CN116426468A - Method for inducing mesenchymal stem cells to differentiate into islet-like cell masses - Google Patents

Method for inducing mesenchymal stem cells to differentiate into islet-like cell masses Download PDF

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CN116426468A
CN116426468A CN202310649162.7A CN202310649162A CN116426468A CN 116426468 A CN116426468 A CN 116426468A CN 202310649162 A CN202310649162 A CN 202310649162A CN 116426468 A CN116426468 A CN 116426468A
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differentiation
mesenchymal stem
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荣耀星
钱云臻
于艳秋
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Shenyang Huizhi Cell Industry Technology Innovation Research Institute Co ltd
Shenyang Cell Therapy Engineering Technology Research And Development Center Co ltd
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Shenyang Huizhi Cell Industry Technology Innovation Research Institute Co ltd
Shenyang Cell Therapy Engineering Technology Research And Development Center Co ltd
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Abstract

The invention discloses a method for inducing mesenchymal stem cells to differentiate into islet cell clusters, which comprises the following steps: (1) Taking mesenchymal stem cells, inoculating the mesenchymal stem cells into a culture plate, adding a stem cell basal medium, and culturing for 1 day; (2) Entering a differentiation stage 1, changing the culture solution into a DMEM/F12 culture medium containing 10% of fetal bovine serum, and culturing for 3 days, wherein the liquid change is carried out for 1 time in the culturing process; (3) Entering a differentiation stage 2, changing the liquid into a nerve cell conditioned medium, culturing for 5 days, and changing the liquid for 1 time in the culturing process; (4) Entering a differentiation stage 3, changing the liquid into a differentiation culture medium, and culturing for 9 days, wherein the liquid is changed for 1 time every 2 days in the culturing process; obtaining islet-like cell mass. The method for inducing mesenchymal stem cells to differentiate into islet-like cell clusters can be completed in only 3 differentiation stages for 17 days, and only one differentiation medium is needed, so that the effect is obvious and the method has a good application prospect.

Description

Method for inducing mesenchymal stem cells to differentiate into islet-like cell masses
Technical Field
The invention relates to a method for inducing mesenchymal stem cells to differentiate into islet-like cell clusters, belonging to the technical field of embryo development and tissue regeneration medicine.
Background
Diabetes is a systemic metabolic disease, can cause multiple organ complications, including diabetic nephropathy, fundus angiopathy, diabetic foot and the like, is the disease with the most known complications, and seriously affects the life quality of patients. If the blood sugar control of diabetics is poor, ketoacidosis and hypertonic hyperglycaemia can be induced, and the lives of the diabetics are threatened. Among them, type I diabetes is caused by insulin secretion deficiency caused by insulin beta cell function deficiency, and type II diabetes is caused by relative insulin deficiency caused by insulin resistance in vivo. The chronic management of diabetics is lengthy and tedious, including long-term life interventions, blood glucose testing, oral medication and continuous insulin replacement therapy, and the cumbersome course of treatment reduces patient compliance. Therefore, many studies have been made in recent years to explore the feasibility of cell replacement therapy.
Islet direct transplantation is the earliest cell replacement therapy, and foreign researchers obtain pancreatic samples from patients with just-dead brain, inject collagenase into the main pancreatic duct for digestion, sort islets under a microscope, and transplant to diabetics. However, such a method has significant drawbacks: first, eligible brain death donors are very rare and thus the source of islets is inadequate; secondly, the time for digesting the pancreas by collagenase is difficult to grasp, and the operation of manually separating the pancreatic islets under a microscope is difficult, so that the operation level of an operator is high; again, the islets of the donor, after entering the diabetic patient, are attacked by the host immune system and have poor survival rates due to immune rejection.
The stem cells are abundant in source, and a new idea is developed for the cell replacement therapy of diabetes. Currently, there are hundreds of registered clinical trials of stem cells for treating diabetes worldwide. Some researchers directly inject stem cells into blood vessels or livers of diabetics, and some researchers induce the stem cells to differentiate into islet-like cell clusters with insulin secretion function in vitro and then transplant the islet-like cell clusters into the diabetics in a microcapsule wrapping manner. In 2006, kevin et al established a method of inducing ESCs into islet-like cell masses, which first completed the induction of stem cells in vitro and resulted in cell products capable of secreting insulin, glucagon, somatostatin, etc., with a milestone significance. In 2014, douglas et al established a method of inducing ipscs into islet-like cell mass and demonstrated that this cell mass can secrete insulin into the blood circulation after implantation under the kidney capsule of mice. The U.S. Food and Drug Administration (FDA) approved VX-264 to initiate clinical trials on day 3 and 10 2023, and VX-264 was a stem cell-derived islet encapsulated by an immunoprotection device, with the potential to functionally cure type I diabetes without the use of immunosuppressants.
However, both Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs) present safety concerns, namely potential tumorigenicity. In 2022, 3 months, a tumor hospital researcher in Yunnan province reported a case of immature teratomas after receiving treatment with iPSC-derived cells, and the teratomas were more invasive than the general ones. Stem cells are being noted and valued for their multipotent differentiation potential and also for their differentiation uncertainty. In 2013, wang Ling et al suggested that ESCs are a population of heterogeneous cells, and that residual phenomena during differentiation may be an inherent property. While iPSC activates "oncogenes" such as c-Myc and the like during the preparation of cells. In addition, seed cell sources may also be involved in ethical issues when preparing ESC cells.
The ideal stem cell derived islets should meet the following four conditions: 1. stem cells, which are the origin of differentiation, should be of sufficient origin, easy to prepare, and not involve ethical problems; 2. the islet-like cell mass as the differentiation end point should have sufficient safety guarantee, and will not cause tumor after transplantation; 3. the differentiation process is simple, the operability is strong, and the culture medium of each differentiation stage is easy to prepare; 4. the differentiation effect is stable, and there is sufficient evidence that islet-like cell mass has the function of secreting insulin. However, most of the current schemes for preparing stem cell derived islets use iPSC or ESC, and have the defects of large differentiation uncertainty, difficult directional differentiation, high tumorigenic risk, multiple and complicated differentiation steps, low differentiation efficiency and the like.
The Chinese patent application CN 110423720A discloses a method for inducing and differentiating human amniotic epithelial stem cells into islet beta cells with biological functions, which comprises the following steps: 1) Isolation, culture, expansion and identification of amniotic epithelial stem cells; 2) In vitro induced differentiation: the amniotic epithelial stem cells of the 2 nd to 3 rd generation are selected in the experiment, and induced differentiation is carried out by using a culture medium containing niacinamide. The insulin secretion cell which can express the specific marker of the islet beta cells and has the function of normal islet beta cells is obtained by 14 days in vitro differentiation; 3) And (3) in vivo transplantation: the induced insulin secretion cells are transplanted into the body of a type 1 diabetes mouse, so that the hyperglycemia state of the mouse can be obviously relieved. Two induction media were used for differentiation.
Disclosure of Invention
The invention provides a method for inducing mesenchymal stem cells to differentiate into islet-like cell masses.
The invention is realized by the following technical scheme:
a method of inducing differentiation of mesenchymal stem cells into islet-like cell mass comprising the steps of:
(1) Taking mesenchymal stem cells, inoculating into a culture plate, adding stem cell basal medium, and adding 5% CO at 37deg.C 2 Culturing for 1 day under the condition;
(2) After 1 day of the above culture, the cells adhere to the wall and enter differentiation stage 1, and the culture solution is changed into DMEM/F12 medium containing 10% (volume percent) Fetal Bovine Serum (FBS), 37 ℃ and 5% CO 2 Co-culturing for 3 days under the condition, and changing the liquid for 1 time in the culturing process;
(3) After 3 days of co-culture, the mixture enters differentiation stage 2, and the culture medium is replaced by nerve cell conditioned medium (neural-conditioned medium, NCM) at 37deg.C and 5% CO 2 Co-culturing for 5 days under the condition, and changing the liquid for 1 time in the culture process;
(4) After 5 days of co-culture, the culture enters a differentiation stage 3,changing liquid into differentiation medium, 37 deg.C, 5% CO 2 Co-culturing for 9 days under the condition, wherein 1 liquid exchange is carried out every 2 days in the culture process, and the total addition of the differentiation culture medium is carried out 5 times; obtaining islet-like cell mass;
the differentiation medium consists of the following components: 15 to 20 percent of mM glucose, 8 to 12 percent of mM nicotinamide, 8 to 12 percent of exendin-4 (exenatide), 8 to 12 percent of nM pentapeptide gastrin, 80 to 120 percent of pM hepatocyte growth factor, 8 to 12 mu M of 5-hydroxytryptamine (5-HT), 8 to 12 mu M of butyramide, 0.08 to 0.12 percent mg/ml TPO (thrombopoietin), 1 to 5 percent (volume percent) of B27 serum-free additive and the balance of DMEM/F12 culture medium.
Further, in the step (1), the mesenchymal stem cells are umbilical cord mesenchymal stem cells.
Further, in the step (1), the inoculation density is 2×10 5 Cells/wells.
Further, in the step (1), 2 ml of the medium was added to each well.
Further, in the step (1), the stem cell basal medium is a commercial medium existing in the prior art, and may be selected from daceae's daryou mesenchymal stem cell basal medium (cat# 6114011).
Further, in the step (3), the neural cell conditioned medium is prepared by the following method: taking rat brain, placing in DMEM/F12 medium containing 10% FBS, homogenizing, thoroughly grinding the rat brain, and filtering with 50 μm cell filter screen to obtain cell suspension; cell suspension at 37℃in 5% CO 2 Culturing for 20-28 hours under the condition; taking out the culture supernatant, adding DMEM/F12 medium containing 10% FBS into the supernatant, and adding 5% CO at 37deg.C 2 Culturing for 4 days under the condition; collecting the culture supernatant, centrifuging, and filtering with 0.22 μm cell filter screen to obtain the neural cell conditioned medium.
Further, in the step (4), the differentiation medium is composed of the following components: 17.5 mM glucose, 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, 10. Mu.M 5-hydroxytryptamine, 10. Mu.M butyramide, 0.1 mg/ml TPO,2% B27 serum free additive, balance DMEM/F12 medium.
The method for inducing mesenchymal stem cells to differentiate into islet-like cell masses adopts umbilical cord mesenchymal stem cells which are convenient to obtain clinically and rich in sources, establishes a stable mesenchymal stem cell separation and differentiation system, and uses the mesenchymal stem cells as a starting point to induce the mesenchymal stem cells to differentiate into islet-like cell masses. The uncertainty of the mesenchymal stem cell differentiation is relatively small, the clinical application is safer, and the process of extracting the mesenchymal stem cell does not violate the ethical principle. Although researchers also consider the risk of tumorigenesis of mesenchymal stem cells, many years of clinical use have shown that no such event has occurred. The conventional operation flow for inducing ESC or iPSC differentiation is mostly divided into 4-6 differentiation stages, and different differentiation culture mediums are required to be prepared for each differentiation stage for about one month, so that the preparation process is complicated. The flow of inducing the mesenchymal stem cells to differentiate can be completed in only 3 differentiation stages within 17 days, and the method has short time, and is simple and convenient to operate by only preparing a differentiation culture medium. The method for inducing the mesenchymal stem cells to differentiate into islet-like cell clusters has remarkable effect, has good application prospect, and has important significance for development of cell replacement therapy of diabetes.
The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
Fig. 1: mesenchymal stem cell flow cell identification result is schematically shown, wherein, A: CD34-; b: CD45-; c: cd73+; d: cd90+.
Fig. 2: schematic of the results of dithizone staining of islet-like cell mass.
Fig. 3: the results of staining for nestin in differentiated stage 2 cells are schematically shown.
Fig. 4: insulin staining results for differentiation stage 3 islet cell mass are schematically shown.
Fig. 5: immunofluorescent staining of differentiation stage 3 islet cell mass is schematically shown.
Description of the embodiments
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents, materials, etc. used in the examples described below are conventional instruments, reagents, materials, etc. known in the art, and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods, detection methods, and the like that are known in the prior art unless otherwise specified.
EXAMPLE 1 umbilical cord mesenchymal Stem cell harvesting
The method comprises the following steps:
(1) The umbilical cord was washed with 0.9% physiological saline and then placed in a 10 cm dish. Adding 75% ethanol solution into the culture dish, immersing the whole umbilical cord, and soaking for 5 min. Transferring the umbilical cord into a new culture dish, adding normal saline for washing, and repeatedly washing until blood stains and alcohol are cleaned.
(2) Placing the washed umbilical cord into a new culture dish, cutting off ligature parts at two ends, discarding, and cutting the residual umbilical cord into a plurality of segments with the length of 2-3 cm. Adding physiological saline to wash the umbilical cord cut into small segments, and repeating the washing until blood stains are removed, wherein the washing liquid is clear.
(3) Finding umbilical vein, cutting off small umbilical cord along spiral trend of vein blood vessel, and removing artery and vein in umbilical cord. Tearing off the Whatman's jelly by using tissue forceps, transferring the peeled Whatman's jelly into a 50 ml centrifuge tube, and adding a proper amount of physiological saline into the centrifuge tube in advance to prevent the Whatman's jelly from drying. Shearing the Whatman gum into 1-8 mm in a centrifuge tube 3 Is a small block of (a).
(4) Physiological saline was added to the centrifuge tube to 45 ml, centrifuged for 750 g,10min, and the supernatant was discarded.
(5) Physiological saline was added to the centrifuge tube to 45 ml, centrifuged for 750 g,10min, and the supernatant was discarded.
(6) The Wharton's jelly is inoculated into T75 culture flasks for culture, each T75 culture flask is inoculated with 1 g Wharton's jelly, and 4 ml umbilical cord cell complete culture solution is added. CulturingConditions are as follows: at 37 ℃, CO 2 Concentration 5%, saturation humidity. Mesenchymal stem cells can be observed to climb out of the tissue.
(7) The resulting mesenchymal stem cells were cultured with MSCBM medium.
Example 2 identification of mesenchymal Stem cells
Detecting cell surface markers by flow cytometry, mesenchymal stem cell surface markers: cd73+, cd90+, CD34-, CD45-, steps are as follows:
(1) The 1 ml cell suspension was transferred to a 1.5 ml centrifuge tube. Centrifuge at 4℃for 5 min at 300 g, carefully aspirate and discard the supernatant.
(2) The cells were washed with an appropriate amount of PBS buffer, centrifuged at 300 g for 5 minutes at 4℃and carefully aspirated, and the supernatant was discarded.
(3) Resuspension of cells with pre-chilled PBS buffer to adjust the final cell concentration to 1X 10 7 cells/ml, gently swiping and mixing.
(4) 100 μl of the cell suspension was used as a blank control group, 100 μl of the cell suspension was used as a isotype control group, isotype control antibody was added, 200 μl of the test antibody was added as an experimental group, and the mixture was incubated at 4℃for 30 minutes.
(5) Cells were washed with an appropriate amount of PBS buffer, centrifuged at 300 g for 5 minutes at 4℃and carefully aspirated, and the supernatant was discarded.
(6) The cells were resuspended in 500. Mu.l PBS buffer and checked on the machine.
The results are shown in fig. 1, markers for mesenchymal stem cells: cd73+99.98% and cd90+99.26%. The results show that the cells cultured in example 1 are mesenchymal stem cells, highly express CD73+, CD90+, and lowly express CD34-, CD45-.
EXAMPLE 3 preparation of rat brain nerve cell conditioned Medium
The method comprises the following steps:
(1) Seven-day-old rats were sacrificed and the brains were removed and placed in 10 cm dishes, and PBS buffer was added to thoroughly wash out blood stains.
(2) The rat brain was transferred to a new 10 cm dish and DMEM/F12 medium containing 10% FBS was added. After the mouse brain was cut into small pieces with an ophthalmic scissors, the pieces were transferred to a 25 ml-volume glass tissue homogenizer, and 5 ml DMEM/F12 medium containing 10% FBS was added. After thoroughly grinding the rat brain, 10 ml DMEM/F12 medium containing 10% FBS was fed to the glass homogenizer.
(3) A new 10 cm petri dish was taken and a 50 μm pore size cell strainer was placed on top. The cell suspension obtained in the glass homogenizer is sucked by a rubber head dropper, is dripped on a filter screen, and is filtered to obtain the cell suspension. Placing the sample at 37deg.C and CO 2 The culture was carried out at a concentration of 5% for 24 hours.
(4) After 24 hours of culture, the cultures were observed, the supernatant was neural cells, and oligodendrocytes had attached. Therefore, the supernatant was transferred to a new 10 cm dish, and the culture was continued for four days with the addition of 15 ml DMEM/F12 medium containing 10% FBS.
(5) The supernatant was collected into a 50 ml centrifuge tube and centrifuged at 1000 g for 15 minutes. The supernatant obtained by centrifugation was withdrawn in portions using a 20 ml syringe, passed through a 0.22 μm cell filter, and then injected into a new 50 ml centrifuge tube to obtain NCM.
EXAMPLE 4 Induction of mesenchymal Stem cell differentiation
The method comprises the following steps:
(1) Umbilical cord mesenchymal stem cells obtained in example 1 were seeded in six-well plates at a seeding density of 2X 10 5 Cells/well, each well was added with 2 ml stem cell basal medium (Dayou mesenchymal stem cell basal medium for Dakeda, cat# 6114011), 37 ℃, CO 2 Culturing under the condition of 5% concentration.
(2) After the next day of cell attachment, the cells enter a differentiation stage 1, and the cells are replaced by DMEM/F12 medium containing 10% FBS, co-cultured for 3 days, and the cells are replaced for 1 time in the middle.
(3) The medium prepared in example 3 was used as the medium for the differentiation stage 2, and the medium was co-cultured for 5 days with 1 medium change.
(4) And (3) entering a differentiation stage 3, changing the liquid into a differentiation medium, co-culturing for 9 days, and 1 liquid change every 2 days in the middle, and adding the differentiation medium 5 times in total.
The differentiation medium consists of the following components: 17.5 mM glucose, 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, 10. Mu.M 5-hydroxytryptamine (5-HT), 10. Mu.M butyramide, 0.1 mg/ml TPO (thrombopoietin), 2% B27 serum free additive, the balance DMEM/F12 medium.
(5) The cell mass in the six-hole plate is gently picked by a needle point, and the cell mass is the islet-like cell mass.
Example 5 in vitro differentiation Effect observations
When the mesenchymal stem cells are initially inoculated, the mesenchymal stem cells are tiled single-layer cells; after entering the differentiation stage 3, the cells spontaneously aggregate to form islet-like cell clusters with the diameter of 300-2000 mu m. For these cell clusters, corresponding staining can be performed to examine the effect of differentiation in vitro.
Dyeing with (one) dithizone
Dithizone staining is the most common and convenient method for identifying islets. The zinc ions contained in the islet beta cells can be chelated with dithizone to form a cherry red complex.
(1) 10 mg dithizone is dissolved in 1 ml DMSO, and then diluted with PBS buffer solution according to the ratio of 1:100 to obtain dithizone staining working solution.
(2) Dithizone staining working solution, 2 ml per well, was added to the 6-well plate and incubated at 37℃for 30 min.
(3) The coating was performed by washing three times with PBS buffer, then adding glycerol, and observing under a light microscope.
The results are shown in FIG. 2, and the differentiated islet cell mass dithizone staining was positive.
(II) immunohistochemical staining
(1) The cell product obtained by differentiation is picked up, 4% paraformaldehyde solution is added, and the mixture is fixed for 60 min. The immobilized cell product was paraffin-embedded and then serial sections were performed, each section having a thickness of 4 μm, and the sections were attached to a glass slide.
(2) Paraffin sections were successively placed in xylene i 10min, xylene ii 10min, xylene iii 10min, absolute ethanol i 5 min, absolute ethanol ii 5 min,90% ethanol solution 5 min,80% ethanol solution 5 min,70% ethanol solution 5 min,50% ethanol solution 5 min, and gradient dewaxed.
(3) The wells were permeabilized with 0.5% Triton X-100 for 10 min.
(4) The sections were washed 3 times with PBS buffer and then soaked in 3% hydrogen peroxide solution (diluted with methanol) for 15 min to block endogenous peroxidase.
(5) The sections were immersed in 10 mM citric acid solution and heated to 100 ℃ for 10min to repair the antigen. The slices were then left to cool slowly and thoroughly at room temperature along with the citric acid solution.
(6) The sections were washed 3 times with PBS buffer, then transferred into a wet box, 10% Bovine Serum Albumin (BSA) solution was added dropwise to the surface, and blocked at room temperature for one hour.
(7) And (5) throwing off sealing liquid and sucking up water stains. Differentiation of stage 2 cells, drop-in of neuregulin antibodies (CST, 10959s,1:200 diluted in PBS buffer); cell mass at stage 3 was differentiated, insulin antibody (CST, 3014S,1:1000 diluted in PBS buffer) was added dropwise. Incubate overnight at 4 ℃.
(8) After the sections were rewet for one hour at room temperature, the sections were washed 3 times with PBS buffer and then with GTVision TM Detection System/Mo & Rb (Genetech, GK 800511) was developed and incubated at room temperature for 10 minutes.
(9) DAB staining solution was washed with PBS buffer, then hematoxylin was added dropwise, and counterstaining was performed for 90 seconds.
(10) Dewatering by gradient cylinder method, and sealing with neutral resin.
(11) And (5) observing and photographing under a light mirror.
As a result, the cells obtained in the stage 2 were differentiated and the nestin staining was positive as shown in FIGS. 3 and 4. The islet-like cell mass obtained in the differentiation stage 3 was positive for insulin staining.
Analysis of results: at the end of differentiation stage 2, the mesenchymal stem cells have differentiated into neuroendocrine cells. At the end of differentiation stage 3, the mesenchymal stem cells have differentiated into cells with insulin-producing function.
(III) immunofluorescent staining
(1) Fixing, embedding, slicing, dewaxing, perforating, sealing by catalase, and thermally repairing and sealing the sample.
(2) Cell mass at stage 3 was divided into two groups, one group with insulin antibody (CST, 3014S,1:1000 diluted in PBS buffer) and one group with C-peptide antibody (CST, 4593S,1:100 diluted in PBS buffer). Incubate overnight at 4 ℃.
(3) After the sections were rewarmed for one hour at room temperature, the sections were washed 3 times with PBS buffer, and then fluorescent secondary antibodies (Immunoway, RS3211, RS 3811) were added dropwise and incubated for 1 hour at room temperature in the dark.
(4) The sections were washed 3 times with PBS buffer, then DAPI staining solution was added dropwise, and incubated at room temperature for 10min in the dark.
(5) The sections were washed 3 times with PBS buffer, then anti-quenching caplets were added dropwise, and the tablets were capped.
(6) And (5) observing and photographing under a confocal microscope.
The results are shown in FIG. 5, where the islet-like cell mass obtained in stage 3 of differentiation was positive for insulin and C-peptide staining.
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.

Claims (7)

1. A method of inducing differentiation of mesenchymal stem cells into islet-like cell masses, comprising the steps of:
(1) Taking mesenchymal stem cells, inoculating into a culture plate, adding stem cell basal medium, and adding 5% CO at 37deg.C 2 Culturing for 1 day under the condition;
(2) After 1 day of the above culture, the cells adhere to the wall and enter differentiation stage 1, and the culture solution is changed into DMEM/F12 medium containing 10% fetal bovine serum, 37 ℃ and 5% CO 2 Co-culturing for 3 days under the condition, and changing the liquid for 1 time in the culturing process;
(3) After 3 days of co-culture, the mixture enters a differentiation stage 2, and the liquid is changed into a nerve cell conditioned medium with the temperature of 37 ℃ and the concentration of CO of 5 percent 2 Co-culturing for 5 days under the condition, and changing the liquid for 1 time in the culture process;
(4) After 5 days of co-culture, the mixture enters a differentiation stage 3, and the liquid is changed into a differentiation medium at 37 ℃ and 5 percent CO 2 Co-culturing for 9 days under the condition, wherein 1 liquid exchange is carried out every 2 days in the culture process, and the total addition of the differentiation culture medium is carried out 5 times; obtaining islet-like cell mass;
the differentiation medium consists of the following components: 15-20 g mM glucose, 8-12 g mM nicotinamide, 8-12 nM exendin-4, 8-12 g nM pentagastrin, 80-120 g pM hepatocyte growth factor, 8-12 mu M5-hydroxytryptamine, 8-12 mu M butyramide, 0.08-0.12 g mg/ml TPO, 1-5% B27 serum-free additive, the balance DMEM/F12 medium.
2. The method of inducing differentiation of mesenchymal stem cells into islet-like cell mass according to claim 1, wherein: in the step (1), the mesenchymal stem cells are umbilical cord mesenchymal stem cells.
3. The method of inducing differentiation of mesenchymal stem cells into islet-like cell mass according to claim 1, wherein: in the step (1), the inoculation density is 2 multiplied by 10 5 Cells/wells.
4. The method of inducing differentiation of mesenchymal stem cells into islet-like cell mass according to claim 1, wherein: in the step (1), 2 ml of the culture medium was added to each well.
5. The method of inducing differentiation of mesenchymal stem cells into islet-like cell mass according to claim 1, wherein: in the step (1), the stem cell basal medium is a daryou mesenchymal stem cell basal medium.
6. The method of inducing differentiation of mesenchymal stem cells into islet-like cell mass according to claim 1, wherein: in the step (3), the nerve cell conditioned medium is prepared by the following method: rat brain was takenPlacing in DMEM/F12 medium containing 10% FBS, homogenizing, thoroughly grinding rat brain, and filtering with 50 μm cell filter screen to obtain cell suspension; cell suspension at 37℃in 5% CO 2 Culturing for 20-28 hours under the condition; taking out the culture supernatant, adding DMEM/F12 medium containing 10% FBS into the supernatant, and adding 5% CO at 37deg.C 2 Culturing for 4 days under the condition; collecting the culture supernatant, centrifuging, and filtering with 0.22 μm cell filter screen to obtain the neural cell conditioned medium.
7. The method of inducing differentiation of mesenchymal stem cells into islet-like cell mass according to claim 1, wherein: in the step (4), the differentiation medium consists of the following components: 17.5 mM glucose, 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, 10. Mu.M 5-hydroxytryptamine, 10. Mu.M butyramide, 0.1 mg/ml TPO,2% B27 serum free additive, balance DMEM/F12 medium.
CN202310649162.7A 2023-06-02 2023-06-02 Method for inducing mesenchymal stem cells to differentiate into islet-like cell masses Pending CN116426468A (en)

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