with a compound and then with another compound. In some embodiments, the cell is contacted with a compound and the other components and the contact is temporal separated, and in some embodiments, a cell is contacted with a compound and the other components substantially simultaneously.

As used herein, the term “differentiation” or “differentiating” refers to the process by which an unspecialized ( “uncommitted” ) or less specialized cell acquires the features of a specialized cell, for example a nerve cell or a muscle cell. A differentiated cell is one that has taken on a more specialized ( “committed” ) position within the lineage of a cell. The term “committed” , when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and to what cells it can give rise. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.

As used herein, the term “differentiated (…) cells” refers to any primary cell that is not pluripotent in its natural form. That is, the term “differentiated cells” when used in reference to cells made by methods of this invention from pluripotent stem cells refer to cells having reduced potential to differentiate when compared to the parent pluripotent stem cells. The differentiated cells of this invention comprise cells that could differentiate further (i.e., they may not be terminally differentiated) . Without wishing to be bound by theory, during the course of normal ontogeny, pluripotent stem cells can differentiate into endodermal cell types capable of forming pancreatic cells. Early endocrine cells are pancreatic islet precursors. The precursors can then be further differentiated into hormon-producing cells (e.g., functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endodermal cells can also differentiate into other cells of endodermal origin, such as lung, liver, intestine, thymus, etc.

As used herein, the term “pluripotent” refers to a cell type that differentiates into more than one differentiated cell type under various conditions, preferably a cell type characteristic of all three germ cell layers (i.e., endoderm (e.g., gut tissue) , mesoderm (e.g., blood, muscle, and vessels) , and ectoderm (e.g., skin and nerve) ) . Although pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is a description of the ability to differentiate into cells of any of the three germ layers. It should be noted that simply clustering such cells does not in itself indicate that they are pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells, as that term is defined herein) also have the characteristic of long-term pass-ability without losing their ability to proliferate relative to the primary parental cells. The parent cells generally have the ability to divide only a limited number of times in culture.

As used herein, the term “stem cells” refers to cells that retain the ability to renew themselves through mitotic division and can differentiate into a range of specialized cell types. Two broad types of mammalian stem cells are embryonic stem (ES) cells, found in blastocysts, and adult stem cells, found in adult tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem and progenitor cells serve as the body's repair system, replenishing specialized cells and maintaining the normal turnover of regenerating organs, such as blood, skin or gastrointestinal tissues. Stem cells are also characterized by the ability to differentiate in vitro into functional cells of different cell lines of differentiation from several germ layers (endoderm, mesoderm and ectoderm) , as well as after transplantation, give rise to tissues originating from several germ layers and make a significant contribution to the formation of the majority if not all tissues after injection into blastocysts. According to the development potential, stem cells are classified as follows: (1) totipotent, i.e. capable of giving rise to all embryonic and extraembryonic cell types; (2) pluripotent, i.e. capable of giving rise to all embryonic cell types; (3) multipotent, i.e. capable of giving rise to a group of cell lines of differentiation within a particular tissue, organ or physiological system (for example, hematopoietic stem cells (HSC) can produce offspring such as HSC (self-renewal) , oligopotent precursors limited to blood cells, and all types of cells and cell elements (such as platelets) , which are normal components of the blood) ; (4) oligopotent, i.e. capable of giving rise to a more limited set of cell lines of differentiation than multipotent stem cells; and (5) unipotent, i.e. capable of giving rise to a single cell line of differentiation (for example, spermatogenic stem cells) .

As used herein, the term “embryonic stem cell” is used to refer to the pluripotent stem cells in the internal cell population of the blastocyst (U. S. Pat. No. 5, 843, 780; U. S. Pat. No. 6, 200, 806) checking. ) . Such cells can similarly be obtained from internal cell populations of blastocysts obtained from somatic cell nuclear transfer (e.g., U. S. Pat. Nos. 5, 945, 577; 5, 994, 619; U. S. Pat. 6, 235, 970) . Characteristics that distinguish embryonic stem cells define their phenotype. Thus, a cell has the phenotype of an embryonic stem cell if it has one or more of the unique characteristics of an embryonic stem cell. This allows the cell to be distinguished from other cells. Typical distinguishable features of embryonic stem cells include, but are not limited to, gene expression profile, proliferation potential, differentiation potential, karyotype, responsiveness to specific culture conditions, and the like.

As used herein, the term “beta cell (β cell) ” is a pancreatic endocrine cell capable of expressing insulin but not glucagon, somatostatin, ghrelin, and pancreatic polypeptides. In specific embodiments as described herein, said pancreatic endocrine cells are types of human pluripotent stem cell-derived islets (hPSC-islets) . Pancreatic endocrine cells expressing markers characteristic of β-cells are at least of insulin and the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, MAFA, and PAX6. It can be characterized by one. “Pancreatic endocrine cells” as used herein refer to cells capable of secretion at least one of the following hormones: insulin, glucagon, somatostatin, ghrelin, and pancreatic polypeptide. In addition to these hormones, markers characteristic of pancreatic endocrine cells including (but are not limited to) one or more of NeuroD1, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, and PAX6.

As used herein, the terms “mature” , “maturity” , etc. describe the final developmental stage of a cell during differentiation and development. In this regard, a cell that is “more mature” refers to cells that are at least one developmental stage next to the “less mature” cells. Furthermore, it should be understood that cells in adults have the highest level of maturity. The phrase “differentiating into mature cells” indicates that a higher level of maturation of differentiated cells is achieved during lineage specialization, and that the expression of “mature” genes refers to the expression of genes that are important for the function and phenotype of functional cells.

As used herein, the term “derived” or “derived from” “established from” or “differentiated from” when made in reference to any cell disclosed herein refers to a cell that was obtained from (e.g., isolated, purified, etc. ) a parent cell in a cell line, tissue (such as a dissociated embryo, or fluids using any manipulation, such as, without limitation, single cell isolation, cultured in vitro, treatment and/or mutagenesis using for example proteins, chemicals, radiation, infection with virus, transfection with DNA sequences, such as with a morphogen, etc., selection (such as by serial culture) of any cell that is contained in cultured parent cells. A derived cell can be selected from a mixed population by virtue of response to a growth factor, cytokine, selected progression of cytokine treatments, adhesiveness, lack of adhesiveness, sorting procedure, and the like.

As used herein, the term “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, test tubes and cell cultures. On the other hand, the term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neural tube formation, etc.

As used herein, the term “expression” refers to cellular processes, including, but not limited to, transcription, translation, folding, modification, and processing, as applicable, to produce RNA and proteins, and optionally to secrete proteins. means. Otherwise, products from “expression” include polypeptides obtained by translation of RNA transcribed from genes and mRNA transcribed from genes.

As used herein, the terms “selectivity” when applied to the inhibitors provided in the present disclosure refers to inhibitors that selectively act on the specific targets or the family thereof. Among all members of the family, they mainly inhibit specific targets, but have no or little inhibitory effect on other members of the family. In other words, target-selective inhibitors are mainly relative to other members of the family, and the possibility of inhibiting other targets outside the family cannot be exclusive. Target-selective inhibitors can inhibit one or more subtypes of the family.

As used herein, the terms “mature insulin granules” refers to storage compartments within pancreatic beta cells that store and release insulin. These granules are formed through a process known as insulin granule biogenesis, which involves the mobilization of granules from a larger reserve pool and the de novo generation of insulin secretory granule (ISG) . ISG are initially formed at the trans-Golgi network in which proinsulin alongside other ISG cargo are sorted into budding vesicles termed immature ISG. Within the immature ISG, proinsulin is cleaved by prohormone convertase enzymes to bioactive insulin and C-peptide, the former of which is then retained as the immature ISG condenses into the mature ISG. Concurrently, soluble C-peptide has been described to be released from the maturing granule in a constitutive-like manner. Once mature insulin granules are formed, insulin is complexed with zinc and condensed into a crystalline core within the granules. The mature insulin granules serve as the storage site for fully processed insulin, ready to be released in response to signaling events such as glucose levels or hormonal stimuli. These granules play a crucial role in regulating blood glucose levels by releasing insulin into the bloodstream when needed.

As used herein, the terms “cell culture medium” or “culture medium” or “medium” refers to a medium containing nutrients that maintain cell viability and support growth. It is a medium for Said cell culture medium contains, in appropriate combination, any of the following: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes and other ingredients such as peptide growth factors, etc. may be included. Cell culture media commonly used for particular cell types are known to those skilled in the art.

As used herein, the term “apopulation of cells” or “acell population” refers to a group of at least two cells. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells. The population may be a pure population comprising one cell type. Alternatively, the population may comprise more than one cell type, for example a mixed cell population. For example, the term “population” may refer to a cell culture of more than one cell having the same identification characteristic, or may refer to a culture of more than one cell type having different identification characteristics, for example, a population in one context may be a subpopulation in another context. The term “subpopulation” means a subset of a cell culture or population, when used to describe certain cell types within a cell culture or cell population. By “acell population of mature pancreatic β cells” as described herein, it means that said cell population mainly comprises the mature pancreatic β cells of the present disclosure, but not excluding the inclusion of other type of cells.

As used herein, the terms “diabetes” is defined by The World Health Organization as having a fasting plasma glucose level of 7.0 mmol/l (126 mg/dl) (whole blood 6.1 mmol/l or 110 mg/dl) or a 2-hour glucose level of 11.1 mmol/L. A diagnostic value of 200 mg/dL or higher is defined. Other values suggestive of or indicative of high risk for diabetes include: elevated arterial pressure, elevated plasma triglycerides, low HDL-cholesterol, central obesity, great body mass index, and/or microalbuminuria. The term diabetes encompasses all types of diabetes, such as type I, type II and type 1.5. The term “type I diabetes” is defined as the condition in which a subject has, in the presence of autoimmunity towards the pancreatic beta-cell or insulin, a fasting blood glucose or serum glucose concentration greater than 125 mg/dL (6.94 mmol/L) . If a glucose tolerance test is carried out, the blood sugar level of a diabetic will be in excess of 200 mg of glucose per dL (11.1 mmol/l) of plasma 2 hours after 75 g of glucose have been taken on an empty stomach, in the presence of autoimmunity towards the pancreatic beta cell or insulin. In a glucose tolerance test 75 g of glucose are administered orally to the patient being tested after 10-12 hours of fasting and the blood sugar level is recorded immediately before taking the glucose and 1 and 2 hours after taking it. The presence of autoimmunity towards the pancreatic beta-cell may be observed by detection of circulating islet cell autoantibodies [ “type 1A diabetes mellitus” ] , i.e., at least one of: GAD65 [glutamic acid decarboxylase-65] , ICA [islet-cell cytoplasm] , IA-2 [intracytoplasmic domain of the tyrosine phosphatase-like protein IA-2] , ZnT8 [zinc-transporter-8] or anti-insulin; or other signs of autoimmunity without the presence of typical circulating autoantibodies [type 1B diabetes] , i.e. as detected through pancreatic biopsy or imaging) . Typically, a genetic predisposition is present (e.g., HLA, INS VNTR and PTPN22) , but this is not always the case. This type of diabetes can be further classified as immune-mediated or idiopathic. It can affect children or adults but was traditionally termed “juvenile diabetes” because it represents a majority of the diabetes cases in children.

As used herein, the term “treating” or “treatment” refers to administering to a subject an effective amount of a composition, e.g. a composition comprising the cells of the present disclosure, so that the recipient has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. The term “treatment” includes prophylaxis. Treatment is “effective” if the progression of a disease is reduced or halted. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already diagnosed with a condition (e.g., diabetes) , as well as those likely to develop a condition due to genetic susceptibility or other factors such as weight, diet, and health.

As used herein, the terms “administering, ” “introducing” and “transplanting” are used interchangeably in the context of the placement of cells (e.g., insulin-producing cells or pancreatic β-cells or pancreatic β-like cells) of the invention into a subject, by a method or route which results in at least partial localization of the introduced cells at a desired site. The cells (e.g., insulin-producing cells or pancreatic β-cells or pancreatic β-like cells) can be implanted directly to the pancreas, or alternatively be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g. twenty-four hours, to a few days, to as long as several years. In some instances, the cells can also be administered at a non-pancreatic location, such as in the liver or subcutaneously, for example, in a capsule to maintain the implanted cells at the implant location and avoid migration of the implanted cells. By way of example and not limitation, administration (e.g., injection) of the composition may be by intravenous (i.v. ) injection, subcutaneous (s.c. ) injection, intradermal (i.d. ) injection, intraperitoneal (i.p. ) injection, or intramuscular (i.m. ) injection. One or more of these approaches may be used. Parenteral administration may be, for example, by bolus injection or by gradual infusion over time. Alternatively or additionally, administration may be by the oral route. In addition, it may also be administered by surgical deposition of a bolus or bolus of cells or by positioning of a medical device. In embodiments, a composition of the disclosure may comprise an engineered cell or host cell expressing a nucleic acid sequence, or a vector comprising at least one nucleic acid sequence described herein, in an amount effective to treat or prevent a proliferative disorder. The pharmaceutical composition may comprise a population of cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide) ; and a preservative.

As used herein, the term “effective amount” or “therapeutic amount” or an equivalent thereof refers to amount of relevant cells in a population of cells, e.g., insulin-producing cells or pancreatic β-cells or pancreatic β-like cells, or composition comprising insulin-producing cells or pancreatic β-cells or pancreatic β-like cells of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment. For example, an amount of a population of insulin-producing cells or pancreatic β-cells or pancreatic β-like cells administered to a subject that is sufficient to produce a statistically significant, measurable change in at least one symptom of Type 1, Type 1.5 or Type 2 diabetes, such as glycosylated hemoglobin level, fasting blood glucose level, hypoinsulinemia, etc. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.

The terms “subject” and “individual” are used interchangeably herein, and refer to an animal, for example, a human from whom cells can be obtained and/or to whom treatment, including prophylactic treatment, with the cells as described herein, is provided. For treatment of those infections, symptoms or disease conditions specific to a particular animal, e.g., human subject, the term “subject” refers to that particular animal. The term “subject” includes any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. “Non-human animal” and “non-human mammal” are used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. Advantageously, however, said subject is a mammal, such as a human or another mammal, such as a domestic mammal, such as a dog, cat, horse, etc. or a production mammal, such as a cow, sheep, pig, etc.

As used herein, the terms “comprising” refers to compositions, methods and their respective components essential to the invention, whether or not essential, is open to inclusion of unspecified components. On the other hand, the term “consisting of” refers to the compositions, methods, and each component thereof described herein, and is exclusive to any component not listed in that description of the embodiment.

As used in this specification and the appended claims, the singular forms “a” , “an” , and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “the method” includes one or more methods and/or steps of the type described herein, and this may be understood based on reading this disclosure etc. will be clear to those skilled in the art.
II. Differentiation Methods

The present disclosure provides an in vitro method of alleviating or treating abnormal insulin secretion, abnormal glucose metabolism, or diabetes, comprising administering an effective amount of the mature pancreatic β cell described herein, the cell population of mature pancreatic β cells described herein, and/or the culture described herein to a subject in need.

The present disclosure provides a method of promoting insulin secretion and/or achieving diabetes reversal, comprising transplanting the mature pancreatic β cell described herein, the cell population of mature pancreatic β cells described herein, and/or the culture described herein into a host.

Sources of pluripotent stem cells and differentiated immature pancreatic β cells

Any pluripotent stem cells as described herein may be used in the methods of the invention. Pluripotent stem cells are able to give rise to all cell types of the organism. There are two sources for human pluripotent stem cells: embryonic stem cells (ESCs) derived from surplus blastocysts created for in vitro fertilization and induced pluripotent stem cells (iPSCs) generated by reprogramming of somatic cells.

Cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells are also suitable. Induced pluripotent stem cells (iPSCs) , or reprogrammed pluripotent cells, derived from adult somatic cells using forced expression of a number of pluripotent related transcription factors, such as OCT4, NANOG, SOX2, KLF4, and ZFP42 (Annu Rev Genomics Hum Genet 2011, 12: 165-185; see also IPS, Cell, 126 (4) : 663-676) may also be used. ESCs can be purchased from commercially available sources. The human embryonic stem cells (hESC) used in the methods of the invention may also be prepared as described by Thomson et al. (U.S. Pat. No. 5,843,780; Science, 1998, 282: 1145-1147; Curr Top Dev Biol 1998, 38: 133-165; Proc Natl Acad Sci U.S.A. 1995, 92: 7844-7848) . Mutant human embryonic stem cell lines, such as, BG01v (BresaGen, Athens, Ga. ) , or cells derived from adult human somatic cells, such as, cells disclosed in Takahashi et al., Cell 131: 1-12 (2007) may also be used. In certain embodiments, hESCs are derived from embryos that never undergo in vivo development and are less than 14 days after fertilization. In certain embodiments, pluripotent stem cells suitable for use in the present invention may be derived according to the methods described in: Li et al. (Cell Stem Cell 4: 16-19, 2009) ; Maherali et al. (Cell Stem Cell 1: 55-70, 2007) ; Stadtfeld et al. (Cell Stem Cell 2: 230-240) ; Nakagawa et al. (Nature Biotechnol 26: 101-106, 2008) ; Takahashi et al. (Cell 131: 861-872, 2007) ; and U. S. Patent App. Pub. No. 2011/0104805. In certain embodiments, pluripotent stem cells suitable for use in the present invention may be considered and derived according to the methods described in: Gafni et al. (Nature, 504: 282, 2013) , and Ware et al. (PNAS, 111: 4484-4489, 2014) . All of these references, patents, and patent applications are herein incorporated by reference in their entirety, in particular, as they pertain to the isolation, culture, expansion and differentiation of pluripotent cells.

Other sources of pluripotent stem cells include induced pluripotent stem cells (IPS, Cell, 126 (4) : 663-676) . Yet other sources of suitable cells include human umbilical cord tissue-derived cells, human amniotic fluid-derived cells, human placental-derived cells, and human parthenotes. In an embodiment, the umbilical cord tissue-derived cells may be obtained by the method of U.S. Pat. No. 7,510,873. In another embodiment, the placental tissue-derived cells may be obtained using the methods of U.S. Patent App. Pub. No. 2005/0058631. In another embodiment, the amniotic fluid-derived cells may be obtained using the methods of U.S. Patent App. Pub. No. 2007/0122903. The disclosure of each of these patent applications is incorporated in its entirety herein as it pertains to the isolation and characterization of the cells. In certain embodiments, the pluripotent stem cells may be of non-embryonic origins.

The cells differentiated from hPSCs and treated with the method as provided herein may be immature pancreatic β cells. Pancreatic β cells undergo different states during, fetal, neonatal, and juvenile stages. These stages are characterized by varying levels of replication and functionality in βcells. During the fetal stages, pancreatic β cells show a high capacity for replication. It has been shown that β cells readily replicate in these stages, contributing to the growth and development of the pancreas. This replication ability is essential for the expansion of the β cell mass and the development of a functional endocrine pancreas. As for the juvenile stage, pancreatic β cells continue to replicate at a slower rate compared to the fetal and neonatal stages. However, during this stage, β cells also acquire functional capabilities and contribute to the regulation of blood glucose levels through insulin secretion. The regulation of gene expression and chromatin modifications play notable roles in the transition from the neonatal to juvenile stage of β cell development. During the development of maturity of β cells, “immature beta cells” usually refers to pancreatic endocrine cells that do not exhibit glucose-dependent mitochondrial respiration or activation, and are biphasic glucose-stimulated insulin secretion (GSIS) . Immature β cells expressing markers characteristic of β cells may be characterized by their expression of insulin and one or more of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, HB9, MAFA and PAX6.

Characterization of mature pancreatic β cells

The methods disclosed herein result in pancreatic β cells exhibiting improved function, as evidenced by exemplary embodiments described in the following examples.

Compositions and kits

The present disclosure provides compositions, comprising one or more pluripotent stem cells or hPSC-derived, differentiated immature pancreatic β cells, and at least an HDAC inhibitor, e.g. but not limited to Scriptaid, Crotonoside, TH34, and/or Sulforaphane. Accordingly, said composition can comprise sufficient amount of a compound of HDAC inhibitor for inducing the differentiation of a population of pluripotent stem cells of interest into a population of mature pancreatic β cells. Compositions described herein can be contained in cell culture media. For example, the compositions of the cells and the HDAC inhibitor may be treated in adherent culture, or in suspension culture. It is understood that the cells contacted with a compound of HDAC inhibitor (such as TH34) can also be simultaneously or subsequently contacted with another agent, such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further. Similarly, a pluripotent stem cell can be contacted with a compound of HDAC inhibitor (such as TH34) and then with the other components. In some embodiments, the cell is contacted with a compound of HDAC inhibitor (such as TH34) and the other components and the contact is temporal separated, and in some embodiments, a cell is contacted with a compound of HDAC inhibitor (such as TH34) and the other components substantially simultaneously.

In another aspect, the present disclosure provides kits, comprising the compositions and/or the cell culture media as described herein. In some embodiment, the compound (e.g., the HDAC inhibitor) in the kit can be provided in a watertight or gas tight container which in some embodiments is substantially free of other components of the kit. The compound can be supplied in more than one container, e.g., it can be supplied in a container having sufficient reagent for a predetermined number of reactions e.g., 1, 2, 3 or greater number of separate reactions to induce pluripotent stem cells to differentiation into pancreatic β cells, and/or subsequently into mature pancreatic β cells. A compound (s) described herein (e.g., Scriptaid, Crotonoside, TH34, or Sulforaphane) or compounds of HDAC inhibitor (e.g., Scriptaid, Crotonoside, TH34, and/or Sulforaphane) can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that a compound (s) described herein be substantially pure and/or sterile. When a compound (s) described herein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When a compound (s) described herein is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kits. And optionally the composition can further comprise instructions for converting a population of pluripotent stem cells to a population of mature pancreatic β cells using a method described herein.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; and the like.

Statistical analysis

Data were summarized and analyzed using GraphPad Prism software. Statistical significance was determined by t-tests. Throughout the disclosure, n represented the number of biological replicates unless otherwise stated. p values were presented as follows: *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.00005.

Example 1: In vitro small molecule screening for boosting functional maturation of hPSC- β cells.

Characterization of hPSC-islets at Stage 6

hPSC-islet differentiation

Suitable cell culture methods are known to the skilled in the art, for example, in Culture of Animal Cells: A Manual of Basic Techniques (R.I. Freshney ed., Wiley &Sons) ; General Techniques of Cell Culture (M.A. Harrison &I.F. Rae, Cambridge Univ. Press) and the latest edition of Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press) . Suitable tissue culture supplies and reagents are available from, for example, Gibco/BRL, Nalgene-Nunc International, Sigma Chemical Co. and ICN Biomedicals.

In the present invention, adherent hPSCs (at ～80–90%confluence) were incubated with Accutase (EMD Millipore, Cat#SCR005) for 3-5 minutes at 37℃. Detached cells were washed with mTeSR1 and centrifuged at 300 g for 3 minutes. After supernatant removal, the cell pellet was resuspended in mTeSR1 medium containing 10μM Y-27632 (Selleck, Cat#S1049) , and the single cell suspension was seeded at ～1.2-1.5 × 10⁵ cells/cm² on Matrigel-coated cell factory (NEST, Cat#771101) in mTeSR1 supplemented with 10μM Y-27632. On the following day, differentiation was initiated by changing the media to Stage 1 medium; subsequent medium changes were conducted in accordance with the differentiation protocol described below. The composition of differentiation media at each stage is shown below:

Stage 1: Definitive endoderm (4 days) . MCDB131 (Life Technologies, Cat#10372019) supplemented with 1%B-27 (Gibco, Cat#12587010) , 1%GlutaMAX (Gibco, Cat#35050079) , 4.5 mM glucose (Sigma, Cat#G7021) , 1%penicillin/streptomycin (Pen/Strep) , 100ng/mL Activin A, 0.25 mM ascorbic acid, 50nM PI-103, 6μM CHIR-99021 and 10μM Y-27632 on day 1 only. For the following 3 days, cells were fed with fresh medium daily: comprising MCDB131 with 1%B-27, 1%GlutaMAX, 4.5 mM glucose, 1%Pen/Strep, 50 ng/mL Activin A and 0.25 mM ascorbic acid.

Stage 2: Primitive gut tube (2 days) . MCDB131 supplemented with 1%B-27, 1%GlutaMAX, 4.5 mM glucose, 1%Pen/Strep, 50 ng/mL keratinocyte growth factor (KGF) , 0.25 mM ascorbic acid, 100nM Wnt-C59, and 5μM SB431542. Cells were fed with fresh medium daily.

Stage 3: Posterior foregut (4 days) . DMEM basic medium (Gibco, Cat#C11965500CP) further supplemented with 1%B-27, 1%Pen/Strep, 2μM Retinoic Acid (RA) , 0.1μM LDN-193189, 0.25μM SANT-1 and 100nM Wnt-C59. Cells were fed with fresh medium daily. After 4 days of culture, cells were incubated with Accutase for 3-5 minutes at 37℃. Detached cells were washed with Stage 3 medium containing 10μM Y-27632, then centrifuged at 300 g for 3 minutes. Next, cells were seeded at 8 × 10⁶ cells/well in AggreWell^TM400 6-well plates (Stem Cell, Cat#27940) with Stage 4 medium containing 10μM Y-27632, and the plates were centrifuged at 300 g for 5 minutes with the lowest acceleration to allow cells to settle to the bottom of each microwell; then incubated at 37℃ in 5%CO₂ for 24h. Microwell clusters were gently washed down and transferred into low adhesion 6-well plates (Greiner, Cat#657185) for suspension culture in Stage 4 medium. Suspended aggregates were cultured at 37℃ with 5%CO₂ and 85%humidity in an incubator shaker (Infors-HT, Multitron) , using a rotation speed of 90 rpm.

Stage 4: Pancreatic progenitors (5 days) . DMEM basic medium supplemented with 1%B-27, 1%GlutaMAX, 1%Pen/Strep, 100 ng/mL epidermal growth factor (EGF) , 0.2μM α-Amyloid Precursor Protein Modulator (TPB) , 10 mM nicotinamide, 0.25μM SANT-1, and 0.25 mM ascorbic acid. Cells were fed with fresh medium every 3 days.

Stage 5: Pancreatic endocrine (6 days) . MCDB131 supplemented with 1%B-27, 1%GlutaMAX, 1%Pen/Strep, 10μM ALK5 inhibitor II, 0.3μM LDN-193189, 1μM 3, 3’ , 5-Triiodo-L-thyronine (T3) , 10μM ISX-9, 10μg/mL heparin, 0.1 μM γ-secretase inhibitor XX, 100 nM Wnt-C59, 10μM Y-27632 and 0.25 mM ascorbic acid. ISX-9 was only added for the first 3 days.

Stage 6: hPSC-islets (2-4 days) . MCDB131 supplemented with 1%B-27, 1%Pen/Strep, 10μM ALK5 inhibitor II, 1μM T3, 10μg/mL heparin, 0.25 mM ascorbic acid, 0.5μM R428, 2 mM N-Acetyl-L-cysteine (NAC) and 10μM ZnSO₄.

Cryopreservation and thawing of hPSC-islets

Cryopreservation. hPSC-islets were collected into a 15 mL centrifuge tube, then dispersed into single-cell suspension by incubation with Accutase for 12 min at 90 rpm and 37 ℃. Detached single cells were washed with Stage 6 medium supplemented with 10 μM Y-27632 and centrifuged at 300 g for 3 min. The cells were cryopreserved in Stage 6 medium containing 35%fetal bovine serum, 5%dimethyl sulfoxide (Sigma, Cat#D2650) , and 10 μM Y-27632, at a final density of 3 × 10⁷ cells/mL (1 mL/vial) . Next, the cryovials were then transferred to a CryoMed Controlled Rate Freezer (Thermo Scientific, Cat#7451) , and cooled at a rate of 1 ℃ per minute from the initial temperature to -40 ℃and 10 ℃ per minute to the final temperature of -90 ℃, by the manufacturer’s instructions. The cryovials were then transferred into liquid nitrogen for long-term storage.

Thawing. Cryovials were immersed and gently swirled in a 37 ℃ water bath until almost completely thawed. For each cryovial, the cell suspension was transferred into a 15 mL centrifuge tube containing 10 mL MCDB131 supplemented with 1%B-27 and 10 μM Y-27632, then centrifuged at 300 g for 3 min. Cells were resuspended in MCDB131 supplemented with 1%B-27 and 10 μM Y-27632. Recovery and post-thaw viability were verified by Trypan Blue (Thermo Scientific, Cat#T10282) and Countess^TM 3 Automated Cell Counter (Invitrogen^TM, Cat#AMQAX2000) . Subsequently, the cells were seeded at 8 × 10⁶ cells/well in the AggreWell^TM400 6-well plates; the plates were centrifuged at 300 g for 5 min with the lowest acceleration to allow cells to settle to the bottom of each microwell, then incubated at 37℃ with 5%CO₂ for 24 h. Microwell clusters were transferred into low-adhesion 6-well plates containing MCDB131 supplemented with 1%B-27 for suspension culture. Suspended aggregates were cultured at 37 ℃, 5%CO₂, and 85%humidity in an incubator shaker (Infors-HT, Multitron) using a rotation speed of 90 rpm.

Flow Cytometry

Cell clusters were dispersed into single cells by incubation with Accutase, centrifuged, and fixed with 200 μL of Cytofix/Cytoperm Buffer (BD Biosciences, Cat#51-2090KZ) at 4 ℃ for 30 min; they were subsequently washed twice in 1x Perm/Wash Buffer (BD Biosciences, Cat#51-2091KZ) . Fixed cells were incubated overnight at 4 ℃ with 200 μL of primary antibodies diluted in buffer, then incubated for 1 h at 4 ℃ with secondary antibodies diluted in buffer. Stained cells were washed twice in 1x Perm/Wash Buffer and then subjected to flow cytometry (BD FACSCalibur) . The resulting data were analyzed by FlowJo software (version 10) . The antibodies used are listed in Table 1.
Table 1 Antibodies resources

Quantitative RT-PCR

RNA was extracted using a RNeasy Micro Kit (QIAGEN, Cat#74004) with DNase treatment, by the manufacturer’s instructions. cDNA was synthesized using Transcript One-Step GDNA removal and cDNA synthesis SuperMix (TransGen Biotech, Cat#AT311-03) . Quantitative PCR was performed with KAPA FAST Universal qPCR Mix (KAPA Biosystems, Cat#KK4601) on a 7500 Real-Time PCR system (Applied Bio-systems) . Analyses were conducted using the ΔΔCt method with GAPDH normalization. The primer sequences are listed in Table 2.
Table 2 Human primers for qRT-PCR

Results of differentiated hPSC-islets that were chemically induced from human pluripotent stem cells using the established protocol and cryopreserved in single cells at Stage 6 Day 2 as shown in Figures 1A-1C) .

Prediction of candidate small molecules to boost functional maturation of hPSC-β cells

Among the pool of chemical molecules as listed below, HDAC inhibitor was the most enriched class in the pool. (Figure 2A) , which therefore, were screened using recovered hPSC-islets. On the first day, single cells from Stage 6 Day 2 were recovered from the cryotube and reaggregated them into cell clusters in the Aggrewell plates. On the second day, the reaggregated cell clusters were transferred to low-adhesion 6-well plates containing MCDB131 supplemented with 1%B-27. On the third day, individual small molecule candidate was added to each well, which contained fresh culture medium (MCDB131 supplemented with 1%B-27) for screening. Assessments were conducted after 7 days of culture to determine the effects of small molecule candidates on hPSC-derived β cells (Figure 2B) .

List of small molecules used for screening: Estradiol, Trichostatin A, Cyclosporin A, Thioridazine hydrochloride, Raloxifene hydrochloride, Astemizole, Benzo [a] pyrene, Retinoic acid, Tamoxifen, Azacyclonol, Celastrol, Terfenadine, Suloctidil, Mefloquine hydrochloride, Valproic acid sodium, Phenoxybenzamine hydrochloride, Menadione, Lomustine, Calmidazolium chloride, Scriptaid, Pimozide, Fendiline hydrochloride, Zinc sulfate, Thapsigargin, Strophanthidin, Parthenolide, Etoposide, Puromycin dihydrochloride, Methotrexate, Trifluridine, Resveratrol, Dasatinib, 8-Azaguanine, Copper sulfate, Monobenzone, Pirinixic acid, Quercetin, Azacitidine, Deferoxamine mesylate, Troglitazone, Paclitaxel, 4-Hydroxytamoxifen, Latamoxef, Entinostat, Thonzonium bromide, Progesterone, Ciclopirox, Genistein, Sanguinarine chloride, Dexamethasone, Anisomycin, Aflatoxin B1, Pyrvinium pamoate, Lucanthone, Topotecan, Tetradioxin, Prochlorperazine, Vitamin E, Pentetrazol, Methylbenzethonium chloride, 15-Deoxy-Δ-12, 14-prostaglandin J2, CUDC-101, SR-4370, Resminostat hydrochloride, Abexinostat, Fimepinostat, Pracinostat, SKLB-23bb, Ricolinostat, Splitomicin, Droxinostat, Suberoyl bis-hydroxamic acid, Pimelic Diphenylamide 106, HPOB, TMP195, NKL22, RGFP966, Citarinostat, Crotonoside, PCI-34051, BRD73954, EDO-S101, Nexturastat A, TMP269, TH34, Givinostat hydrochloride monohydrate, Vorinostat, BG45, RG2833, CAY10603, Tasquinimod, Sinapinic acid, WT-161, Tubastatin A, BML-210, ACY-738, ACY-775, Santacruzamate A, UF010, Sulforaphane, BRD3308, Sodium 4-phenylbutyrate, 4-Phenylbutyric acid (4-PBA) , Coumestrol.

Glucose-stimulated insulin release (GSIR)

Krebs buffer (KRB) was prepared as follows: 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl₂, 1.2 mM MgCl₂, 1 mM Na₂HPO₄, 1.2 mM KH₂PO₄, 5 mM NaHCO₃, 10 mM HEPES, and 0.1%BSA were dissolved in deionized water and then sterile filtered. KRB (pH 7.3-7.4) containing 2.8 mM, 16.7 mM glucose, and 30 mM KCl was prepared and warmed to 37 ℃ before use.

Static GSIR assays were performed by collecting ～50–100 hPSC-islets or human islets in 24-well plates. Islets were washed twice with 0 mM glucose KRB, then incubated in 2.8 mM glucose KRB for 1 h for equilibration and removal of residual insulin from the prior culture medium. Sequential 30 min incubations in 2.8 mM, 16.7 mM, and then 2.8 mM glucose KRB were conducted to simulate low, high, and then low glucose challenges; supernatant samples were collected at the end of each incubation stage and immediately stored at -80 ℃ until analysis. In Figure 3B, the incubation sequence of KRB was 2.8 mM, 16.7 mM, 2.8 mM, 16.7 mM glucose, and 30 mM KCl for 30 min, respectively; and in Figure S4C, the incubation sequence of KRB was 2.8 mM, 5 mM, 10 mM, 16.7 mM glucose and 30 mM KCl for 30 min, respectively.

Supernatant samples were frozen at -80 ℃ before measurement with the human C-peptide enzyme-linked immunosorbent assay (ELISA) kit (ALPCO, Cat#80-CPTHU-E10) . Cells were dispersed into single cells by Accutase treatment, then counted with Countess^TM 3 Automated Cell Counter, and the number of viable cells in each sample was used to normalize the level of C-peptide secretion levels.

Unbiased screening yielded four primary hits (scriptaid, crotonoside, TH34, and sulforaphane) that increased the glucose-stimulated insulin release (GSIR) index by more than threefold compared with the control group (Figure 3A) . Additionally, TH34-treated hPSC-β cells responded to sequential glucose challenges and were more sensitive to moderate changes in glucose concentration (Figure 3B) . These results suggested that the HDAC inhibitor TH34 could improve hPSC-β cell function across hPSC lines.

Immunofluorescence staining

Cryosections. Clusters or tissues were washed with PBS, then fixed with 4%paraformaldehyde (PFA) (Biosharp, Cat#BL539A) overnight at 4 ℃. PFA was removed by washing three times with PBS, and samples were dehydrated in 30%sucrose solution overnight at 4 ℃. Subsequently, the samples were overlaid with OCT (Sakura, Cat#4583) and rapidly frozen by immersion in liquid nitrogen and stored at -80 ℃. Ten-micrometer sections were generated using a microtome.

Staining. The sections were washed with PBS and permeabilized with PBST solution (0.2%Triton X-100 and 5%donkey serum in PBS) for 2 h at room temperature, then stained with primary antibodies diluted in PBST solution at 4 ℃ overnight, and washed three times with PBS. Sections were stained with secondary antibodies at 4 ℃ for 2 h, then incubated with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature to stain nuclei. Imaging was performed on a Leica TCS SP8 confocal microscope. The antibodies used are listed in Table 1.

Figure 4A illustrates the expression of β cell markers after TH34 treatment by Flow cytometry (via same procedure as applied in Example 1) . Figures 4B-4C illustrates the results conducted to analyze the gene expression of hPSC-β cells before and after TH34 treatment, indicating that genes enriched in mature β cells, such as MAFA, NFIC, UCN3, and IAPP (via same procedure as applied in Example 1, with primer sequences listed in Table 2) , were significantly upregulated. Taken together, these results confirmed that TH34 effectively improved the maturation of hPSC-β cells in vitro.

Calcium imaging

To detect the calcium influx dynamics of hPSC-islets or human islets in different concentrations of glucose, live cell cluster samples were washed with KRB and stained with Cal-520-AM dye (10 μM, 45 min) at 37 ℃ and then incubated for 15 min without the dye. Before imaging, cell cluster samples were loaded into a microchannel chip and then immediately staged on a confocal microscope. A Dragonfly 200 series (Andor) equipped with a Zyla4.2 sCMOS camera (Andor) was used to acquire high-resolution and time series images. The sequence of glucose challenges in KRB during imaging was 2 mM glucose for 10 min, 20 mM glucose for 20 min, 2 mM glucose for 20 min, and 30 mM KCl for 10 min.

Further characterization showed that the TH34-treated group displayed functional improvement in terms of calcium transient, mitochondrial function, insulin processing, and the ability to reverse diabetes, compared with the control group. As GSIR is tightly controlled by electrical impulses, the cytoplasmic Ca²⁺ concentration was recorded, results of which suggest that glucose challenge led to an excessive influx of calcium ions (Figure 5) .

Oxygen consumption rate (OCR) assays

Oxygen consumption of hPSC-islets or human islets was measured using the Seahorse Bioscience XFe24 Analyzer and analyzed with Agilent Wave software v. 2.6.3. Plates with sensors calibrated were calibrated in XF calibration buffer at 37 ℃ overnight without CO₂ supplementation. ～40 hPSC-islets or human islets were dispersed into single cell suspension by incubation with Accutase and 120, 000 single cells were seeded overnight in Matrigel-coated Seahorse XF24 cell culture microplates. Each biological replicate had at least three technical replicates that were averaged to determine OCR. On the following day, the cells were washed and equilibrated for 60 min without CO₂ supplementation at 37 ℃ in basal Seahorse XF medium containing 2.8 mM glucose. The OCR was calculated by sequential addition of a stimulatory nutrient, 2.8 mM glucose, 16.7 mM glucose, 2 μM oligomycin (inhibitor of ATP-synthetase activity) , 1μM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP, mitochondrial uncoupling agent) , and 1 μM rotenone/antimycin A (inhibitor of electron transport chain complexes I and III) in basal Seahorse XF medium. Each experiment was conducted for three cycles with a 3-minute mix, 2-minute incubation, and 3-minute measurement protocol for all data points collected. The ATP-linked respiration and maximal respiration of hPSC-islets and human islets were then calculated.

The glucose-stimulated insulin release is also correlated with glucose-induced mitochondrial respiration, a feature of mature β cells. Measurement of the oxygen consumption rate (OCR) revealed an increase in OCR in the TH34-treated group during glucose challenge, in contrast to the blunt response in the control group (Figure 6) .

Hormone content

hPSC-islets or human islets were dispersed into single cells by Accutase treatment, then counted with Countess^TM 3 Automated Cell Counter, and viable cell counts were used to normalize levels of hormone content. Single cells were resuspended in radioimmunoprecipitation assay (RIPA) buffer on ice and sonicated for a few seconds until cell membranes were dispersed. Cell fragments were subsequently removed by centrifugation and the supernatant was frozen at -80 ℃ until measurement of hormone content using the following ELISA kits: human C-peptide (ALPCO, Cat#80-CPTHU-E10) , human insulin (ALPCO, Cat#80-INSHUU-E10) , total proinsulin (ALPCO, Cat#82-PINHUT-E01) , glucagon (Mercodia, Cat#10-1271-01) , serotonin (Abcam, Cat#ab133053) , and somatostatin (Biomatik, Cat#EKU07410-96T) .

As shown in Figures 7A-7B, the intracellular contents of both insulin and C-peptide were elevated, resulting in a reduced proinsulin/insulin ratio.

Transmission electron microscopy

hPSC-islets or human islets were fixed in fresh fixative, comprising 2.5% (v/v) glutaraldehyde plus 2%paraformaldehyde (w/v) in 0.1 M phosphate buffer (pH 7.4) , and then incubated at room temperature for 4 h and at 4℃ overnight. On the following day, samples were washed three times with 0.1 M phosphate buffer (pH 7.4) , then stained with 2%osmium tetroxide (w/v) plus 1.5%potassium ferricyanide (w/v) in the same buffer for 2 h at 4 ℃. After three times washing with ultrapure water, en-bloc staining with 2%uranyl acetate (w/v) was performed at 4 ℃ overnight. Samples were subsequently dehydrated by exposure to a series of graded ethanol concentrations, then embedded in fresh resin and polymerized at 65 ℃ for 24 h. Ultrathin (70 nm) sections were generated using a Leica UC7 ultramicrotome equipped with a Ditome diamond knife; the sections were post-stained with uranyl acetate and lead citrate. Micrographs were imaged at 120 kV in a JEOL Jem-1400 transmission electron microscope using a CMOS camera (XAROSA, EMSIS) .

Moreover, the number of crystallized dense-core insulin granules was also significantly increased (Figure 8) .

Ceramides analysis

hPSC-islets were dispersed into single cells by incubation with Accutase, then counted with Countess^TM 3 Automated Cell Counter. Single cells were resuspended in 100 μL H₂O on ice; the H₂O contained containing 10 μg/mL 19: 0 Ceramides (Avanti Polar Lipids, Alabaster, AL, USA) as the internal standard. Samples were vortexed for 30 s and sonicated for a few seconds until cell membranes had been disrupted. Ceramides were extracted with a cold chloroform: methanol (2: 1) solution. After samples had been vortexed for 1 min, they were incubated at room temperature for 30 min and then centrifuged at 13, 000 rpm for 5 min. The lower organic phase of each sample was collected and evaporated. The dried residue was reconstituted in 50 μL of isopropyl alcohol: acetonitrile (1: 1) for LC-MS/MS analysis. Samples were analyzed by Eksigent LC100 coupled with the AB SCIEX Triple TOF 5600 system. Separation was performed using a Waters XBridge Peptide BEH C18 column (3.5 mm, 2.13100 mm) . The elution phases contained water with 10 mM ammonium formate and 0.1%formic acid (phase A) , and 49.95%acetonitrile and 49.95%isopropanol with 10 mM ammonium formate and 0.1%formic acid (phase B) . The following gradient conditions were used as follows: 35%B at 0 min, 80%B at 2 min, 100%B at 9 min, 100%B at 15 min, 35%B at 16 min, and 35%B at 20 min. The flow rate was 0.4 mL/min, and the injection volume was 2 μL. All the lipids were identified using the publicly available database Lipidmaps (http: //www. lipidmaps. org) and through the comparison with the standards (including retention time, parent ion mass, and MS/MS fragmentations) . Peak extraction and integration were performed with PeakView1.2 software (AB SCIEX, Washington D.C., USA) . For lipid metabolite quantitation, data were analyzed using MultiQuant2.1 software (AB SCIEX, Washington D. C., USA) . Ceramide standards (C16: 0, C20: 0, C22: 0) were obtained from Avanti Polar Lipids (Alabaster, AL, USA) .

Sphingolipids constitute a group of bioactive lipids that provide structural integrity to plasma membranes and regulate major cellular processes. In sphingolipid metabolism, ceramides occupy a central position and is the precursor of most sphingolipids. LC-MS/MS analysis showed that the intracellular content of ceramides was significantly decreased after TH34 treatment (Figure 9A) . In previous studies, C16-and C20-ceramide were reported related to β cell dysfunction and diabetes risk (like Yaribeygi, H., et al. (2020) . Diabet Med 37, 11-19. ) . Along with the decreased accumulation of intracellular ceramides, endoplasmic reticulum (ER) stress was alleviated (Figure 9B) . In contrast, exogenous supplementation of ceramide in the culture medium eliminated the effects of TH34 on the functional improvement of hPSC-β cells (Figure 9C) . To investigate the linkage between TH34 and ceramide metabolism, we first analyzed the expression of genes involved in anabolic and catabolic metabolism of ceramide by qRT-PCR (Figure 9D) . Found that the SPTLC1, SPTLC3 and CERS family genes, rate-limiting enzymes for ceramide synthesis, were significantly downregulated after TH34 treatment. In addition, SMase, such as SMPD4 and ENPP7, which converts sphingomyelin to ceramide and phosphocholine, was also significantly downregulated. Furthermore, other enzymes that convert ceramides to other sphingolipids, such as CREK, ASAH2, SPHK2 and SGPL1, were also down-regulated. Collectively, these results suggested that TH34 treatment led to the significant downregulation of the genes related to ceramide synthesis and the decrease in the intracellular ceramides improved hPSC-β cell function. The primer sequences are listed in Table 2.

Example 2: hPSC-islet transplantation under the mouse kidney capsule

Method

All experimental mouse procedures were performed in accordance with the Animal Protection Guidelines of Peking University, China. Male mice (SCID/Beige) at ages of 6–8 weeks old were obtained for the present example. Diabetes model was induced by intraperitoneal injection of STZ (Selleck, Cat#S1312) for five consecutive days at a dose of 75 mg/kg. One week after STZ administration, mice with three consecutive measurements of fasting blood glucose > 16.7 mM were considered diabetic. STZ-treated diabetic and non-STZ-treated SCID/Beige male mice were used in this study. Mice were anesthetized with avertin by injection, and approximately 3×10⁶ hPSC-islets cells were transplanted under the left kidney capsule. Mice were monitored for up to 56 days after transplantation by performing intraperitoneal glucose tolerance tests (IPGTT) and in vivo GSIR, during which mice were fasted for 16 h and then intraperitoneally injected with glucose (2 g/kg, 30%solution) . Fasting blood glucose levels were measured with a handheld glucometer (ROCHE, Cat#06870279001) via tail bleed. Serum samples were separated by microvessels and frozen at -80℃ until analysis.

Results

As shown in Figure 10, after hPSC-β cells had been transplanted into diabetic mice, diabetes reversal was more rapid in the TH34-treated group than in the control group. The alleviated glycemic control at an early stage also led to improved body weight in diabetic mice.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A method of differentiating human pluripotent stem cells (hPSCs) into mature pancreatic β cells, comprising contacting hPSC-derived, differentiated immature pancreatic β cells with a histone deacetylase (HDAC) inhibitor.
The method of claim 1, wherein said differentiated immature pancreatic β cells are pancreatic β cells presenting a fetal and neonatal islet state.
The method of claim 1 or 2, wherein said hPSCs are human induced pluripotent stem cells (hiPSCs) and/or human embryonic stem cells (hESCs) .
The method of any one of claims 1-3, which is an in vitro method.
The method of any one of claims 1-4, which is capable of promoting the acquisition of functional maturity of said hPSC-derived, differentiated immature pancreatic β cells.
The method of claim 5, wherein said acquisition of functional maturity of pancreatic β cells comprises one or more of the functional characteristics described below:

a. said functionally mature pancreatic β cells have a glucose-stimulated insulin release (GSIR) index that is increased threefold or more than threefold compared to said differentiated immature pancreatic β cells;

b. said functionally mature pancreatic β cells undergo a decrease in ceramide accumulation compared to said differentiated immature pancreatic β cell;

c. gene expression levels of MAFA, NFIC, UCN3 and/or IAPP in said functionally mature pancreatic β cells undergo upregulation compared to said differentiated immature pancreatic β-cells;

d. calcium ion fluorescence signal of said functionally mature pancreatic β cells is enhanced compared to said differentiated immature pancreatic β cells;

e. the mitochondrial oxidative respiration function of said functionally mature pancreatic β cells is improved compared to said differentiated immature pancreatic βcells;

f. insulin intracellular proteins are more normally sheared, folded and processed in said functionally mature pancreatic β cells;

g. number of mature insulin granules in said functionally mature pancreatic β cells is improved twice or more than twice compared to said differentiated immature pancreatic β cells

h. after hPSC-islets have been transplanted in vivo, diabetes reversal is more rapid in said functionally mature pancreatic β cells from said hPSC-islets.
The method of any one of claims 1-6, wherein said HDAC inhibitor comprises Scriptaid, Crotonoside, TH34, and/or Sulforaphane.
The method of any one of claims 1-7, wherein said HDAC inhibitor is capable of presenting selectivity for inhibiting HDAC6, HDAC8, and HDAC10.
The method of any one of claims 1-8, wherein said HDAC inhibitor comprises TH34.
The method of any one of claims 1-9, which comprises providing an in vitro culture environment comprising said HDAC inhibitor, and culturing said differentiated immature pancreatic β cells in said in vitro culture environment.
The method of claim 10, wherein said in vitro culture environment is a culture medium.
The method of claim 10 or 11, wherein said HDAC inhibitor comprises TH34, and micromolar concentration of said TH34 within said culture medium is from about 2.0 μM to about 20.0 μM.
The method of claim 12, wherein said TH34 has a micromolar concentration of about 10 μM.
A mature pancreatic β cell obtained by the method of any one of claims 1-13.
A cell population of mature pancreatic β cells obtained by the method of any one of claims 1-13.
A culture comprising mature pancreatic β cells obtained by the method of any one of claims 1-13.
Use of the mature pancreatic β cell of claim 14, the cell population of mature pancreatic β cells of claim 15, and/or the culture of claim 16 for promoting insulin secretion and/or achieving diabetes reversal.
Use of the mature pancreatic β cell of claim 14, the cell population of mature pancreatic β cells of claim 15, and/or the culture of claim 16 for alleviating or treating abnormal insulin secretion, abnormal glucose metabolism, or diabetes.
The use of claim 17 or 18, wherein said diabetes is type I diabetes.
The use of claims 17-19, wherein said use is via one or more of the in vitro achievements described below:

a. glucose-stimulated insulin release (GSIR) index is increased threefold or more than threefold;

b. ceramide accumulation is decreased;

c. gene expression levels of MAFA, NFIC, UCN3 and/or IAPP undergo upregulation;

d. calcium ion fluorescence signal is enhanced;

e. the mitochondrial oxidative respiration function is improved;

f. insulin intracellular proteins are more normally sheared, folded and processed;

g. number of mature insulin granules is improved twice or more than twice;

h. after hPSC-islets have been transplanted in vivo, diabetes reversal is more rapid.