Induction medium and induction method for directionally differentiating mesenchymal stem cells into insulin-secreting cells

Abstract

The invention discloses an induction culture medium for directionally differentiating mesenchymal stem cells into insulin-secreting cells. The induction culture medium consists of a differential culture medium I, a differential culture medium II and a differential culture medium III. The invention further discloses an induction method for directionally differentiating the mesenchymal stem cells into the insulin-secreting cells. The induction culture medium for directionally differentiating the mesenchymal stem cells into the insulin-secreting cells is used for carrying out differentiation culture. According to the induction culture medium and the induction method for directionally differentiating the mesenchymal stem cells into the insulin-secreting cells, the intestinal bioelectricity environment is simulated by using electrical stimulation while chemical induction is used, cell contraction is promoted to achieve the effect of simulating movement, and active small-molecule polypeptide of spidermoss is added to protect cell membranes, so that the insulin-secreting cells are directionally differentiated into the insulin-secreting cells. The success rate of original intestinal canal differentiation is improved; and the IPCs differentiation efficiency is improved. The induction culture medium and the induction method have important application value in treating diseases such as diabetes mellitus and the like by clinically applying the MSC.

Description

Technical Field

[0001] The invention relates to an induction culture medium and an induction method for directed differentiation of mesenchymal stem cells into insulin-secreting cells, belonging to the technical field of mesenchymal stem cell culture and differentiation. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of stem cell that has the ability to self-renew, multidirectionally differentiate, and regulate immunity. MSCs can be isolated from a variety of tissues, such as fat, bone marrow, placenta, umbilical cord, and gums. Based on their easy access, easy culture, multiple immunoregulatory abilities, and low immunogenicity, MSCs have gradually become an important cell source for tissue engineering and clinical cell therapy.

[0003] Insulin deficiency is an important link and one of the decisive factors in the onset of diabetes. At present, islet transplantation is a new technical means that is expected to completely cure diabetes, especially for patients with type 1 diabetes. However, clinical islet transplantation still faces two major problems, namely the limited source of pancreatic organs and the rejection of organ transplantation. Inducing mesenchymal stem cells to differentiate and secrete insulin through multiple steps to become insulin-producing cells (IPCs) with similar functions to β cells can become an alternative treatment for islet transplantation. The process of mesenchymal stem cells differentiating into primitive intestinal cells is one of the key difficult steps for successful differentiation into IPCs. Improving the efficiency of the differentiation step into primitive intestinal tubes can effectively improve the efficiency of the final differentiation into IPCs. Therefore, exploring an effective and efficient induction method and induction culture medium is crucial for the clinical application of MSC in the treatment of diabetes.

[0004] Previous studies have found that mesenchymal stem cells are easy to differentiate into mesodermal tissues such as cartilage, osteoblasts, myoblasts and adipocytes, and also have the ability to differentiate into endoderm cells and primitive intestinal tubes, and further differentiate into IPCs, but the existing differentiation methods have the problem of low differentiation success rate. Improving the efficiency of mesenchymal stem cells to differentiate into primitive intestinal tubes and IPCs has great application value for the clinical application of MSC to treat diseases such as diabetes. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides an induction medium and an induction method for directed differentiation of mesenchymal stem cells into insulin-secreting cells.

[0006] The present invention is achieved through the following technical solutions:

[0007] An induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells, comprising differentiation medium 1, differentiation medium 2 and differentiation medium 3, wherein:

[0008] The differentiation medium 1 is composed of a basic cell culture medium and the following components at the following concentrations: 0.4-0.6 mmol / L of β-thionicotinamide;

[0009] The differentiation medium II is composed of a basic cell culture medium and the following components: 0.5% to 1.5% (volume percentage, the same below) of a non-essential amino acid solution, 50 to 70 ng / mL of bFGF (basic fibroblast growth factor), 30 to 50 ng / mL of EGF (epidermal growth factor), 0.5% to 1.5% of a small molecule peptide solution of the rusty spider hair moss, and 1.5% to 2.5% of a B27 additive (B-27 Serum-Free Supplement, a serum-free additive); the concentration of the small molecule peptide solution of the rusty spider hair moss is 2 to 4 mg / mL;

[0010] The differentiation medium three is composed of a basic cell culture medium and components of the following concentrations: 20-40 ng / mL of betacellulin (BTC), 90-110 ng / mL of activin A (ACV-A), and 8-12 mmol / L of nicotinamide.

[0011] Furthermore, in the differentiation medium 1, the concentration of β-mercaptoethanol is preferably 0.5 mmol / L.

[0012] Furthermore, in the differentiation medium 2, the concentration of each component is preferably: 1% non-essential amino acids, 60 ng / mL bFGF, 40 ng / mL EGF, 1% small molecule peptide of Aralia rustata, and 2% B27 additive.

[0013] Furthermore, in the differentiation medium three, the concentration of each component is preferably: 30 ng / mL of beta-cell killer, 100 ng / mL of activin A, and 10 mmol / L of nicotinamide.

[0014] Furthermore, the rusty spider hair moss small molecule peptide solution is prepared by the following method: under sterile conditions, using sterile scissors to cut the rusty spider hair moss leaves into small pieces, placing them in 8 to 12 times (weight multiples) of water, adding 2% to 6% of the weight of the leaves The complex enzyme is hydrolyzed at 55 to 65°C and pH 7.0 for 20 to 40 minutes; collecting the supernatant by centrifugation, placing the supernatant in a 30KD ultrafiltration tube for centrifugation, and collecting the liquid phase to obtain the rusty spider hair moss active small molecule polypeptide solution. After measurement, the concentration of the rusty spider hair moss small molecule peptide is 2.8 mg / mL; the complex enzyme is composed of neutral protease and alkaline protease, and the weight ratio of the two is (0.8 to 1.2):1, preferably 1:1.

[0015] Furthermore, the basal cell culture medium is a culture medium for culturing cells available in the prior art, such as serum-free high-glucose DMEM culture medium.

[0016] A method for inducing directed differentiation of mesenchymal stem cells into insulin-secreting cells, using the above-mentioned induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells for differentiation culture, comprising the following steps:

[0017] (1) Differentiation stage 1: mesenchymal stem cells were placed in differentiation medium 1 and cultured at 37°C and 5% CO2 for 3 days;

[0018] (2) Differentiation stage II: After the above culture, the supernatant was discarded, and differentiation medium II was added, and cultured at 37°C and 5% CO2 for 6 days. During the culture process, electrical stimulation was performed once a day;

[0019] (3) Differentiation stage three: After the above culture, the supernatant was discarded, and differentiation medium three was added, and cultured at 37° C. and 5% CO 2 for 8 days to obtain insulin-secreting cells.

[0020] Furthermore, in step (1), the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0021] Furthermore, in step (2), the conditions of electrical stimulation are: stimulation intensity 32V, stimulation time 30min, frequency 1Hz, 20ms.

[0022] Furthermore, in the step (2), the time of electrical stimulation is consistent every day.

[0023] The induction medium and induction method for directed differentiation of mesenchymal stem cells into insulin-secreting cells of the present invention improve the differentiation efficiency of mesenchymal stem cells into IPCs. While using chemical induction, electrical stimulation is used to simulate the intestinal bioelectric environment to promote cell contraction to achieve the effect of simulating movement, and the active small molecule polypeptide of rusty spider hair moss is added (the spider hair moss has strong antioxidant activity, can play antioxidant, anti-inflammatory and anti-aging properties in cell culture, and protect the cell membrane from possible damage caused by electric current) to protect the cell membrane, improve the success rate of primitive intestinal differentiation, and thus improve the differentiation efficiency of IPCs. The induction medium and induction method of the present invention have important application value for clinical application of MSC to treat diseases such as diabetes.

[0024] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 :Schematic diagram of RT-PCR detection results of primitive intestinal marker transcription factors.

[0026] Figure 2 : Schematic diagram of WB detection results of primitive intestinal marker transcription factors.

[0027] Figure 3 : Schematic diagram of dithizone staining detection results (100X), where A: blank group; B: control group; C: experimental group.

[0028] Figure 4 : Schematic diagram of ELISA test results, where A: insulin secretion; B: c-peptide secretion.

[0029] Figure 5 : Schematic diagram of immunofluorescence detection results, where A: DAPI staining of the blank group; B: insulin staining of the blank group; C: DAPI staining of the control group; D: insulin staining of the control group; E: DAPI staining of the experimental group; F: insulin staining of the experimental group. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0031] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0032] The serum-free high-glucose DMEM culture medium used in the present invention is a commercial culture medium available in the prior art: DMEM high-glucose culture medium, purchased from Keygene Biotechnology, item number: KGM12800N-500.

[0033] The non-essential amino acid solution used in the present invention is a mixed solution of 7 kinds of amino acids, namely L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline and L-serine, which is a cell culture medium additive available in the prior art. The product is provided in the form of 100X working solution; MEM NEAA is purchased from Gibco, item number: 11140-050.

[0034] Example 1 Induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells

[0035] It consists of differentiation medium 1, differentiation medium 2 and differentiation medium 3.

[0036] Differentiation medium 1 consisted of serum-free high-glucose DMEM culture medium and the following components at the following concentrations: 0.5 mmol / L β-mercaptoethanol.

[0037] Differentiation medium 2 consisted of serum-free high-glucose DMEM culture medium and the following components: 1% non-essential amino acids, 60 ng / mL bFGF, 40 ng / mL EGF, 1% small molecule peptide from Aralia rustata, and 2% B27 additive.

[0038] Differentiation medium 3 consisted of serum-free high-glucose DMEM culture medium and the following components at the following concentrations: 30 ng / mL beta-cell membrane protein, 100 ng / mL activin A, and 10 mmol / L nicotinamide.

[0039] The rust-colored spider hair moss small molecule peptide solution is prepared by the following method: under sterile conditions, using sterile scissors to cut the rust-colored spider hair moss leaves into small pieces, placing them in 10 times the amount of water, adding 4% of the weight of the leaves as a composite enzyme, and shaking hydrolyzing them at 55-65°C and pH 7.0 for 30 minutes; collecting the supernatant by centrifugation, placing the supernatant in a 30KD ultrafiltration tube for centrifugation, collecting the liquid phase, and obtaining the rust-colored spider hair moss active small molecule polypeptide solution. After measurement, the concentration of the rust-colored spider hair moss small molecule peptide is 2.8 mg / mL; the composite enzyme is composed of a neutral protease and an alkaline protease, and the weight ratio of the two is 1:1.

[0040] The active small molecule peptides obtained after hydrolysis and ultrafiltration of the leaves of the rusty spider hair moss have more superior water solubility. The structure of the small molecule peptide makes it more conducive to cell absorption and conversion than ordinary rusty spider hair moss leaf extracts. It can better protect the cell membrane during electrical stimulation and reduce the risk of cell damage.

[0041] Experiment 1: Induction of differentiation of mesenchymal stem cells into IPCs

[0042] Here are the steps:

[0043] (1) Primary human umbilical cord mesenchymal stem cells were isolated from human umbilical cord tissue according to conventional methods, and were inoculated into 6-well plates after culture. When the cultured cells reached 80% confluence, they were washed with phosphate buffered saline (PBS).

[0044] (2) Differentiation stage 1: The cultured human umbilical cord mesenchymal stem cells were seeded in a 6-well plate, with 1×10 5 The cells were placed in 2 ml differentiation medium and cultured at 37°C and 5% CO2 for 3 days.

[0045] (3) Differentiation stage II: After the above culture, centrifuge (1500r, 10min), discard the supernatant, add 2ml differentiation medium II, and culture at 37°C, 5% CO2 for 6 days. During the culture process, electrical stimulation is performed once a day at a fixed time. The conditions for electrical stimulation are: adjust the electrical stimulator to a stimulation intensity of 32V, a stimulation time of 30min, a frequency of 1Hz, 20ms, and the operation method of electrical stimulation is: insert the electrical stimulation plate into the six-well plate, ensure that the electrode contacts the cell culture medium, and put the six-well plate together with the electrical stimulation plate into the cell culture incubator, and electrical stimulation is performed once a day at a fixed time.

[0046] (4) Differentiation stage III: After the above culture, the supernatant was discarded, 2 ml of differentiation medium III was added, and cultured at 37° C. and 5% CO 2 for 8 days.

[0047] Experiment 2: Differentiation efficiency detection

[0048] 1 Method

[0049] 1.1 PT-PCR detection of primitive intestinal marker transcription factors

[0050] In order to verify the effect of the method of the present invention on the differentiation efficiency of mesenchymal stem cells into primitive intestinal tube, an experimental group, a control group induced by traditional chemical induction and a blank group were set up.

[0051] Experimental group: cells cultured according to the method of Experiment 1.

[0052] Control group: The chemical induction reagent and culture conditions were the same as those of the experimental group, except that no electric stimulator was used for stimulation during the culture process of differentiation stage II, and no small molecule peptide of Aralia rustata was added.

[0053] Blank group: human umbilical cord mesenchymal stem cells were seeded in 6-well plates and cultured in DMEM / F12 containing 10% FBS for 1 day.

[0054] The cells after the completion of differentiation stage 2 in the experimental group and the control group were taken to extract total mRNA (the test samples of the blank group were cells cultured for 9 days, and total mRNA was extracted), and cDNA was prepared using a reverse transcription kit. The cDNA samples were tested by RT-PCR. The reaction system was: cDNA 1μL; 1μL of upstream and downstream primers (10μM), 10μL of PT-PCR Master Mix, and 8μL of RNA freewater; the reaction conditions were: 95℃10min, 95℃15s, 60℃1min, 40 cycles; HNF1β, HNF4α were detected, and β-Actin was used as an internal reference. The nucleotide sequences of the primers are shown in Table 1.

[0055] Table 1 Nucleotide sequences of primers

[0056]

[0057] 1.2 Western blot detection of primitive intestinal marker transcription factors

[0058] After the completion of the second stage of differentiation in the experimental and control groups (after 9 days of routine culture in the blank group), the culture medium was removed from each group, the cells were washed with PBS, 200 μl of RIPA lysis buffer and 2 μl of PMSF (phenylmethylsulfonyl fluoride) were added to each well to allow the lysis buffer and cells to fully contact, and the lysis buffer was collected after a few seconds; the lysed samples were centrifuged (12000 g, 4 minutes), and the supernatant was taken for BCA protein quantification.

[0059] After adjusting each group of proteins to the same concentration, SDS-PAGE electrophoresis (100V, 60min) was performed, and the SDS-PAGE gel was transferred to a PVDF membrane of the same size, and transferred at 200mA for 30min. After blocking with skim milk, HNF1β, HNF4α, and β-Actin specific primary antibodies were used for blocking overnight, and after washing, the secondary antibody was incubated at room temperature for 1h. After washing the secondary antibody, ECL was evenly sprinkled on the PVDF membrane, and imaging analysis was performed after a few seconds of reaction.

[0060] 1.3 In vitro differentiation effect detection by dithizone staining

[0061] After the completion of the third differentiation stage culture in the experimental and control groups (17 days of conventional culture in the blank group), IPCs differentiated from MSCs were stained with the zinc chelating dye dithizone (DTZ).

[0062] The preparation method of DTZ stock solution is as follows: dissolve 10 mg of DTZ in 1 ml of dimethyl sulfoxide (DMSO) and filter through a 0.2 mm filter; use 10 μl of DTZ stock solution to stain cells and incubate at 37°C for 30 minutes; then wash the cells three times with PBS and observe the staining results under a microscope.

[0063] 1.4 ELISA test of in vitro differentiation effect

[0064] After the completion of the third differentiation stage culture in the experimental group and the control group (after 17 days of conventional culture in the blank group), the cells in each group were washed twice with PBS and incubated in DMEM low-glucose fasting medium containing 0.5% BSA for 12 hours; the DMEM medium containing 25mM glucose and 10% FBS was replaced and incubated for 2 hours, and the levels of secreted insulin and c-peptide in the culture medium were determined using human insulin and c-peptide ELISA kits.

[0065] 1.5 Immunofluorescence detection of in vitro differentiation effect

[0066] After the completion of the third culture of the differentiation stage of the experimental group and the control group (after 17 days of conventional culture in the blank group), the cells in each group were washed three times with PBS, fixed with 4% paraformaldehyde solution for 10 minutes at room temperature, and permeabilized in 0.1% Triton X-100 for 10 minutes at room temperature. The cells were washed three times with PBS and blocked with 2% BSA at room temperature for 60 minutes. The blocked cells were incubated with human insulin antibodies and bound at 37°C for 2 hours. Washed three times with PBS and incubated with fluorescent Alexa Fluor 488-labeled secondary antibodies at room temperature in the dark for 1 hour. After washing the cells with PBS, incubated with DAPI for 10 minutes, and the images of fluorescently labeled cells were captured using a fluorescence microscope.

[0067] 2 Results

[0068] 2.1 Results of RT-PCR detection of primitive intestinal marker transcription factors

[0069] RT-PCR test results Figure 1 As shown, the results of RT-PCR test showed that the mRNA expression levels of primitive intestinal marker transcription factors HNF1β and HNF4α in the experimental group were significantly higher than those in the control group, and the expression levels of HNF1β and HNF4α in the blank group were zero.

[0070] 2.2 Western blot results of primitive intestinal marker transcription factors

[0071] WB test results Figure 2 As shown, compared with the control group, the expression levels of HNF1β and HNF4α factor proteins in the experimental group were greatly increased, indicating that the differentiation ability of mesenchymal stem cells cultured into primitive intestinal tubes using the method of the present invention was greatly increased compared with the conventional differentiation culture method.

[0072] 2.3 Results of in vitro differentiation effect dithizone staining test

[0073] The results of dithizone staining were as follows Figure 3As shown, the islet-like cell clusters differentiated from the control group and the experimental group were stained dark red by dithizone, showing dithizone staining positive, while no staining was observed in the blank group.

[0074] 2.4 ELISA test results of in vitro differentiation effect

[0075] ELISA test results Figure 4 As shown, the expression of insulin and C-peptide can be monitored in the culture medium of both the control group and the experimental group. The secretion of insulin and C-peptide in the experimental group was significantly higher than that in the control group, indicating that the efficiency of IPCs in secreting insulin and C-peptide in the experimental group was higher.

[0076] 2.5 Results of immunofluorescence detection of in vitro differentiation effect

[0077] Immunofluorescence test results Figure 5 As shown, the IPCs in the control group and the experimental group were positive for insulin staining, indicating that insulin was produced. The positive rate of IPCs in the experimental group was higher than that in the control group, indicating that the experimental group had a higher proportion of IPCs induced to differentiate into insulin secreting function.

[0078] 3 Conclusion

[0079] The method of the present invention can improve the success rate of primitive intestinal differentiation and improve the differentiation efficiency of IPCs.

[0080] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. An induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells, characterized in that: It is composed of differentiation medium 1, differentiation medium 2 and differentiation medium 3, wherein: The differentiation medium 1 is composed of a basic cell culture medium and the following components at the following concentrations: 0.4 to 0.6 mmol / L of β-mercaptoethanol; The differentiation medium II is composed of a basic cell culture medium and the following components: 0.5% to 1.5% of a non-essential amino acid solution, 50 to 70 ng / mL of bFGF, 30 to 50 ng / mL of EGF, 0.5% to 1.5% of a small molecule peptide solution of the rusty spider hair moss, and 1.5% to 2.5% of a B27 additive; The differentiation medium three is composed of a basic cell culture medium and components of the following concentrations: 20-40 ng / mL of beta-cellulin, 90-110 ng / mL of activin A, and 8-12 mmol / L of nicotinamide.

2. The induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells according to claim 1, characterized in that: In the differentiation medium 1, the concentration of β-mercaptoethanol is 0.5 mmol / L.

3. The induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells according to claim 1, characterized in that: In the differentiation medium II, the concentrations of the components are: 1% of non-essential amino acids, 60 ng / mL of bFGF, 40 ng / mL of EGF, 1% of the small molecule peptide of Aralia rustata, and 2% of the B27 additive.

4. The induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells according to claim 1, characterized in that: In the differentiation medium three, the concentrations of the components are: 30 ng / mL of beta-cell killer, 100 ng / mL of activin A, and 10 mmol / L of nicotinamide.

5. The induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells according to claim 1, characterized in that: The small molecule peptide solution of the rusty spider hair moss is prepared by the following method: cutting the rusty spider hair moss leaves into small pieces, placing them in water, adding a complex enzyme, and performing a shaking hydrolysis treatment; The supernatant is collected by centrifugation, and the supernatant is placed in an ultrafiltration tube for centrifugation, and the liquid phase is collected to obtain an active small molecule polypeptide solution of the rusty spider hair moss. The complex enzyme is composed of a neutral protease and an alkaline protease, and the weight ratio of the two is (0.8-1.2):

1.

6. The induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells according to claim 1, characterized in that: The basic cell culture medium is serum-free high-glucose DMEM culture fluid.

7. A method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells, characterized in that: The method of using the induction medium for directed differentiation of mesenchymal stem cells into insulin-secreting cells according to any one of claims 1 to 6 for differentiation culture comprises the following steps: (1) Differentiation stage 1: mesenchymal stem cells were placed in differentiation medium 1 and cultured at 37°C and 5% CO2 for 3 days; (2) Differentiation stage II: After the above culture, the supernatant was discarded, and differentiation medium II was added, and cultured at 37°C and 5% CO2 for 6 days. During the culture process, electrical stimulation was performed once a day; (3) Differentiation stage three: After the above culture, the supernatant was discarded, and differentiation medium three was added, and cultured at 37° C. and 5% CO 2 for 8 days to obtain insulin-secreting cells.

8. The method for inducing directed differentiation of mesenchymal stem cells into insulin-secreting cells according to claim 7, characterized in that: In the step (1), the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

9. The method for inducing directed differentiation of mesenchymal stem cells into insulin-secreting cells according to claim 7, characterized in that: In the step (2), the conditions of electrical stimulation are: stimulation intensity 32V, stimulation time 30min, frequency 1Hz, 20ms.

10. The method for inducing directed differentiation of mesenchymal stem cells into insulin-secreting cells according to claim 7, characterized in that: In the step (2), the time of electrical stimulation is consistent every day.

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