Preparation and method for inducing differentiation of mesenchymal stem cells into islet cells

Abstract

Provided are a preparation and method for inducing the differentiation of mesenchymal stem cells into islet cells. Extracellular vesicles are provided to carry transcription factors to induce the differentiation of mesenchymal stem cells into islet cells. The extracellular vesicles can transfer correctly folded protein molecules in cells, and therefore the protein molecules have better physiological activity, thereby promoting the differentiation of mesenchymal stem cells more effectively.

Description

Preparation and method for inducing mesenchymal stem cells to differentiate into pancreatic islet cells Technical Field

[0001] The present invention relates to the field of cell culture technology, and in particular to a preparation and method for inducing mesenchymal stem cells to differentiate into pancreatic islet cells. Background Art

[0002] Diabetes is a metabolic disease characterized by patients' blood sugar levels being higher than the standard value for a long time, and medication is required to control blood sugar within the normal range. Diabetes is divided into type I and type II. Type I diabetes (T1DM) is an autoimmune disease that often occurs in adolescence. The patient's immune system attacks its own pancreatic islets, causing apoptosis of pancreatic beta cells, and ultimately unable to produce enough insulin. Pathologically, it is called insulin-dependent diabetes. Type II diabetes (T2DM) is non-insulin-dependent diabetes that begins with insulin resistance. In the early stages of the disease, the number of pancreatic beta cells in patients is reduced or normal, but as the disease progresses, the patient's pancreatic function may gradually be impaired. Changes in metabolic status caused by obesity and chronic inflammation can also increase the risk of cancer. Therefore, how to restore the ability of patients with T1DM and T2DM in the late stage of the disease to regulate their own blood sugar has always been a difficult problem and goal in the field of diabetes treatment.

[0003] In clinical practice, patients with T1DM, severe T2DM, and diabetes requiring organ transplantation who experience severe imbalances in blood sugar control can undergo pancreatic islet cell transplantation to restore endogenous insulin secretion, reduce secondary complications, and improve quality of life. Currently, the islets used in clinical practice are derived from deceased organ donors with healthy islets. Transplantation efficacy is correlated with the number of islets, but the availability of islet cell donors is extremely limited. Furthermore, allogeneic transplantation can result in rejection, and immunosuppressive drugs can cause chronic toxicity to islets, reducing long-term survival rates after transplantation. Therefore, the development of alternative sources of islets is urgent.

[0004] With the deepening of stem cell research, the development of insulin-producing cells (IPCs) derived from stem cells, i.e., insulin-secreting pancreatic β-cells, has become a new research direction. There are two main sources of induced β-cells: iPSCs and MSCs. iPSCs are pluripotent stem cells that are reprogrammed from somatic cells to possess differentiation capabilities similar to those of embryonic stem cells. Consequently, they have a strong differentiation capacity. There have been reports of iPSCs differentiating into β-cells. A 2022 study reported that iPSC-derived β-cells transplanted into patients with T1DM failed to significantly regulate blood sugar and instead developed invasive teratomas at the transplant site. MSCs, on the other hand, are mesodermal cells with a rich source. They can be isolated from tissues such as adipose tissue, umbilical cord, and dental pulp. They have low HLA expression, low immunogenicity, and require no matching. They also possess both differentiation potential and immunomodulatory functions. Therefore, MSCs have been widely used in a variety of novel strategies for tissue regeneration and immune system diseases, and no serious adverse events have been reported from MSCs themselves. MSCs are also used in organoid differentiation due to their potential for differentiation.

[0005] Methods for MSC differentiation into β-cell-like cells reported in the literature include small molecule differentiation and genetic engineering differentiation. The small molecule differentiation method involves inducing MSCs into insulin-secreting β-cell-like cells by adding small molecule reagents during culture, while the genetic engineering method involves direct differentiation and insulin expression by transfecting the cells with the PDX-1 gene. Pancreatic β-cells develop from the endoderm, and MSC differentiation into β-cells involves trans-embryonic differentiation, which is much more challenging than iPSC differentiation. While genetic engineering differentiation methods offer higher differentiation efficiencies than small molecule induction methods, they may carry a risk of tumorigenicity. While small molecule differentiation methods avoid genetic modification, their differentiation efficiency is lower. Therefore, the current challenge in MSC differentiation into β-cell-like cells lies in improving differentiation efficiency while ensuring safety.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a preparation and method for efficiently inducing mesenchymal stem cells to differentiate into pancreatic islet cells.

[0008] The present invention provides extracellular vesicles loaded with transcription factors; the transcription factor is at least one of PDX1, MAFA, Pax6 and Glis3.

[0009] In some embodiments, the transcription factor loaded in the extracellular vesicles is PDX1.

[0010] In other embodiments, the transcription factor loaded in the extracellular vesicles is MAFA.

[0011] In other embodiments, the transcription factor loaded in the extracellular vesicles is Pax6.

[0012] In other embodiments, the transcription factor loaded in the extracellular vesicles is Glis3.

[0013] In other embodiments, the transcription factors loaded into the extracellular vesicles are PDX1 and Glis3.

[0014] In other embodiments, the transcription factors loaded in the extracellular vesicles are PDX1 and MAFA.

[0015] In other embodiments, the transcription factors loaded in the extracellular vesicles are PDX1 and Pax6.

[0016] In other embodiments, the transcription factors loaded into the extracellular vesicles are MAFA and Glis3.

[0017] In other embodiments, the transcription factors loaded into the extracellular vesicles are MAFA and Pax6.

[0018] In other embodiments, the transcription factors loaded into the extracellular vesicles are Glis3 and Pax6.

[0019] In other embodiments, the transcription factors loaded into the extracellular vesicles are MAFA, Pax6 and Glis3.

[0020] In other embodiments, the transcription factors loaded in the extracellular vesicles are PDX1, Pax6 and MAFA.

[0021] In other embodiments, the transcription factors loaded in the extracellular vesicles are PDX1, Glis3 and Pax6.

[0022] In other embodiments, the transcription factors loaded into the extracellular vesicles are PDX1, MAFA, Pax6 and Glis3.

[0023] The present invention provides extracellular vesicles containing transcription factors, thereby achieving effective delivery of correctly folded protein molecules, and cell-derived nanovesicles (NV) can efficiently deliver active protein molecules into cells. Therefore, the present invention uses HEK293 cell-derived NV to deliver one or a combination of transcription factor proteins PDX1, PAX6, MAFA and Glis3 to promote MSC differentiation into beta-cell-like cells. In order to achieve tracing and / or quantification, the extracellular vesicles described in the present invention can also carry proteins with labeling effects, such as green fluorescent protein, red fluorescent protein, nanoLuc protein, etc.

[0024] Furthermore, the present invention provides a method for preparing extracellular vesicles as described above, comprising: transfecting cells with a plasmid vector containing a nucleic acid encoding a transcription factor, and obtaining a product containing extracellular vesicles after culturing.

[0025] In the embodiment of the present invention, the backbone of the plasmid vector is pHEK293 Ultra Expression Vector I, and the cells are HEK 293F cells.

[0026] Furthermore, the present invention also provides a composition comprising at least one of extracellular vesicles loaded with PDX1, extracellular vesicles loaded with MAFA, extracellular vesicles loaded with Pax6, and extracellular vesicles loaded with Glis3.

[0027] In some embodiments, the composition comprises extracellular vesicles loaded with PDX1 and extracellular vesicles loaded with Glis3.

[0028] In other embodiments, the composition includes extracellular vesicles loaded with PDX1 and extracellular vesicles loaded with MAFA.

[0029] In other embodiments, the composition includes extracellular vesicles loaded with PDX1 and extracellular vesicles loaded with Pax6.

[0030] In other embodiments, the composition includes extracellular vesicles loaded with MAFA and extracellular vesicles loaded with Glis3.

[0031] In other embodiments, the composition includes extracellular vesicles loaded with MAFA and extracellular vesicles loaded with Pax6.

[0032] In other embodiments, the composition includes extracellular vesicles loaded with Glis3 and extracellular vesicles loaded with Pax6.

[0033] In other embodiments, the composition includes extracellular vesicles loaded with MAFA, extracellular vesicles loaded with Pax6, and extracellular vesicles loaded with Glis3.

[0034] In other embodiments, the composition includes extracellular vesicles loaded with PDX1, extracellular vesicles loaded with Pax6, and extracellular vesicles loaded with MAFA.

[0035] In other embodiments, the composition includes extracellular vesicles loaded with PDX1, extracellular vesicles loaded with Glis3, and extracellular vesicles loaded with Pax6.

[0036] In other embodiments, the composition includes extracellular vesicles loaded with PDX1, extracellular vesicles loaded with MAFA, extracellular vesicles loaded with Pax6, and extracellular vesicles loaded with Glis3.

[0037] Furthermore, the present invention provides a culture medium comprising a basal culture medium and at least one of the following I) or II):

[0038] 1) extracellular vesicles as described above;

[0039] II) A composition as described above.

[0040] Furthermore, the present invention also provides a culture medium combination:

[0041] Includes Medium 1 and Medium 2:

[0042] Culture medium 1 includes basic culture medium 1 and the extracellular vesicles as described above or the composition as described above; the basic culture medium 1 includes DMEM / F12, FBS, EGF and B27.

[0043] Culture medium 2 includes basal culture medium 2 and the extracellular vesicles as described above or the composition as described above; the basal culture medium 2 includes DMEM / F12, FBS, GLP-1, β-cellulin, HGF, nicotinamide, B27 and β-mercaptoethanol.

[0044] In the embodiment of the present invention,

[0045] The culture medium 1 comprises: DMEM / F12, FBS, EGF, B27 and the composition as described above;

[0046] The culture medium 2 includes: DMEM / F12, FBS, GLP-1, β-cellulin, HGF, nicotinamide, B27, β-mercaptoethanol and the composition as described above.

[0047] In some embodiments,

[0048] The culture medium 1 comprises: DMEM / F12, 1% to 5% FBS, 80 to 120 ng / ml EGF, 1% to 5% B27 and the above-mentioned combination; preferably, the culture medium 1 comprises: DMEM / F12, 2% FBS, 100 ng / ml EGF, 2% B27 and the above-mentioned combination;

[0049] The culture medium 2 comprises: DMEM / F12, 1%-5% FBS, 8-12 ng / ml GLP-1, 8-12 ng / ml β-cellulin, 8-12 ng / ml HGF, 8-12 mmol / l nicotinamide, 1%-5% B27, 0.1 mmol / l β-mercaptoethanol, and the above-mentioned combination. Preferably, the culture medium 2 comprises: DMEM / F12, 2% FBS, 10 ng / ml GLP-1, 10 ng / ml β-cellulin, 10 ng / ml HGF, 10 mmol / l nicotinamide, 2% B27, 0.1 mmol / l β-mercaptoethanol, and the above-mentioned combination.

[0050] In some specific embodiments,

[0051] The density of extracellular vesicles in the culture medium 1 is 1×10 8 ~1×10 10 / mL; preferably, the density of extracellular vesicles in the culture medium 1 is 1×10 9 / mL;

[0052] The density of extracellular vesicles in the culture medium 2 is 1×10 8 ~1×10 10 As an example, the density of extracellular vesicles in the culture medium 2 is 1×10 9 pieces / mL.

[0053] Furthermore, the present invention also provides the use of the aforementioned culture medium combination in inducing MSCs to differentiate into β cells.

[0054] Furthermore, the method of inducing MSCs to differentiate into β cells according to the present invention comprises: culturing MSCs in sequence with the culture medium 1 and the culture medium 2 in the culture medium combination as described above.

[0055] In some embodiments, the culture conditions of medium 1 include a seeding density of 40,000 cells / well, medium replacement every other day, and culture for 7 days.

[0056] In some embodiments, the culture medium 2 culture conditions include changing the medium every other day and culturing for 10 days.

[0057] In some embodiments, the MSCs are passage 2 to passage 4 MSCs.

[0058] In some embodiments, the sources of the MSCs include umbilical cord, fat, bone marrow, placenta, dental pulp, cornea, skeletal muscle, gland, skin, or embryo.

[0059] In the present invention, umbilical cord mesenchymal stem cells are used as an example to verify the differentiation effect. Similar effects are obtained when mesenchymal stem cells from other sources are induced with the culture medium of the present invention.

[0060] The present invention provides a method for inducing mesenchymal stem cells to differentiate into pancreatic islet cells by using extracellular vesicles (EVs) loaded with transcription factors. EVs can deliver properly folded protein molecules to cells, resulting in enhanced physiological activity and, therefore, more effectively promoting the differentiation of MSCs. Experiments have shown that after 17 days of induction, the present invention significantly increased the expression of the glucagon gene secreted by MSCα cells by over 100-fold, and also significantly increased the secretion of insulin secreted by β cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 shows NVnanoluc enzyme activity detection;

[0062] Figure 2 shows the morphological changes of cells induced by differentiation;

[0063] Figure 3 shows the changes in islet-related gene expression detected by RT-qPCR;

[0064] Figure 4 shows the insulin concentration test results of each group;

[0065] FIG5 shows the results of insulin secretion in each group. DETAILED DESCRIPTION

[0066] The present invention provides preparations and methods for inducing efficient differentiation of mesenchymal stem cells into pancreatic islet cells. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0067] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0068] The sequences involved in the embodiments of the present invention include:

[0069] PDX1 sequence:

[0070] PAX6 sequence:

[0071] MAFA sequence:

[0072] Glis3 sequence:

[0073] nanoluc sequence:

[0074] The nucleic acid encoding the transcription factor is codon-optimized, wherein

[0075] The nucleic acid sequence encoding PDX1 is:

[0076] The nucleic acid sequence encoding Pax6 is

[0077] The nucleic acid sequence encoding MAFA is

[0078] The nucleic acid sequence encoding Glis3 is

[0079] The nucleic acid sequence encoding nanoluc is

[0080] The present invention will be further described below in conjunction with the embodiments:

[0081] Example 1:

[0082] Objective: To load the transcription factors PDX1 (SEQ ID NO: 1), Pax6 (SEQ ID NO: 2), MAFA (SEQ ID NO: 3) and Glis3 (SEQ ID NO: 4) proteins into HEK293-derived NV by transient transfection with plasmids.

[0083] method:

[0084] Transcription factor loading: The CDS region of the relevant transcription factor gene was inserted into the multiple cloning site of the HEK 293 overexpression plasmid pHEK293 Ultra Expression Vector I. The nanoLuc gene was then added to the C-terminus of the transcription factor to enable identification of PDX1, Pax6, MAFA, and Glis protein loading in extracellular vesicles and NV delivery of PDX1, Pax6, MAFA, and Glis proteins to MSCs. The constructed expression vector was transfected into cells, and the cells were harvested to prepare NVs. NV NanoLuc enzyme activity was measured to verify NV loading of the transcription factor-nanoLuc protein. [Note: The nanoLuc fusion protein is only used for tracing and quantitative analysis. In actual use, nanoLuc protein loading is not required; overexpression of the transcription factor protein alone is sufficient.]

[0085] Results: HEK293F cells were transiently transfected with PDX1-Nanoluc, Pax6-Nanoluc, MAFA-Nanoluc and Glis-Nanoluc expression plasmids 48 hours after cell collection and purification of NV. The nanoluc enzyme activity of 1E+07 NVs was detected using a nanoluc detection kit. The results showed that all four NVs had nanoluc enzyme activity, indicating that PDX1-Nanoluc, Pax6-Nanoluc, MAFA-Nanoluc and Glis-Nanoluc proteins were loaded in NVs, and the expression level of MAFA-Nanoluc was higher than that of PDX1-Nanoluc, higher than that of Pax6-Nanoluc and higher than that of Glis-Nanoluc (Figure 1).

[0086] Example 2 Inducing differentiation of MSCs into β-cell-like cells

[0087] Objective: To deliver transcription factor proteins to MSCs by loading them with NVs to promote their differentiation into β cells. Furthermore, the differentiation of MSCs into β cells was verified by measuring changes in cell morphology during differentiation, changes in the expression of pancreatic islet-related genes at the end of differentiation, and cell glucose response activity.

[0088] method:

[0089] 1) Cell differentiation induction

[0090] Human umbilical cord mesenchymal stem cells (UC-MSCs) from passage 2 to 4 were seeded into six-well plates at 40,000 cells / well. The medium for stage 1 basal induction was changed on the second day and then every other day. The medium was changed to stage 2 basal induction medium on the seventh day. Stage 2 induction was continued for 10 days, during which the medium was changed every other day.

[0091] Scheme 1 is to add a final concentration of 1×10 9 PDX1 / mL + NV;

[0092] Option 2 is to add PDX1 to the basal induction medium + NV (final concentration was 1×10 9 cells / mL) and PAX6 + NV (final concentration was 1×10 9 cells / mL);

[0093] Solution 3 is to add PDX1 to the basal induction medium + NV (final concentration was 1×10 9 cells / mL) and MAFA + NV (final concentration was 1×10 9 cells / mL);

[0094] Solution 4 is to add PDX1 to the basal induction medium + NV (final concentration was 1×10 9 cells / mL) and Glis3 + NV (final concentration was 1×10 9 / mL).

[0095] Induction basal medium:

[0096] 2) RT-qPCR detection of islet-related gene expression changes in differentiated end-point cells: remove the supernatant, extract RNA according to the instructions of the RNA extraction kit, perform reverse transcription according to the instructions of the reverse transcription kit, and perform qPCR according to the instructions of the qPCR kit to detect the expression fold changes of the key gene glucagon for α cells, the key gene insulin for β cells, the key gene SST for δ cells, and the key gene PP for PP cells in the islets.

[0097] 3) Glucose stimulation experiment: Remove the cell supernatant, wash once with PBS, add 2.8 mM glucose to stimulate for 1 hour, and store the supernatant at -20°C. Add 17.8 mM glucose to stimulate for 1 hour, and store the supernatant at -20°C. Detect insulin concentration in the sample according to the ELISA kit instructions.

[0098] result

[0099] 1) Photos of the cell differentiation process: MSCs begin as spindle-shaped, fibrous cells during induced differentiation. As differentiation progresses, the cells gradually transform from a spindle-shaped cell to a rounded shape, which remains constant until the end of differentiation. This conforms to the reported β-cell-like morphology. This suggests that NV loading and delivery of transcription factors promotes the transformation of MSCs into β-cell-like morphology (Figure 2).

[0100] 2) RT-qPCR analysis of related gene expression changes: Results showed that NV loading and transcription factor delivery significantly upregulated the expression of the glucagon gene secreted by MSCα cells by more than 100-fold, and that insulin secretion by MSCβ cells was also significantly upregulated. This suggests that NV-delivered transcription factors can significantly promote trans-germ layer differentiation of MSCs (Figure 3).

[0101] 3) Glucose stimulation experiment: After induction of differentiation, the cell supernatant was collected and the supernatant was collected after stimulating the cells with different concentrations of glucose to detect insulin secretion. The results showed that insulin secretion was found in the cell culture supernatant after induction with different schemes. The induced cells showed obvious response activity to different concentrations of glucose stimulation, and the higher the glucose concentration, the higher the insulin secretion of the cells. The cells induced by Scheme 3 and Scheme 2 had slightly stronger response activity to glucose. This shows that NV-delivered transcription factors promote MSC-induced differentiation of β-cells, which can respond to glucose stimulation and secrete insulin. (Figure 4)

[0102] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Extracellular vesicles loaded with transcription factors; The transcription factor is at least one of PDX1, MAFA, Pax6 and Glis3.

2. The method for preparing the extracellular vesicles according to claim 1, comprising: The plasmid vector containing the nucleic acid encoding the transcription factor is transfected into the cells, and the product containing the extracellular vesicles is obtained after culture.

3. The preparation method according to claim 2, characterized in that: The skeleton of the plasmid vector is pHEK293 Ultra Expression Vector I, and the cell is HEK 293F cell.

4. A composition comprising: At least one of extracellular vesicles loaded with PDX1, extracellular vesicles loaded with MAFA, extracellular vesicles loaded with Pax6, and extracellular vesicles loaded with Glis3.

5. The composition according to claim 4, characterized in that Including extracellular vesicles loaded with PDX1 and extracellular vesicles loaded with MAFA; or including PDX1-loaded extracellular vesicles and Pax6-loaded extracellular vesicles; Or include PDX1-loaded extracellular vesicles and Glis3-loaded extracellular vesicles.

6. A culture medium comprising a basal medium and at least one of the following I) or II): 1) The extracellular vesicle according to claim 1; II) The composition according to claim 4 or 5.

7. Culture medium combination, including culture medium 1 and culture medium 2: Culture medium 1 comprises a basal culture medium 1 and the extracellular vesicles according to claim 1 or the composition according to claim 4 or 5; Culture medium 2 comprises basal culture medium 2 and the extracellular vesicles according to claim 1 or the composition according to claim 4 or 5; The basic culture medium 1 includes DMEM / F12, FBS, EGF and B27; The basic culture medium 2 includes DMEM / F12, FBS, GLP-1, β-cell killer, HGF, nicotinamide, B27 and β-mercaptoethanol.

8. The culture medium combination according to claim 7, characterized in that: The culture medium 1 comprises: DMEM / F12, FBS, EGF, B27 and the composition according to claim 4 or 5; The culture medium 2 includes: DMEM / F12, FBS, GLP-1, β-cell factor, HGF, Nicotinamide, B27, β-mercaptoethanol and the composition of claim 4 or 5.

9. The culture medium combination according to claim 8, characterized in that: The culture medium 1 comprises: DMEM / F12, 1% to 5% FBS, 80 to 120 ng / ml EGF, 1% to 5% B27 and the composition of claim 4 or 5; The culture medium 2 comprises: DMEM / F12, 1%-5% FBS, 8-12 ng / ml GLP-1, 8-12 ng / ml β-cellulin, 8-12 ng / ml HGF, 8-12 mmol / l nicotinamide, 1%-5% B27, 0.1 mmol / l β-mercaptoethanol and the composition according to claim 4 or 5.

10. The culture medium combination according to claim 9, characterized in that: The density of the extracellular vesicles in the culture medium 1 is 1×10 8 ~1×10 10 Pieces / mL; The density of the extracellular vesicles in the culture medium 2 is 1×10 8 ~1×10 10 Pieces / mL.

11. Use of the culture medium combination according to any one of claims 7 to 10 in inducing MSCs to differentiate into β cells.

12. A method for inducing MSCs to differentiate into β cells, comprising: MSCs are cultured sequentially with the culture medium 1 and the culture medium 2 in the culture medium combination according to any one of claims 7 to 10.

13. The method according to claim 12, characterized in that The conditions for culture in medium 1 included a seeding density of 40,000 cells / well, medium replacement every other day, and culture for 7 days; The conditions for culture in medium 2 included changing the medium every other day and culturing for 10 days.

14. The method according to claim 12 or 13, characterized in that The MSCs are MSCs of the second to fourth generations.

15. The method according to claim 14, characterized in that The MSCs are umbilical cord mesenchymal stem cells.

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