WO2024008614A1

WO2024008614A1 - Use of 25-hydroxycholesterol for diabetic treatment and/or prevention

Info

Publication number: WO2024008614A1
Application number: PCT/EP2023/068160
Authority: WO
Inventors: Maria MARQUES DE LIMA; Marine KRAUS; Denis Marcel Barron; Yann RATINAUD; Daniel Pipeleers
Original assignee: Société des Produits Nestlé S.A.; Vrije Universiteit Brussel
Priority date: 2022-07-08
Filing date: 2023-07-03
Publication date: 2024-01-11

Abstract

Methods and compositions for diabetic treatment and/or prevention are disclosed. One exemplary method includes administering a composition comprising 25-hydroxycholesterol to a region of the subject (e.g., a portion of the subject's pancreas comprising defective pancreatic beta-cells). The composition may be administered to the subject by injecting into a bloodstream associated with the region of the subject. The composition is administered in an effective amount to cause a proliferation of healthy pancreatic beta-cells in the subject. For example, the composition may be administered to the subject in a dosage amount of 0.1 - 50 mg/kg.

Description

TITLE

USE OF 25-HYDROXYCHOLESTEROL FOR DIABETIC TREATMENT AND/OR PREVENTION

TECHNICAL FIELD

[0001] Certain aspects of the present disclosure generally relate to diabetic treatment and/or prevention, and more specifically to the use of 25-hydroxycholesterol for diabetic treatment and/or prevention.

BACKGROUND

[0002] Diabetes is a global health imperative affecting hundreds of millions of people worldwide. Diabetes may be a result of the pancreatic beta cells not making enough insulin, or cells of the body improperly responding to insulin. Insulin secretion from the pancreatic beta-cells, in response to elevated blood glucose levels, may be responsible for controlling blood glucose homeostasis. Therefore, a reduction in beta-cell mass may contribute to the development of type 1 and type 2 diabetes (Butler et al., 2003, Pipeleers et al., 1992). In the adult, beta-cell mass can be maintained by a low replication rate (e.g., of less than 0.5%, Meier et al., 2008). Replenishment of the beta-cell mass in diabetic patients could provide an alternative therapy strategy and a promising path towards restoring glucose control.

[0003] However, efforts at replenishing pancreatic beta-cells have resulted in one or more shortcomings. For example, while genetic manipulation of cell-cycle effectors in human beta-cells may result in efficient induction of beta-cell replication, it has been found that the resulting beta-cells often revert to an immature state, which reduces their functionality (Puri et al., 2018). In another example, it has been found, via high throughput screening assays, that small molecules can induce beta-cell proliferation without the need of genetic manipulation (Ackeifi et al., 2020; Akbib et al., 2019; Assefa et al., 2016; Dirice et al., 2016; Wang et al., 2019). However, while these small molecules may be able to efficiently induce beta-cell proliferation in murine betacells, the efficiency of the small molecules is significantly lower in human beta-cells. Harmine and its analogue 5-lodoturbecidin (5-IT) are compounds that have been described as inducers of proliferation in human beta-cells, increasing their proliferation rate up to 1 .0 % (Dirice et al., 2016). Despite the significant progress achieved with the discovery of the ability of harmine or 5-IT to promote beta-cell proliferation, their proliferation rate of <1 % is low, and their impact in blood glucose normalization and therapy for diabetes is unclear.

[0004] Various embodiments of the present disclosure address one or more of the shortcomings presented above. For example, the present disclosure provides methods and compositions that facilitate the generation of pancreatic beta cells, as this may help to treat and/or prevent diabetes.

SUMMARY

[0005] The present disclosure presents new and innovative methods and compositions for diabetic treatment and/or prevention.

[0006] For example, in at least one embodiment, a method is provided for diabetic treatment and/or prevention. One method involves administering a composition comprising 25- hydroxycholesterol to a region of the subject. The region may comprise, for example, a pancreatic region, or a portal vein of a liver region of the subject. In one embodiment, the pancreatic region may comprise a plurality of defective pancreatic beta-cells. The administering may involve injecting, into a bloodstream associated with the pancreatic region of the subject, the composition comprising the 25-hydroxycholesterol. For example, a dosage amount of 0.1-50 mg/kg of the composition comprising the 25-hydroxycholesterol may be injected into the bloodstream, e.g., at a desired frequency. In some embodiments, the composition may further comprise a plurality of healthy pancreatic beta-cells (e.g., as a healthy pancreatic beta-cell mass). The 25- hydroxycholesterol may cause the proliferation of the healthy pancreatic beta-cells. In such embodiments, the administering the composition may involve transplanting, into the pancreatic region of the subject, the composition comprising the 25-hydroxycholesterol and further comprising the healthy pancreatic beta-cells.

[0007] In some embodiments, the method may involve synthesizing a composition comprising the 25-hydroxycholesterol and further comprising a healthy pancreatic beta-cell mass. The synthesizing may involve using a plurality of human induced pluripotent stem cells (hiPSCs) from the subject or a donor. For example, the method may include receiving, in a plurality of wells, hiPSCs, which may subsequently differentiate, to pancreatic beta-cells (hiPSC-derived pancreatic beta-cells). A dose of 25-hydroxycholesterol may be applied to the plurality of wells (e.g., resulting in a composition having a concentration of 1-50 uM 25-hydroxycholesterol). The method may further include identifying, in at least a portion of the plurality of wells, a marked proliferation of the hiPSC-derived pancreatic beta-cells. The method may further include forming a composition comprising a pancreatic beta-cell mass using the pancreatic beta-cells of the at least the portion of the plurality of wells, the portion corresponding to those wells showing a marked proliferation of the pancreatic beta-cells. In some aspects, the formed composition may include additional or residual 25-hydroxycholesterol.

[0008] Also or alternatively, the synthesizing may include receiving, in a plurality of wells, the plurality of hiPSCs from the subject or donor. The method may further include causing, via contact with one or more inducers, an in vitro differentiation of at least a portion of the plurality of hiPSCs into hiPSC-derived pancreatic beta-cells. A dose of 25-hydroxycholesterol to the plurality of wells. The method may further include identifying, in at least a portion of the plurality of wells, a marked proliferation of the hiPSC-derived pancreatic beta-cells. The method may further include forming a composition comprising a pancreatic beta-cell mass using the pancreatic beta-cells of the at least the portion of the plurality of wells, the portion corresponding to those wells showing a marked proliferation of the pancreatic beta-cells. In some aspects, the formed composition may include additional or residual 25-hydroxycholesterol. [0009] In at least one embodiment, a composition is disclosed for the prevention and/or treatment of diabetes. For example, a composition may include a pancreatic beta-cell mass comprising a plurality of human induced pluripotent stem cell (hiPSC)-derived pancreatic betacells and 25-hydroxycholesterol. In one embodiment, the concentration of the 25- hydroxycholesterol in the composition may be 1-50 uM. The composition may further comprise a portion comprising undifferentiated pluripotent stem cells. For example, the portion comprising the undifferentiated pluripotent stem cells is at most 10% weight of the composition. The composition may be administered to a subject (e.g., via injection to a bloodstream associated with a pancreatic region or via a transplant procedure to the pancreatic region) for use in prevention or treatment of diabetes in the subject. For example, a transplant procedure may replace previously existing defective pancreatic beta-cells in the pancreatic region and/or may replenish the pancreatic region with healthy pancreatic beta-cells. The administration of the pancreatic betacell mass to the subject may result in increased insulin production by the pancreatic region of the subject, relative to insulin production prior to the administration.

[0010] In a further embodiment, another method is provided for the prevention and/or treatment of diabetes in a subject. The method may include receiving, in a plurality of wells, human induced pluripotent stem cells (hiPSCs) from a subject or a donor. The hiPSCs may subsequently differentiate to pancreatic beta-cells (hiPSC-derived pancreatic beta-cells) in vitro. The method may further include applying a dose of 25-hydroxycholesterol to the plurality of wells containing the hiPSC-derived pancreatic beta-cells; and causing, via contact with the 25-hydroxycholesterol, an in vitro proliferation of at least a portion of the hiPSC-derived pancreatic beta-cells. For example, the dose of 25-hydroxycholesterol applied to the hiPSC-derived pancreatic beta-cells may result in a concentration of 1-50 uM 25-hydroxycholesterol. The method may further include generating a healthy pancreatic beta-cell mass resulting from the proliferated hiPSC-derived pancreatic beta-cells. The method may include transplanting the healthy pancreatic beta-cell mass to a pancreatic region of the subject. The transplanting may cause an increase in insulin production by the pancreatic region of the subject, relative to insulin production by the pancreatic region prior to the transplant.

[0011] In some embodiments, receiving hiPSC-derived pancreatic beta-cells may comprise treating a plurality of pluripotent stem cells (PSCs) associated with a subject with a dose of one or more inducers to cause a differentiation of the pluripotent stem cells to the hiPSC- derived pancreatic beta-cells.

[0012] In some embodiments, causing the in vitro proliferation may comprise identifying, among at least the portion of the plurality of wells, a marked proliferation of the hiPSC- derived pancreatic beta-cells. For example, the identifying may involve measuring, in the plurality of wells, insulin levels at predetermined intervals over a predetermined period of time; and identifying an insulin level above a predetermined threshold for at least the portion of the plurality of wells. Furthermore, the healthy pancreatic beta-cell mass may be generated from hiPSC- derived pancreatic beta-cells contained in at least the portion of the plurality of wells.

[0013] The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the figures and description. Moreover, it should be noted that the language used in the specification has been principally selected for readability and instructional purposes, and not to limit the scope of the inventive subject matter.

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 is an illustration of human induced pluripotent stem cells (hiPSCs) being prepared for treatment with 25-hydroxycholesterol, according to an exemplary embodiment of the present disclosure.

[0015] FIG. 2 is a chart of an example screening assay process for the differentiation and proliferation of pancreatic beta-cells, according to an exemplary embodiment of the present disclosure. [0016] FIG. 3 is a graph indicating a validation of the screening assay based on a positive control compound, according to an exemplary embodiment of the present disclosure.

[0017] FIG. 4 is a set of graphs showing a dose-response curve of 25- hydroxycholesterol on human induced pluripotent stem cell (hiPSC) derived pancreatic beta-cells, according to an exemplary embodiment of the present disclosure.

[0018] FIG. 5 is a graph indicating a validation of 25-hydroxycholesterol on primary human pancreatic beta-cells, according to an exemplary embodiment of the present disclosure.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

[0019] As discussed previously, diabetes is a metabolic disorder that may be a result of the pancreatic beta cells not making enough insulin, or cells of the body improperly responding to insulin. Since pancreatic beta-cells secrete insulin in response to elevated blood glucose levels, and control blood glucose levels, a reduction in beta-cell mass may contribute to the development of diabetes. A replenishment of the pancreatic beta-cell mass can be a promising path towards restoring glucose control, and therefore treating and/or preventing diabetes.

[0020] Various embodiments presented herein discuss the identification and use of compositions that can more effectively induce human pancreatic beta-cell proliferation, normalize blood glucose levels, and show promising results in diabetes treatment and prevention.

[0021] The present disclosure relates generally to a composition comprising 25- hydroxycholesterol (25-OH), which is a cholesterol derivative compound, and the use of the composition thereof, as a method of treatment and/or prevention for diabetes in a subject. In some examples, the composition comprises or consists of 25-hydroxycholesterol (25-OH), e.g., for administration to a subject for the treatment and/or prevention of diabetes. For example, the composition may be injected into a bloodstream associated with a region of a subject seeking treatment and/or prevention of diabetes. The region may be, for example, a pancreatic region of the subject or a liver region of the subject. In some embodiments, the composition may be administered orally.

[0022] In another example, the composition may further comprise a healthy pancreatic beta-cell mass. The healthy pancreatic beta-cell mass may be a result of a marked proliferation of pancreatic beta-cells caused by 25-hydroxycholesterol. Such a composition may be administered to the subject by way of a transplant procedure in a region of the subject. For example, that region may include at least a portion of the pancreas of the subject, as the pancreas includes the pancreatic beta-cells typically responsible for producing insulin in response to increased blood glucose levels. The region may include defective beta-cells of the pancreas, as may be characteristic for diabetic patients. A pancreatic beta-cell may be defective based on a diminished ability to sufficiently produce insulin in response to increased blood glucose, a diminished ability to properly synthesize insulin, or a combination thereof. The transplanted healthy pancreatic beta-cell mass may replace defective pancreatic beta cells from the region of the subject, and/or may replenish the region with healthy pancreatic beta-cells. The administration of the pancreatic beta-cell mass to the subject may result in increased insulin production by the region of the subject, relative to insulin production prior to the administration. The healthy pancreatic beta-cells may restore normal levels of insulin production in the subject, thus treating and/or preventing (e.g., a further degradation of) diabetes.

[0023] In some embodiments, administering the composition to the subject may involve transplanting the composition to a portal vein of the liver of the subject during an islet cell transplant procedure.

[0024] In some aspects, human induced pluripotent stem cells (hiPSCs) may be leveraged to derive the composition comprising the healthy pancreatic beta cell mass. For example, pluripotent stem cells may be received from the subject or a donor. The pluripotent stem cells may comprise cells that have the ability to differentiate into one of multiple types of cells (e.g., including pancreatic beta-cells). In some aspects, the pluripotent stem cell may already be within an advanced stage of differentiation such that it is relatively more likely to be differentiated to a pancreatic beta-cell if provided with appropriate treatment of inducers. The pluripotent stem cells may be harvested from the subject ora donor from various regions of the subject’s or donor’s body (e.g., muscle, bone marrow, etc.), or may be induced from adult stem cells (e.g., human induced pluripotent stem cells (hiPSCs) of the subject or donor.

[0025] For example, pancreatic beta-cells may be derived from (e.g., differentiated from) hiPSCs harvested from the patient or donor. As will be described in relation to FIG. 1 , the human induced pluripotent stem cell (hiPSC)-derived pancreatic beta-cells may be placed within a plurality of wells within a plate device. A predetermined dose of 25-hydroxycholesterol may be applied to the hiPSC-derived pancreatic beta-cells to cause proliferation of the hiPSC-derived pancreatic beta-cells. The applied dose of 25-hydroxycholesterol may result in a composition comprising 25-hydroxycholesterol at a concentration of 1-50 uM.

[0026] Also or alternatively, pluripotent stem cells may be harvested from the subject and may be placed within a plurality of wells within a plate device. For example, the wells may receive a predetermined amount of an inducer (e.g., in periodic treatments). As discussed herein, the inducer may cause the pluripotent stem cells to differentiate into pancreatic beta-cells. The inducer may be applied to a portion (e.g., a designated portion of wells) of the pancreatic beta cells.. The inducer may be a compound that regulates gene expression of the pluripotent stem cell, such that it causes the pluripotent stem cell to differentiate itself to pancreatic beta-cells.

[0027] Furthermore, a dose of 25-hydroxycholesterol may be applied to the wells, for example, to result in a composition of hiPSC-derived pancreatic beta-cells having 25- hydroxycholesterol at a concentration of 1-50 uM. The 25-hydroxycholesterol may cause an in vitro proliferation of the hiPSC-derived pancreatic beta-cells. As will be discussed herein, the efficacy of 25-hydroxycholesterol as a proliferator has been proven by comparing the effects of 25-hydroxycholesterol on the pluripotent stem cell (e.g., proliferation rate of pancreatic beta-cells) to other compounds (e.g., harmine, 5-IT). [0028] Among the plurality of wells, the marked proliferation of the pancreatic betacells may be identified. For example, as will be discussed in relation to FIG. 3, markers signifying the presence of pancreatic beta-cells may include insulin or the transcription factor, Nkx6.1 , present within the wells. Thus, the amount of insulin or Nkx6.1 may be tied to the proliferation of the pancreatic beta-cells, thus providing a way to measure the rate of proliferation of the pancreatic beta-cells. In one aspect, the marked proliferation may be identified by measuring insulin and/or Nkx6.1 levels at predetermined intervals (e.g., daily, weekly, etc.) over a predetermined period of time (e.g., up to one month). The marked proliferation may be helpful in the isolation of wells with effective proliferation of pancreatic beta-cells from those wells where the pluripotent stem cells did not proliferate as effectively (e.g., due to an absence of an inducer or 25-hydroxycholesterol, a failure of an inducer or 25-hydroxycholesterol, environmental factors, etc.).

[0029] A healthy pancreatic beta-cell mass may thus be generated for transplant, e.g., by receiving, from those wells, hiPSC-derived pancreatic beta-cells that proliferated. For example, those wells that have a high rate of proliferation of the pancreatic beta-cells may be used to form a pancreatic beta-cell mass. The resulting pancreatic beta-cell mass may be prepared for eventual transplant into the subject. In some aspects, the healthy pancreatic beta-cell mass may include some residual or additional 25-hydroxycholesterol. For example, the pancreatic beta-cell mass may include a concentration of 25-hydroxycholesterol at1-50 uM (e.g., 20-50 uM). Also or alternatively, the healthy pancreatic beta-cell mass may include a portion comprising residual and/or additional pluripotent stem cells. Such additional inducers and/or pluripotent stem cells may ensure further differentiation and proliferation even after transplant.

Examples

[0030] A screening process was used to identify 25-hydroxycholesterol as a compound with a particularly effective capability of stimulating human pancreatic beta-cell proliferation. HiPSC-derived beta-cells were adapt to screening conditions. The hiPSC-derived beta-cells were transferred to 384-well plates, and the effect of the previously described compound 5-IT on beta-cell proliferation was assessed as a positive control of the assay (e.g., as shown in, and described in relation to, FIGS. 1 and 2). After the screening conditions were established, a plurality of compounds (e.g., 1680 compounds in a known commercial library) were tested under the same assay conditions (e.g., as shown FIGS. 2 through 5).

[0031] The compound 25-hydroxycholesterol (25-OH) was found to be the most effective inducer in the differentiation and proliferation of human pancreatic beta-cells. For example, 25-OH was able to increase proliferation of hiPSC-derived beta-cells for up to 10% (e.g., as shown in FIG. 4) and up to 1.5% in primary beta-cells (e.g., as shown in FIG. 5). This is the first time that 25-OH is associated with a beneficial effect in beta-cell or pancreatic proliferation and also with diabetes. Furthermore, the effect of the compound is superior to that observed for 5-IT (0.5-1 % in primary human islets) (e.g., as shown in FIG. 3).

[0032] FIG. 1 is an illustration of human induced pluripotent stem cell (hiPSC)- derived pancreatic beta-cells being prepared for treatment with 25-hydroxycholesterol, according to an exemplary embodiment of the present disclosure. For example, marker 102 shows pancreatic beta-cells from the subject derived from hiPSCs harvested from the subject. In some embodiments, the harvested hiPSC-derived pancreatic beta-cells may include one or both of human embryonic stem cells (ESCs). A dissociation process 104 may be performed (e.g., via a dissociation reagent) to isolate the hiPSC-derived pancreatic beta-cells. The resulting isolated hiPSC-derived pancreatic beta-cells 106 may be prepared for the process of proliferation of pancreatic beta-cells, as will be shown and described in relation to FIG. 2. For example, the resulting isolated hiPSC-derived pancreatic beta-cells 106 may be “plated” (e.g., inserted into microplates, wells, or other containers) for the purpose of treatment with one or more proliferation inducers and/or control substances over a period of time. The plating may occur as part of the initial process (e.g., “Day 0”) in this period of time. [0033] FIG. 2 is a chart of an example screening process for the proliferation of hiPSC- derived pancreatic beta-cells. As shown in FIG. 2, the screening assay process may occur through an eight day period, and may be based on a standard assay procedure 202 or an automated assay procedure 204. For example the isolated human induced pluripotent stem cells 106 described in relation to FIG. 1 may be plated into a 384-well plate at day zero, as shown by marker 206. At least a portion of the wells containing the hiPSC-derived pancreatic beta-cells may be treated with a compound of interest to test the compound’s efficacy in causing an in vitro proliferation of the hiPS-derived pancreatic beta-cells. In a standard process, the compound of interest may be applied at day 1 (e.g., as shown by marker 208) whereas, in the automated screening assay process, the compound of interest may be applied at day 2 (e.g., as shown by marker 210). As will be discussed herein, experiments involving different compounds of interest have shown that 25- hydroxy cholesterol is a particularly effective compound for the differentiation of human induced pluripotent stem cells to, and proliferation of, pancreatic beta cells. However, other compounds of interest for inducers may include, for example, harmine or 5-IT.

[0034] In some embodiments, different inducers may be administered to different portions (e.g., wells) of the pluripotent stem cells to determine the efficacy of the inducer at causing the desired proliferation of the hiPSC-derived pancreatic beta-cells. For example, if the wells containing the hiPSC-derived pancreatic beta-cells were divided into portions A, B, C, and D, portion A may receive proliferation inducer harmine, portion B may receive the proliferation inducer 5-IT, portion C may be a control that does not receive any inducers, and portion D may receive the proliferation inducer 25-hydroxycholesterol, whose relatively superior efficacy is described herein.

[0035] As shown by marker 212, a proliferation label (e.g., 5-Ethynyl-2-deoxyuridine (Edll)) may be applied at day 3, at least in the standard assay process. For example, nascent DNA of the human induced pluripotent stem cells may be labeled with 5-Ethynyl-2-deoxyuridine (Edll). The proliferation (e.g., replication) of the hiPSC-derived pancreatic beta-cells may cause DNA to be replicated, and the Edll may be used to easily determine the proliferation rate of hiPS- derived pancreatic beta-cells. The proliferation rate of different wells treated with different compounds of interest may be used to determine which compound of interest is most effective. In some embodiments, other proliferation labels (e.g., 5-bromo-2-deoxyuridine (Brdll)), and/or other methods of measuring a proliferation rate, may be used.

[0036] At the end of the screening assay process, the wells (e.g., containing differentiated and/or undifferentiated pluripotent stem cells, or the hiPSC-derived pancreatic betacells) may be fixed and stained for the specific pancreatic beta-cell markers (e.g., as shown by marker 314). In some aspects, such pancreatic beta-cell markers may include one or more of Insulin or Nkx6.1 . The pancreatic beta-cell markers may indicate the presence of pancreatic betacells (e.g., derived from the human induced pluripotent stem cells).

[0037] FIG. 3 is a graph indicating a validation of the screening assay based on a positive control compound, according to an exemplary embodiment of the present disclosure. Specifically, the graph shows the results of proliferation of hiPSC-derived pancreatic beta cells using the inducer, 5-IT 304, when compared to those pluripotent stem cells having a positive control substance (e.g., No T 302). The proliferation can be measured based on proliferation labels such as Edll (“EdU+”), and pancreatic beta-cell markers, such as insulin (“INS+”) and NKX6.1 (“NKX6.1+”). The graphs show that 5-IT, when compared to the positive control substance (e.g., No T 302), is somewhat effective at causing an in vitro proliferation of human induced pluripotent stem cells. However, as will be shown in relation to FIGS. 4 and 5, 25- hydroxycholesterol is a more effective compound of interest.

[0038] FIG. 4 is a set of graphs showing a dose-response curve of 25- hydroxycholesterol on human induced pluripotent stem cell derived pancreatic beta-cells (hiPSC- derived pancreatic beta-cells), according to an exemplary embodiment of the present disclosure. As shown in FIG. 4, the structure of the compound 25-hydroxycholesterol 406 is characterized by its unique hydroxyl group on the 25th carbon on a hydrocarbon tail, a central sterol nucleus made of four hydrocarbon rings, and a hydroxyl group typical of cholesterol compounds. Specifically, FIG. 4 shows two batches (e.g., wells or groups of wells) of human induced pluripotent stem cells treated with varying concentrations of 25-hydroxycholesterol. In Batch A, as the concentration of 25-hydroxycholesterol 404A increases, the pancreatic beta-cell proliferation 402A, represented by the blue and green dots, also increases. Similarly, in Batch B, as the concentration of 25- hydroxycholesterol 404B increases, the pancreatic beta-cell proliferation 402B also increases. As previously discussed in relation to FIG. 3, the proliferation of pancreatic beta-cells may be measured using markers such as insulin and Nkx6.1. The green and blue dots indicate two independent experiments, respectively.

[0039] FIG. 5 is a graph indicating a validation of 25-hydroxycholesterol on primary human pancreatic beta-cells, according to an exemplary embodiment of the present disclosure. As shown in FIG. 5, a primary human pancreatic beta-cells are treated with varying concentrations of 25-hydroxycholesterol 504. A score is provided based on the percent increase in human pancreatic beta-cell proliferation (e.g., “percentage scoring” 502). The results in FIG. 5 show that the percentage increased with increasing concentrations of 25-hydroxycholesterol. Thus, the results validate the efficacy of using 25-hydroxycholesterol on primary human beta-cells, proving that the effect is reproducible on primary human tissue.

[0040] It should be understood that various changes and modifications to the examples described here will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

1 . A method of prevention or treatment of diabetes in a subject in need thereof, the method comprising: administering a composition comprising 25-hydroxycholesterol to a region of the subject.

2. The method of claim 1 , wherein the region of the subject comprises: a pancreatic region of the subject, or a portal vein of a liver of the subject.

3. The method of claim 1 , wherein the region comprises a plurality of defective pancreatic beta-cells.

4. The method of claim 1 , wherein the administering comprises: injecting, into a bloodstream associated with the region of the subject, the composition comprising the 25-hydroxycholesterol.

5. The method of claim 1 , wherein the administering comprises: orally administering, by the subject, the composition comprising the 25- hy d roxy ch o I este ro I .

6. The method of claim 1 , wherein the composition is administered to the region of the subject in a dosage amount of 0.1 - 50 mg/kg.

7. The method of claim 1 , wherein the composition is administered in an effective amount to cause a proliferation of healthy pancreatic beta-cells in the subject.

8. The method of claim 1 , wherein the administering comprises: transplanting, into the region of the subject, the composition comprising the 25- hy d roxy ch o I este ro I .

9. The method of claim 1 , wherein the composition further comprises a healthy pancreatic beta-cell mass derived from a plurality of human induced pluripotent stem cells (hiPSCs) from the subject.

10. The method of claim 1 , further comprising: providing a plurality of human induced pluripotent stem cells (hiPSCs) from the subject; causing an in vitro differentiation of at least a portion of the plurality of hiPSCs into hiPSC- derived pancreatic beta-cells; adding 25-hydroxycholesterol to the hiPSC-derived pancreatic beta-cells; and forming, using the hiPSC-derived pancreatic beta-cells and the 25-hydroxyhcholesterol, a healthy pancreatic beta-cell mass.

11. A composition comprising a plurality of human induced pluripotent stem cell (hiPSC)- derived pancreatic beta-cells and 25-hydroxycholesterol.

12. The composition of claim 11 , wherein a concentration of the 25-hydroxycholesterol is 1- 50 uM.

13. The composition of claim 11 , further comprising a portion comprising undifferentiated pluripotent stem cells.

14. The composition of claim 13, wherein the portion comprising the undifferentiated pluripotent stem cells is at most 10 % of the composition.

15. A method comprising: receiving human induced pluripotent stem cell (hiPSC)- derived pancreatic beta-cells, wherein the hiPSC-derived pancreatic beta-cells are derived from hiPSCs received from a subject; applying a dose of 25-hydroxycholesterol to the hiPSC-derived pancreatic beta-cells; causing, via contact with the 25-hydroxycholesterol, an in vitro proliferation of at least a portion of the hiPSC-derived pancreatic beta-cells; and generating a healthy pancreatic beta-cell mass resulting from the proliferated hiPSC- derived pancreatic beta-cells.

16. The method of claim 15, wherein the applying of the dose of 25-hydroxycholesterol to the hiPSC-derived pancreatic beta-cells results in a composition having 1-50 uM 25- hydroxycholesterol. .

17. The method of claim 15, wherein the receiving of the hiPSC-derived pancreatic beta-cells comprises: treating a plurality of pluripotent stem cells (PSCs) associated with a subject with a dose of one or more inducers to cause a differentiation of the pluripotent stem cells to the hiPSC- derived pancreatic beta-cells.

18. The method of claim 15, wherein the causing of the in vitro proliferation comprises: identifying proliferation of the hiPSC-derived pancreatic beta-cells.

19. The method of claim 18, wherein the identifying of the proliferation of the hiPSC-derived pancreatic beta-cells comprises: measuring insulin levels at predetermined intervals over a predetermined period of time; and identifying an insulin level above a predetermined threshold.

20. The method of claim 15, further comprising: transplanting the healthy pancreatic beta-cell mass to a pancreatic region of the subject in an effective amount to cause an increase in insulin production by the pancreatic region of the subject, relative to insulin production by the pancreatic region prior to the transplanting.