CN115044534B - Method for producing islet beta cells in vitro by utilizing BMP7 factor, obtained islet beta cells and application - Google Patents
Method for producing islet beta cells in vitro by utilizing BMP7 factor, obtained islet beta cells and application Download PDFInfo
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Abstract
The invention discloses a method for producing islet beta cells in vitro by utilizing BMP7 factor, the obtained islet beta cells and application thereof, and belongs to the technical field of biomedicine. To provide a method for utilizing bile duct and pancreatic duct-derived epithelial cells and BMP7 factor and C 17 H 22 N 2 O 4 S methods for obtaining islet cells. The invention provides a method for culturing biliary tract organoid or pancreatic duct organoid in proliferation medium containing BMP7 factor to obtain multipotent pancreatic progenitor cells, and then placing multipotent pancreatic progenitor cells in proliferation medium containing C 17 H 22 N 2 O 4 Islet beta cells are obtained from the differentiation medium of S. The method can be used for preparing artificial islet system transplantation for treating diabetes.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a method for producing islet beta cells in vitro by utilizing BMP7 factor, the obtained islet beta cells and application.
Background
Cell replacement therapy can maintain a healthy life for diabetic patients with beta cell destruction. However, the lack of a pancreatic donor has made islet transplantation a dilemma in the treatment of diabetes, and some evidence suggests that progenitor cells capable of differentiating into insulin-secreting cells exist in adult pancreatic ducts. However, for patients in need of cell transplantation, immune rejection of donor tissue and xenogeneic cells still needs to be overcome.
Recent studies have shown that progenitor cells removed from the patient's own bile duct by microsurgery can be expanded to support organoids and that biliary tract diseases can be cured after implantation through the bile duct or hepatic portal vein. These findings emphasize a pathway for repairing damaged gastrointestinal organs using the patient's own progenitor cells. The challenge with using ductal progenitor cells is that the efficiency of beta cell differentiation is quite low both in vivo and in vitro. A recent report by hund M showed that only 1% of insulin positive cells were derived from organoids grown from epcam+tsq-pancreatic ductal progenitors. Over the past few decades, a variety of cytokines and signal inhibitors have been explored to increase beta cell differentiation, including nicotinamide, noggin, retinoic acid, and Exendin 4 have been shown to increase insulin expression in ES or iPS-derived pancreatic progenitor cells, whereas these molecules promote beta cell differentiation only after conversion of ductal cells into endocrine cells.
Chemical methods have proven to be useful tools for promoting committed differentiation of stem/progenitor cells. In particular, in the field of embryonic stem cells, chemical small molecules have become a powerful strategy for inducing ES or iPS cells to differentiate hepatocytes efficiently. In addition to those known activators and inhibitors of pancreatic developmental cell signaling pathways, there is still a lack of clear chemical molecules to modulate unknown pathways that enhance beta cell differentiation.
Disclosure of Invention
The invention aims to provide a method for utilizing epithelial cells derived from bile duct and pancreatic duct and BMP7 factor and C 17 H 22 N 2 O 4 S methods for obtaining islet cells.
The invention provides a method for producing islet beta cells in vitro, which comprises the steps of placing organoids in a culture medium containing BMP7 factor for culture, and obtaining islet beta cells.
Further defined, the culturing step is: the method comprises the steps of culturing the pancreatic progenitor cells in a proliferation medium containing BMP7 factor to obtain the pluripotent pancreatic progenitor cells, and culturing the pluripotent pancreatic progenitor cells in a differentiation medium to obtain islet beta cells.
Further defined, the organoid is a biliary organoid or a pancreatic ductal organoid.
Further defined, the concentration of said BMP7 factor is 25-200ng/mL.
Further defined, the medium further comprises small molecule C17H 22 N 2 O 4 S。
Further defined, the C 17 H 22 N 2 O 4 The concentration of S is 1 mu M-10 mu M.
The invention provides islet beta cells obtained by the above method.
The invention provides application of the islet beta cells in preparing a cell medicine for treating diabetes or preparing a cell medicine for reducing blood sugar.
Further defined, the cytodrug comprises an extracellular matrix compatible with the cells and a pharmaceutical carrier.
Further defined, the cytodrug comprises an extracellular matrix compatible with the cells and a pharmaceutical carrier.
Further defined, the pharmaceutical carrier is a nanomaterial or a microfluidic.
Differentiation medium: mouse pancreatic differentiation medium (mouse pancreatic differentiation medium, mDM) formulation: includes high sugar DMEM medium, 1% Penicillin-Streptomycin Solution (Hyclone; SV 30010), 1% glutamine additive, 1%B27 Supplement,1%ITS (Thermo; 51500056), 50ng/mL VEGF,50ng/mL Exendin 4, 25ng/mL NOGGIN,750ng/mL RSPO1,1 μ M T3 (TOCRIS; 6666), 10 μ M A8301,5 μM DAPT,10 μg/mL heparin 100ng/mL Activin A (PEPTEOCH, 120-14P), 20ng/mL FGF2,4.4mM Nicotinamide (Sigma; N0636) and 1 μM Retinoic acid.
Human pancreas differentiation medium (human pancreatic differentiation medium, hDM 9) formulation: comprises high sugar DMEM medium, 1% Penicillin-Streptomycin Solution,1% glutamine additive, 2%B27 Supplement,1%N2 Supplement,1%ITS,50ng/mL VEGF,50ng/mL Exendin 4, 25ng/mL NOGGIN,750ng/mL RSPO1,1 μ M T3, 10 μ M A8301,5 μM DAPT,10 μg/mL heparin, 100ng/mL Activin A,20ng/mL FGF2, 20ng/mL,4.4mM Nicotinamide and 1 μM Retinoic acid.
Proliferation medium: human proliferation medium (human expansion medium, hEM) formulation: 1% Penicillin-Streptomycin Solution,1% glutamine additive, 1%B27 without vitamin A (Thermo; 12587010), 50ng/mL EGF,50ng/mL FGF10, 25ng/mL NOGGIN,1% N2, 10nM gamin, 3 μM PGE2 (SIGMA; P0409), 5 μ M A83-01,1mol/L Nicotinamide,1mol/L N-acetyl-L-cysteine, 100ng/mL R-Spondin-1, and D/F12 to 40mL;
mouse proliferation medium (mouse expansion medium, mEM) formulation: 1% Penicillin-Streptomycin Solution,1% glutamine additive, 1%B27 without vitamin A,50ng/mL EGF,50ng/mL FGF10, 25ng/mL NOGGIN,1mol/L Nicotinamide,1mol/L N-acetyl-L-cysteine, 100ng/mL R-Spondin-1 plus D/F12 to 40mL;
the beneficial effects are that: progenitor cell populations with long-term organogenesis ability were identified from mouse bile ducts (intrahepatic and extrahepatic) and pancreatic ducts and demonstrated similar self-renewal and multipotent potential. BMP7 is a potent cytokine that converts the ductal progenitor cells into a pluripotent state and enhances the secretory differentiation inwards. By screening chemical molecules, TLY142 was found to greatly increase the efficiency of differentiation of mBDO into insulin-secreting cells based on BMP7 starting conditions. Human bile duct progenitor cells have long-term organogenesis and pluripotency, TLY142 enhances the differentiation of hBDO to insulin secreting cells by more than 20%, and transplantation of differentiated mBDO and hBDO in vivo can be effective in stimulating glucose and curing hyperglycemia in diabetic mice.
The results of the present invention extend the use of bile duct epithelial cells as a potential source of beta cells in diabetes cell replacement therapies. Progenitor cells isolated from autologous bile ducts have been shown to form organoids and to be able to repair damaged bile ducts by feedback transplantation. The bile duct progenitor cells can be isolated from bile duct epithelial cells using markers, and these epithelial cells are more readily obtained from autologous bile ducts as well as donated bile ducts by microsurgery.
The differentiated biliary organoids have similar endocrine cell lineages to the pancreatic ductal organoids, particularly insulin secreting cells (fig. 4C and 4D). Transplantation of differentiated human BDO effectively heals diabetes in model mice, demonstrating that biliary progenitor cell-derived organoids are a reliable source of cell transplantation.
The high proportion of beta cells differentiated from catheter organoids that deal with glucose changes provides a viable source of beta cells for cell transplantation therapy for diabetes. BMP7 and the chemical small molecule TLY142 are critical to enhance organoid differentiation into beta cells. Supplementing BMP7 is important for organoid conversion to ngn3+ endocrine progenitor state, consistent with previous reports that BMP7 significantly increased expression of Ngn 3. Thus, after BMP7 withdrawal we defined a DM9 medium that significantly increased the proportion of insulin secreting cells to 7% of total cells, higher than the control group without BMP7 added (1%).
The present invention has found a chemical molecule named TLY142 that significantly increases beta cell differentiation from 7% to over 20%. In addition, differentiated insulin secreting cells can respond to glucose stimulation in vitro and in vivo. An increase in the percentage of insulin secreting cells from mouse or human BDO ensures a hypoglycemic effect following kidney subcapsular transplantation.
In summary, epithelial cells in pancreatic or bile ducts are of multiple differentiation potential and can form long-term cultured organoids in vitro. They can be used to treat diabetes by differentiating into a high proportion of functional beta cells by a defined medium, acting as replacement islets to reduce blood glucose.
Drawings
FIG. 1 is a graph of the results of organoids prepared using human bile duct epithelium, mouse bile duct, and pancreatic duct epithelium, respectively. The pictures show cell patterns of prepared human biliary organoids hdpo, mouse biliary organoids mbpo and mouse pancreatic organoids mPDO after 10 passages.
FIG. 2 is a graph showing the results of immunostaining for identifying organoid pluripotency-pancreatic progenitor cell specific gene expression, wherein A is the detection of PDX1, EPCAM expression in hBDO, mBDO and mPDO; b is immunostaining to detect SOX9, E-cadherin expression in hBDO, mBDO and mPDO.
FIG. 3 is a graph showing the results of immunostaining for identification of organoid pluripotency, liver and gall progenitor cell specific gene expression, wherein A is the detection of HNF1B, E-cadherein expression in hBDO, mBDO and mPDO; b is the detection of HNF4A, E-cadherein expression in hBDO, mBDO and mPDO by immunostaining.
FIG. 4 is a graph showing the results of the differentiation of BMP7 in combination with mDM to promote secretion of mBDO and mPDOinto the pancreas, wherein A is the detection of NGN3 expression in mBDO and mPDO7 days after induction of BMP7 by quantitative PCR; b is quantitative PCR detection of mRNA expression of pancreatic exocrine cell specific genes in mBDO and mPDO after induced differentiation of BMP7 in combination with mDM9 for 21 days; c is the expression of pancreatic exocrine cell specific gene proteins in mBDO after 21 days of induced differentiation by combining immunostaining with BMP7 and mDM 9; d is the expression of pancreatic exocrine cell specific gene proteins in mPDOs 21 days after immunostaining detection of BMP7 in combination with mDM9 induced differentiation.
FIG. 5 is a graph showing the results of BMP7 in combination with TLY 142-containing mDM in promoting differentiation of mBDO into pancreatic endocrine cells, wherein A is the quantitative PCR assay of mRNA expression of pancreatic endocrine cell specific genes in mBDO 21 days after induced differentiation of BMP7 in combination with TLY 142-containing mDM 9; b is the expression of pancreatic endocrine cell specific gene proteins in mBDO 21 days after immunostaining for detection of BMP7 in combination with TLY 142-containing mDM9 induction differentiation.
FIG. 6 shows the results of differentiation efficiency and physiological cell function assays of mBDO and mPDOs induced by BMP7 in combination with mDM9, wherein A is the ratio of beta cell differentiation in mBDO and mPDOs after 21 days of induced differentiation by BMP7 in combination with mDM 9; b is a statistical graph of the differentiation proportion of beta cells in the mBDO and the mPDO after the induced differentiation of BMP7 and mDM is detected by flow cytometry for 21 days; c is the secretion of peptide C by Elisa, stimulated with glucose by mBDO and mPDO 21 days after induced differentiation of BMP7 in combination with mDM 9.
FIG. 7 shows the results of the differentiation efficiency and physiological function of cells after induction of mBDO by BMP7 in combination with mDM containing TLY142, wherein A is the differentiation ratio of beta cells in mBDO after 21 days of induced differentiation of BMP7 in combination with mDM containing TLY 142; b is a statistical graph of the differentiation proportion of beta cells in the mBDO after 21 days of induced differentiation of BMP7 in combination with mDM9 containing TLY 142; c is BMP7 and after 21 days of induced differentiation in combination with mDM containing TLY142, mBDO was stimulated with glucose and elas detected for C peptide secretion.
FIG. 8 shows TLY142 (C 17 H 22 N 2 O 4 S) chemical structure diagram.
FIG. 9 is a graph showing the results of activity assays in transplanted diabetic mice after induced differentiation of mBDO by BMP7 in combination with mDM or mDM containing TLY142, wherein A is the graph showing the change in blood glucose after induced encapsulation of kidney of mice from mBDOs to STZ after induced differentiation of BMP7 in combination with mDM containing TLY142 for 21 days; b and C are glucose tolerance changes in mice after transplantation; d is the change in the level of ins in mouse serum; e is 8 weeks after transplantation of BMP7 under the kidney capsule of mBDOs after 21 days of induced differentiation in combination with mDM containing TLY142, transplanted kidney was removed and immunostained for detection of insulin and glucon expression.
FIG. 10 is a graph showing the results of BMP7 in combination with TLY 142-containing hDM to promote differentiation of hBDO into pancreatic endocrine cells; wherein, quantitative PCR detects hBDO NGN3 expression after induced by BMP7 with different concentration for 7 days; b is quantitative PCR detection of mRNA expression of pancreatic endocrine cell specific genes in hBDO 21 days after induced differentiation of BMP7 in combination with hDM9 containing 1 μMTLY 142; c is the expression of pancreatic endocrine cell specific gene proteins in hBDO 21 days after immunostaining to detect BMP7 in combination with mDM9 containing 1 μm TLY 142; d is quantitative PCR to detect INS gene mRNA expression in hBDO 21 days after BMP7 induced differentiation in combination with hDM9 containing 10 μmtly 142.
FIG. 11 shows the results of the differentiation efficiency and physiological cell function assays of hBDO after induction by BMP7 in combination with hDM containing TLY142, wherein A is the proportion of beta cells in hBDO after 21 days of induced differentiation by flow cytometry to detect BMP7 in combination with hDM containing TLY 142; b is a statistical graph of the differentiation proportion of beta cells in hBDO after flow cytometry detection of the induced differentiation of BMP7 in combination with hDM9 containing TLY142 for 21 days; c is BMP7 and after 21 days of induced differentiation in combination with hDM containing TLY142, hBDO was stimulated with glucose and elas detected for C peptide secretion.
FIG. 12 shows the results of activity assays in mice transplanted with diabetes after induced differentiation of hBDO with BMP7 in combination with hDM containing TLY142, wherein A is a graph showing the change in blood glucose after induced encapsulation of kidney of mice transplanted with diabetes by hBDOs to STZ after 21 days of induced differentiation of BMP7 in combination with mDM containing TLY 142; b and C are glucose tolerance changes in mice after transplantation; d is the change in the level of ins in mouse serum. E is that after 8 weeks of subcapsular transplantation of hBDOs kidney after induction of differentiation of A BMP7 in combination with TLY 142-containing hDM, transplanted kidney was removed and examined for expression of insulin and glucagon by immunostaining.
Detailed Description
1- (3, 4-xylyl) sulfonyl-5- (pyrrolidine-1-carbonyl) pyrrolidin-2-one with the English name 1- (3, 4-Dimethylphenyl) sulfophenyl-5- (pyrrosidine-1-carboyl) pyrrosidin-2-one, pubchem CID 53008166, molecular formula C 17 H 22 N 2 O 4 S, named TLY142 in the present invention, is shown in FIG. 8.
The brand and trade name of recombinant human bone morphogenic protein-7 (BMP 7) was purchased from Biyun (Recombinant Human BMP-7, P5772-100 μg).
Bile duct tissue of animals and humans
C57BL/6N and NU/NU immunodeficiency mice were purchased from Experimental animal technologies limited, beijing, vitrenly, raised in SPF laboratory at the university of forestry, university of northeast student, and raising conditions strictly followed the SPF laboratory.
Intrahepatic and extrahepatic bile ducts were collected from adjacent non-tumor tissue of patients obtained from hepatectomy in a second affiliated hospital of the university of halbine medical science. All patients participating in the present invention provided informed consent. The invention is approved by the research ethics committee.
Differentiation of PDO/BDO into pancreatic cell lineages
The well-grown mPDOs or mPDOs were taken and cultured with mEM (noggin-free) containing 100ng/mL BMP7 for 7 days after passage, and replaced with fresh mDM9 for 14 days. The medium was changed every other day. And after differentiation is finished, RNA samples and immunostained samples are collected for identification, and the differentiation efficiency is detected by a flow cytometer. The main components of mDM include high sugar DMEM medium, 1% Glutamax, 1% penicillin-streptomycin, 2% B27 supplement,1% ITS,50ng/mL VEGF,50ng/mL Exendin 4, 25ng/mL Noggin,750ng/mL RSPO1,1 μ M T3, 10 μ M A8301,5 μ DAPT,10 μ g/mL heparin, 100ng/mL Activin A,20ng/mL FGF2,4.4mM Nicotinamide and 1 μ M retinoic acid. hBDO was subjected to the same pancreatic differentiation procedure as mice using human pancreatic beta cell differentiation medium (hDM 9) based on mDM9 and 1% N-2 supplement.
Extraction of Total RNA and Synthesis of cDNA
The Trizol method is used to extract total RNA of cells. According toQSelect RT SuperMix Kit (catalog number R233-01). The synthesized cDNA was aliquoted and stored at-80℃for quantitative real-time PCR (qPCR).
Real-time PCR
The primers are shown in Table 1, the reaction system and condition reference ChamQ Universal SYBR qPCR Master Mix Kit (Cat. No. Q711-02), 3 repeats are set for each gene, beta-actin is used as reference gene, and the relative expression level of the target gene is calculated by Ct method.
Frozen section and organoid patch
Frozen section preparation
The tissue or cultured organoids were fixed with 4% pfa at room temperature for 15min. The tissue or organoids were then dehydrated overnight at 4 ℃ in 30% sucrose, then embedded in OCT and approximately 10 μm sections were prepared using a constant temperature cryomicrotome.
Preparation of organoid patches
The cultured organoids were digested with dispersing enzymes, matrigel was removed, then fixed with 4% pfa, and washed with cold PBS to ensure matrigel clearance. During the washing process, the organoids may naturally settle overnight at 4 ℃ to ensure organoid recovery. The recovered organoids were resuspended in PBS, and were dropped onto a glass slide, dried at 37℃and immunofluorescent stained after drying.
Immunofluorescent staining
The wells were punched with 0.3% TritonX-100 and 10% horse serum (Meta fir gold bridge, ZLI 9024) was incubated at 37℃for 1h for blocking. Primary antibodies (table 2) were incubated overnight at 4 ℃. The secondary antibodies (Table 3) were incubated at room temperature for 2h in the dark. Nuclear staining was performed using Hoechst 33342, and after sealing with anti-fluorescence quenching sealing tablet, fluorescence detection results were observed using high resolution cell imager Delta vision.
Flow cytometry-analysis
The cultured organoids were blown with pre-chilled high-sugar DMEM medium, centrifuged to remove Matrigel, and repeated to ensure Matrigel clearance. Organoids were digested into single cells using 0.25% trypsin. At least 5X 10 was taken from each set of experiments 5 Individual cells. Cells were fixed on ice using 4% pfa, perforated with 0.1% triton x-100 and blocked with 5% bsa. Primary antibody diluted in 1% bsa was added and incubated on ice for 40min with isotype antibody dilution as control. Dilution of the secondary antibody in 1% BSA1:100) and incubated on ice for 30min in the absence of light. After washing the cells with PBS, the cells were resuspended in PBS and loaded for detection.
Glucose stimulated C-peptide secretion
The matrigel-removed organoids were resuspended in sugarless Krebs solution, washed 2 to 3 times, and then incubated in a low-adsorption culture plate overnight. The next day, krebs solution containing 2mM glucose was added to re-suspend the organoids after removal by sugar-free Krebs centrifugation. After incubation for 10 minutes, the supernatant was collected by centrifugation. Cells were washed with sugarless Krebs, then resuspended in 20mM glucose Krebs solution and incubated for 10 minutes, and the supernatant was collected by centrifugation. Samples of the supernatant were analyzed for C-peptide levels using a mouse/human C-peptide ELISA kit (mlbrio, ml-1015542) according to standard protocols.
Animal model
Nude mice starved for 16h or more were injected with STZ at a concentration of 160mg/kg and blood glucose levels were measured after 72 hours. Mice with blood glucose exceeding 16.8mmol/L for 3 consecutive days were used for kidney capsule inferior transplantation surgery, and blood glucose was then monitored.
Organ transplantation under kidney capsule of diabetic mice
After differentiation culture, matrigel around organoids was removed using pre-chilled high glucose DMEM medium. After centrifugation, organoids were collected for subcapsular kidney transplantation. About 10 grafts per mouse 6 Individual cells and organoids were resuspended using 30% matrige for transplantation. Eight weeks after transplantation, kidney of transplanted cells was removed, PFA was fixed, sucrose was dehydrated, and frozen sections and immunostaining were performed.
IPGTT glucose tolerance test
Abdominal glucose tolerance test (IPGTT), mice fasted for 16h and were free to drink. The tail of the fully awake mice was collected with baseline blood samples, and then D-glucose (2 g/kg body weight) was intraperitoneally injected, and blood glucose was monitored with a rogowski glucometer for 15, 30, 60, 90, 120min tail vein blood samples after glucose administration. Area under the curve (AUC) of IPGTT was determined using the trapezoidal rule. Serum samples before and after glucose injection were collected at the same time, and the secretion of insulin was measured by ELISA.
Statistical analysis
Three replicates were run for each experiment. Analysis was performed using one-way analysis of variance (ANOVA), comparing two or more doses, and then the Dunnett test was performed for multiple group comparison (as applicable) with a single control group. * P <0.05, < P <0.01, < P <0.001 are considered statistically significant.
Preparation of organoids from mouse bile duct and pancreatic main duct
Mouse bile duct (mBD) and mouse pancreatic duct (mouse pancreatic duct, mPD) were obtained by direct dissection under a dissecting microscope, then digested with collagenase IV for 20min at 7 ℃, then mechanically sheared, washed and centrifuged to resuspend cells in Matrigel. (Corning, 54234) and 600. Mu.L of mouse amplification medium (mEM) were added to 24-well plates. All cells were incubated in a 37℃humidified incubator with 95% air and 5% CO2 and the freshly prepared medium was kept at 4℃for 2 weeks. The main components of the mouse amplification medium (mEM) included D/F12 basal medium, 1% Glutamax, 1% penicillin-streptomycin, 2% B27 supplement (vitamin A removed), 30% Wnt3A conditioned medium, 1mM N-acetylcysteine, 50ng/mL EGF, 100ng/mL R-Spondin-1 (RSPO 1), 25ng/mL Noggin, 100ng/mL FGF10, 10mM Nicotinamide, and 10. Mu. M Y27632. After obtaining the luminal-like organoids, the medium was replaced with EM without Wnt3A and Y27632.
Cultured mouse biliary organoids (BDO) and pancreatic ductorganoids (mPDO) were changed 1 time every other day at 1:3 were passaged 1 time.
Preparation of human bile duct organoids
A donated human bile duct sample (approximately 1 cm long) was taken for preparation of human bile duct organoids (hBDO). Briefly, samples were placed in 10cm petri dishes containing sterile PBS, the catheter was cut longitudinally with dissecting scissors, and the catheter surface was scraped with a sterile slide. After rinsing the catheter with sterile PBS, the rinse was collected, and the pellet was resuspended in Matrigel and allowed to stand at 37℃for 10 minutes. After solidification, hEM medium containing 30% Wnt3A and 10. Mu. M Y-27632 was added and incubated at 37℃under 5% CO 2. When cells proliferated into lumen-like structures, they were switched to normal hEM medium and fresh hEM medium needed to be changed every other day. Passaging is carried out 2 times per week, and the passaging ratio is 1:3-1:5. To the human amplification medium (hEM), 10nm gartin, 3. Mu.M PGE2, 5. Mu. M A8301 were added.
Human proliferation medium (human expansion medium, hEM) formulation: 1% Penicillin-Streptomycin Solution (HyClone; SV 30010), 1% glutamine additive (gibco; 35050-061), 1%B27 without vitamin A (Thermo; 12587010), 50ng/mL EGF (MCE; HY-P7067), 50ng/mL FGF10 (PeproTech; 100-26-25), 25ng/mL NOGGIN (PeproTech; 120-10C-20), 1% N2 (Thermo; 17502001), 10nM gamin (SIGMA; G9145), 3. Mu.M PGE2 (SISH; P0409), 5. Mu. M A83-01 (MCE; HY-10432), 1mol/L Nicotinamide (Sigma; N0636), 1mol/L N-acetyl-L-cysteine (Sigma; A9165), 100ng/mL R-Spondin-1 (R & D; 7150-RS), and D/F12 to 40mL (HyClone);
mouse proliferation medium (mouse expansion medium, mEM) formulation: 1% Penicillin-Streptomycin Solution (HyClone; SV 30010), 1% glutamine additive (gibco; 35050-061), 1%B27 without vitamin A (Thermo; 12587010), 50ng/mL EGF (MCE; HY-P7067), 50ng/mL FGF10 (PeproTech; 100-26-25), 25ng/mL NOGGIN (PeproTech; 120-10C-20), 1mol/L Nicotinamide (Sigma; N0636), 1mol/L N-acetyl-L-cysteine (Sigma; A9165), 100ng/mL R-Spondin-1 (R & D;7150-RS, D/F12 added to 40mL (HyClone; SH 30023.01);
pluripotent pancreatic progenitor cell culture medium: the above EM (proliferation medium) medium contains BMP7 (Biyundian; P5772) at final concentration of 25, 50, 100, 200ng/mL
Mouse pancreatic differentiation medium (mouse pancreatic differentiation medium, mDM) formulation: including high sugar DMEM medium (HyClone; SH 30022.01), 1% Penicillin-Streptomycin Solution (HyClone; SV 30010), 1% Glutamine additive (gibco; 35050-061), 1% B27 Supplement (Thermo; 17504044), 1% ITS (Thermo; 51500056), 50ng/mL VEGF (PeproTech; 100-20B), 50ng/mL Exendin 4 (MCE, HY-13443), 25ng/mL NOGGIN (PeproTech; 120-10C-20), 750ng/mL RSPO1 (R & D; 7150-RS), 1 μ M T (TOCRIS; 6666), 10 μ M A8301 (MCE; HY-10432), 5 μM DAPT (MCE; HY-13027), 10 μg/mL heparin (SIGMA; H49), 100ng/mL Actin A (ROTECH, 120-14P), 20/mL (FGF; 32) and Niamid (Sigma-7335) 1. Mu.6. Mu.M 2; sigma-7335).
Human pancreas differentiation medium (human pancreatic differentiation medium, hDM 9) formulation: including high sugar DMEM medium (HyClone; SH 30022.01), 1% Penicillin-Streptomycin Solution (HyClone; SV 30010), 1% Glutamine additive (gibco; 35050-061), 1% B27 Supplement (Thermo; 17504044), 1% N2 Supplement (Thermo; 17502001), 1% ITS (Thermo; 51500056), 50ng/mL VEGF (PeproTech; 100-20B), 50ng/mL Exendin 4 (MCE, HY-13443), 25ng/mL NOGGIN (PeproTech; 120-10C-20), 750ng/mL RSPO1 (R & D; 7150-RS), 1 μ M T (TOCRIS; 6666), 10 μ 79 8301 (MCE; HY-10432), 5 μ M T (HY-13027), 10 μg/mL (SIG; H3149), 100ng/mL VEGF (PeproTech; 100-20B), 50ng/mL Exendin 4 (MCE, HY-13443), 25ng/mL NOGGIN (PeproTech; 120-10C-20), 750ng/mL RSPO1 (R & D; 7150-RS), 1 μ M A (TOCRIS; 66), 10 μ 8301 (MCE; HY-10432), 5 μ M T (HY-13027), 10 μg/mL (SIG/mL), 100/mL, and 100 mg/mL heparin (P).
Example 1 method for preparing epithelial cells
1. Bile duct (mBD) and pancreatic duct (mPD) of mice:
after the mice are killed by the cervical dislocation method, the mice are soaked in a beaker with 75% alcohol, and the beaker is quickly placed into a sterilized biosafety cabinet after the surface of the beaker is sterilized by alcohol cotton. The mice were sterilized by soaking for about 5min. Taking out the mice, draining the liquid, placing on a sterile anatomical frame, sequentially cutting off fur and peritoneum along the center line of the abdomen, and exposing the abdominal cavity. The celiac-opened mice were placed under an anatomic scope, the liver, pancreas, and common bile duct (extrahepatic bile duct) connecting the two were found, taken together, and placed in a 10cm dish containing sterile PBS. The method is characterized in that the intrahepatic bile duct and the pancreatic duct are directly peeled off along the common bile duct under a microscope by using clock forceps, the main duct of the pancreas is mainly positioned at the head part of the pancreas, and the head part is the widest part of the pancreas, takes the shape of a disc and is positioned at the concave part of the duodenum. When the pancreas head is also connected with the duodenum, the pancreas head is gently resected, so that the pancreas main duct can be better separated in the later period. For ease of observation, the connection of the biliary-pancreatic duct was maintained until the dissection was completed, and the bile duct (mBD, extrahepatic duct and intrahepatic duct) and pancreatic duct (mouse pancreatic duct, mPD) were separated again after the dissection was completed using a scalpel. The bile duct and pancreatic duct were placed in separate 15mL centrifuge tubes, respectively, and 2mg/mL collagenase IV was digested at 37℃for 15min to separate the remaining tissue around the duct, shaking the centrifuge tubes vigorously every 5min. The digested hepatobiliary and pancreatic ducts were replaced in a 10cm dish containing sterile PBS. The tissue remaining around the catheter was stripped off using a dissecting scope, and PBS flushed to ensure removal of excess tissue. Placing the hepatobiliary pancreatic duct into different sterile 1.5mL centrifuge tubes, cutting the hepatobiliary pancreatic duct into pieces as much as possible with dissecting scissors, adding 1mL of 2mg/mL collagenase IV, digesting at 37deg.C for 20min, taking out every 5min, and repeatedly blowing with 1mL gun head. After passing the digested catheter cells through a 70 μm screen, the cells were centrifuged horizontally at 300g for 5min. The precipitate is the epithelial cells.
2. Bile duct of person (mBD)
Human bile duct (hBD) samples of human bile ducts (intrahepatic and extrahepatic bile ducts) were taken in 10cm petri dishes containing sterile PBS, the catheter was longitudinally sheared with dissecting scissors, and the catheter was deployed in a square with dissecting forceps. One hand holds the deployed catheter with dissecting forceps (note holding the inside of the catheter-the epithelial cell side up) and the other hand scrapes the catheter surface with a sterile glass slide. The side of the slide in contact with the catheter and the catheter after handling was rinsed with sterile PBS and the rinse was directly retained in a 10cm dish. Collecting PBS containing human common bile duct epithelial cells in a 10cm culture dish, centrifuging 300g for 5min, and collecting precipitate. The precipitate is the epithelial cells.
Example 2 method of preparing organoids
The obtained human/mouse epithelial cells were resuspended in 50. Mu.L Matrigel (Corning, 54234), and then dropped into a 24-well plate and allowed to stand at 37℃for 5 minutes to coagulate Matrigel. mu.L of mouse proliferation medium (mouse expansion medium, mEM) or human proliferation medium (human expansion medium, hEM) was added to each well for cultivation.
The prepared human biliary organoids (human bile duct organoid, hdpo), mouse biliary organoids (mouse bile duct organoid, mbpo) and mouse pancreatic organoids (mouse pancreatic duct organoid, mppo) can all be serially subcultured for more than 10 passages (fig. 1). Immunostaining results showed that each of the prepared hdpo, mBDO and mPDO expressed biliary/pancreatic progenitor marker gene (Sox 9, PDX 1), hepatobiliary progenitor marker gene (HNF 1B, HNF a), multipotent stem cell marker gene (EpCAM) and epithelial specific gene (E-cadheren) (results are shown in fig. 2 and 3), demonstrating that hdpo, mBDO and mPDO are progenitor cells with similar differentiation potential, and that the antibodies used are shown in table 1.
TABLE 1
Example 3A method of obtaining islet cells
Culturing the bile duct and pancreatic duct organoids of the mice: the bile duct and pancreatic duct organoids of the mice were cultured separately using mEM medium containing BMP7 at a final concentration of 100ng/mL for an induction time of 7 days, and quantitative PCR results showed that pancreatic endocrine progenitor cell-specific genes in mbmos and mpos were significantly up-regulated for expression (fig. 4A), resulting in pancreatic progenitor cells, and the whole procedure for obtaining islet beta cells required replacement of fresh medium every other day, with replacement mDM for 14 days.
Example 4A method of obtaining islet cells
Culturing the bile duct and pancreatic duct organoids of the mice: the bile duct and pancreatic duct organoids of the mice were cultured separately using mEM medium containing BMP7 at a final concentration of 100ng/mL for an induction time of 7 days, and quantitative PCR results showed that pancreatic endocrine progenitor cell-specific genes in mbmos and mpos were significantly up-regulated for expression (fig. 4A), resulting in pancreatic progenitor cells, and the replacement of mDM containing TLY142 at a final concentration of 1 μm was continued for 14 days, and finally the whole procedure for obtaining islet beta cells required replacement of fresh medium every day.
The experimental effect was verified by using the following experiment:
(1) Fluorescent quantitative PCR: after addition of BMP7 to mbmos and mpos for 7 days or stepwise continuous induction for 21 days, total RNA was extracted by centrifugation and lysis in TRIZOL. Using Primerstript RT The master kit (Vazyme, R323-01) inverts the total RNA into cDNA. The reaction system and conditions were as described in ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) (primers see Table 2) and tested using a Roche Light Cycle fluorescent quantitative PCR apparatus. Analysis Ct values were calculated by Abs Quant/2nd Derivative Max using the Light Cycler 480 self-contained software analysis module and using 2 -ΔΔCt The relative expression level of mRNA was calculated by the method.
TABLE 2 mouse primer sequences
(2) Immunostaining: after 21 days of staged induction culture in mBDOs and mPDOs, mBDOs and mPDOs were washed with PBS, respectively, fixed with 4% PFA for 20min, repeated washing with cold PBS 3 times, and cell suspension was dropped onto the slide, dried at 37℃until organoids were fixed on the slide. Punching with 0.3% Triton X-100deg.C for 1 hr, repairing antigen retrieval liquid (Biyun Tian, P0090) at room temperature for 10min, sealing with 10% horse serum at 37deg.C for 1 hr, adding primary antibody for incubation at 4deg.C overnight, secondary antibody for incubation at room temperature for 2 hr, and Hochest 33342 (1:1000) staining for 15min, and quenching with anti-fluorescence sealing plate (Biyun Tian, P0126). Fluorescence photography was performed using the high resolution live cell imaging system DeltaVision. Antibody cargo numbers and use concentrations are shown in table 3.
TABLE 3 antibody Source and use concentration
Results: after BMP7 was cultured for 7D, expression of multipotent pancreatic progenitor marker gene ngn3 in mbmos and mpos was significantly upregulated (fig. 4A), BMP 7-induced mbmos and mpos had multipotent differentiation potential, and gene and protein expression of pancreatic exocrine markers of mbmos and mpos was significantly upregulated after mDM-induced differentiation (fig. 4B-D).
Results: TLY142 was effective in promoting differentiation of mBDOs into pancreatic cells in vitro, and gene and protein expression of the secretion markers inside and outside the pancreas of the differentiated mBDOs were significantly up-regulated (FIGS. 5A-B).
(3) Flow cytometry: differentiated mBDOs and mPDOs were digested with trypsin into single cells, fixed on 4% PFA ice for 15min, PBS washed, 0.1% Triton X-100 punched at room temperature for 10min, PBS washed, 5% BSA blocked at room temperature for 15min, left at room temperature for 15min, 3% BSA diluted primary antibody (C-peptide, 1:200), incubated on ice for 40min, control added isotype antibody diluent, PBS washed, 3% BSA diluted secondary antibody (donkey anti-rabbit 488, 1:100), incubated on ice protected from light for 30min, PBS washed, and 500. Mu.L resuspended on machine for detection.
(4) Glucose stimulates C peptide secretion: the differentiated organoids were resuspended using sugar-free Krebs solution, washed 2-3 times, placed in low-adsorption plates and incubated overnight. Krebs solution containing 2mM glucose was added, incubated for 10min, and the supernatant was collected by centrifugation. After the cells were washed with sugar-free Krebs solution containing 20mM glucose, the cells were resuspended, incubated for 10min, and the supernatant was collected by centrifugation. The supernatant samples were analyzed for C-peptide levels using a mouse C-peptide ELISA kit (mlbrio, ml-1015542) according to standard protocols.
Results: BMP7 is effective to promote differentiation of both mbmos and mpos into functional beta cells, flow cytometry indicates that the C-peptide+ cells in both mbos and mpos after differentiation account for approximately 8.49±0.45% and 7.23±0.63% (fig. 6A-B), while being able to secrete C-peptide in response to glucose stimulation (fig. 6C), TLY142 can increase the differentiation rate of mbos to pancreatic beta cells by 19.90±0.62% (fig. 7A-B), and that the cells after differentiation have a physiological response to glucose stimulation of mature beta cells (fig. 7C). The structural formula of TLY142 is shown in FIG. 8.
(5) In vivo transplantation experiments: nu/Nu mice were induced by intraperitoneal injection of streptozotocin (160 mg/kg) 7 days prior to transplantation. The blood glucose meter measures non-fasting blood glucose of the tail vein sample, and the blood glucose level is selected to rise above 16.8mMAs a model mouse for diabetes. The mBDOs after 2-stage induced differentiation are beaten into single cells by pancreatin according to the ratio of 10 6 Individual cells/kidney capsule transplanted into recipient mice, regular non-fasting blood glucose was measured every 7 days after transplantation. At week 8 of implantation, a nephrectomy was performed to examine the effect of removal of transplanted organoids or islets on glycemic improvement.
(6) Glucose tolerance test: glucose tolerance test was performed according to standard protocol, mice were starved overnight, were intraperitoneally injected with 1g/kg glucose, blood glucose levels were measured for 0, 15, 30, 60, 90 and 120min, serum was collected before and after glucose injection, and insulin content changes were measured by ELISA.
(7) Immunostaining of transplanted kidney frozen sections: the kidney of the transplanted organoid obtained by nephrectomy was fixed overnight with PFA, dehydrated with 30% sucrose, and then a tissue section 10 μm thick was obtained by frozen section technique, and the differentiation of the transplanted organoid was examined by immunostaining. After the sections were dried at room temperature, wax circles were drawn, washed 3 times with PBS to remove embedding medium, fixed for 20min with 4% PFA, repeatedly washed 3 times with cold PBS, punched 30min at 37℃with 0.3% Triton X-100, repaired 5min at room temperature with antigen retrieval solution (Biyun, P0090), blocked 1h at 37℃with 10% horse serum, incubated overnight at 4℃with primary antibody, incubated 2h at room temperature with secondary antibody, stained for 15min with Hochest 33342 (1:1000), and blocked with anti-fluorescence quenching (Biyun, P0126). Fluorescence photography was performed using the high resolution live cell imaging system DeltaVision.
Results: the mice transplanted with 2-stage induced differentiated mbmos into STZ-induced diabetic mice kidney capsule were significantly observed to have a decrease in blood glucose, and TLY 142-induced differentiated mbos had a more similar effect on relieving hyperglycemia as the positive control group of islet transplantation (fig. 9A), and glucose tolerance experiments showed that TLY 142-induced differentiated mbos mice had glucose tolerance similar to the positive control group of islet transplantation (fig. 9B-C), while serum levels of instrin were increased (fig. 9D). The blood glucose levels in mice were significantly elevated after the transplanted nephrectomy (fig. 9A), further demonstrating that blood glucose was indeed reduced by transplanted mbnos. Immunostaining demonstrated that transplanted TLY142 induced differentiated mBDOs contained β cells expressing insulin and α cells expressing glucagon (FIG. 9E).
2. Culturing human bile duct organoids
Inoculating epithelial cells of human bile duct (intrahepatic duct and extrahepatic duct) obtained by mechanical scraping method into extracellular matrix, culturing with hEM culture medium at 37deg.C and 5% CO 2 Human biliary organoids (human bile duct organoid, hbdes) were formed after culture in incubator conditions.
(1) Subculturing human bile duct organoids (human extrahepatic bile duct organoid, hbdus): organoids were resuspended by blowing them up using pre-chilled high-sugar DMEM medium (HyClone; SH 30022.01), and after centrifugation to remove the supernatant, the pellet was subcultured with Matrigel and hEM. Passage was performed at a ratio of 1:3 to 1:5 every 7 to 8 days according to cell density.
(2) Cryopreservation of human bile duct organoids (human bile duct organoid, hbdus): the organoids were resuspended by blowing them up using a pre-chilled high-sugar DMEM medium (HyClone; SH 30022.01), centrifuging to remove the supernatant, then adding 1mL of pre-chilled organoid cryopreservation solution, transferring the resuspended organoids into a cell cryopreservation tube, placing the cryopreservation tube in a cryopreservation box, then transferring the tube into a refrigerator at-80℃overnight, and transferring the cryopreservation tube into liquid nitrogen for storage the next day.
The obtained hBDOs can be passaged for a long period of time, have stable proliferation and passaging ability, and can maintain good cell state even when passed to the 10 th generation, and the result is shown in FIG. 1. The structural formula of TLY142 is shown in FIG. 7A. The human biliary organoids were cultured in hEM medium containing BMP7 at final concentrations of 25, 50, 100, 200ng/mL for 7 days, and hDM containing TLY142 at final concentrations of 1 or 10 μm was replaced for further 14 days to obtain islet cells. The whole process needs to change fresh culture medium every other day.
The following experiment was used to verify the experimental effect:
(1) Fluorescent quantitative PCR: after 21 days of stepwise continuous induction culture of hbdus, total RNA was extracted by centrifugation and lysis in TRIZOL. Using Primerstript RT The master kit (Vazyme, R323-01) inverts the total RNA into cDNA. Reaction system and condition reference ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) (primers see Table 4) and detected using a Roche Light Cycle 480 fluorescent quantitative PCR apparatus. Analysis Ct values were calculated by Abs Quant/2nd Derivative Max using the Light Cycler 480 self-contained software analysis module and using 2 -ΔΔCt The relative expression level of mRNA was calculated by the method.
TABLE 4 human primer sequences
(2) Immunostaining: after 21 days of staged induction culture of hBDOs, the hBDOs were washed with PBS, fixed with 4% PFA for 20min, washed 3 times with cold PBS repeatedly, and the cell suspension was dropped onto a slide, dried at 37℃until the organoids were fixed on the slide. Punching with 0.3% Triton X-100deg.C for 1 hr, repairing antigen retrieval liquid (Biyun Tian, P0090) at room temperature for 10min, sealing with 10% horse serum at 37deg.C for 1 hr, adding primary antibody for incubation at 4deg.C overnight, secondary antibody for incubation at room temperature for 2 hr, and Hochest 33342 (1:1000) staining for 15min, and quenching with anti-fluorescence sealing plate (Biyun Tian, P0126). Fluorescence photography was performed using the high resolution live cell imaging system DeltaVision. Antibody cargo numbers and use concentrations are shown in table 2.
Results: 25.50, 100 and 200ng/mL BMP7 can up-regulate the expression of pancreatic endocrine progenitor gene NGN3 after culturing hBDO 7 for 7 days respectively (figure 10A), 1 mu M TLY142 can effectively promote differentiation of hBDOs into islet cells in vitro, and mRNA and protein expression of differentiated hBDOs pancreatic endocrine markers are up-regulated obviously (figures 10B-C), and meanwhile, 10 mu M TLY142 also has the effect of promoting up-regulated expression of INS gene surfaces (figure 10D).
(3) Flow cytometry: the differentiated hBDOs are digested into single cells by trypsin, fixed on 4% PFA ice for 15min, PBS washed, 0.1% Triton X-100 for 10min at room temperature, PBS washed, 5% BSA blocked for 15min at room temperature, 3% BSA diluted primary antibody (C-peptide, 1:200) incubated on ice for 40min, control group added with isotype antibody diluent, PBS washed, 3% BSA diluted secondary antibody (donkey anti-rabbit 488, 1:100), incubated on ice for 30min in the absence of light, PBS washed, and 500 μl resuspended on machine for detection.
(4) Glucose stimulates C peptide secretion: the differentiated organoids were resuspended using sugar-free Krebs solution, washed 2-3 times, placed in low-adsorption plates and incubated overnight. Krebs solution containing 2mM glucose was added, incubated for 10min, and the supernatant was collected by centrifugation. After the cells were washed with sugar-free Krebs solution containing 20mM glucose, the cells were resuspended, incubated for 10min, and the supernatant was collected by centrifugation. The supernatant samples were analyzed for C-peptide levels using a mouse C-peptide ELISA kit (mlbrio, ml-1015542) according to standard protocols.
Results: BMP7 in combination with TLY142 was effective in promoting differentiation of hbdus into functional beta cells, and flow cytometry showed that the differentiated hbdus had a C-peptide+ cell ratio of about 25.50±4.82 (fig. 11A-B), while being able to secrete C-peptide in response to glucose stimulation (fig. 11C), with the physiological response of glucose stimulation of mature beta cells.
(5) In vivo transplantation experiments: nu/Nu mice were induced by intraperitoneal injection of streptozotocin (160 mg/kg) 7 days prior to transplantation. The glucometer measures non-fasting blood glucose in tail vein samples, and a diabetic model mouse with a blood glucose level elevated above 16.8mM was selected. Beating hBDOs after stage induced differentiation into single cell with pancreatin according to 10 6 Individual cells/kidney capsule transplanted into recipient mice, regular non-fasting blood glucose was measured every 7 days after transplantation. At week 8 of implantation, a nephrectomy was performed to examine the effect of removal of transplanted organoids or islets on glycemic improvement.
(6) Glucose tolerance test: glucose tolerance test was performed according to standard protocol, mice were starved overnight, were intraperitoneally injected with 1g/kg glucose, blood glucose levels were measured for 0, 15, 30, 60, 90 and 120min, serum was collected before and after glucose injection, and insulin content changes were measured by ELISA.
(7) Immunostaining of transplanted kidney frozen sections: the kidney of the transplanted organoid obtained by nephrectomy was fixed overnight with PFA, dehydrated with 30% sucrose, and then a tissue section 10 μm thick was obtained by frozen section technique, and the differentiation of the transplanted organoid was examined by immunostaining. After the sections were dried at room temperature, wax circles were drawn, washed 3 times with PBS to remove embedding medium, fixed for 20min with 4% PFA, repeatedly washed 3 times with cold PBS, punched 30min at 37℃with 0.3% Triton X-100, repaired 5min at room temperature with antigen retrieval solution (Biyun, P0090), blocked 1h at 37℃with 10% horse serum, incubated overnight at 4℃with primary antibody, incubated 2h at room temperature with secondary antibody, stained for 15min with Hochest 33342 (1:1000), and blocked with anti-fluorescence quenching (Biyun, P0126). Fluorescence photography was performed using the high resolution live cell imaging system DeltaVision.
Results: BMP7 in combination with TLY142 induced differentiated hbmos transplanted into STZ-induced kidney capsule of diabetic mice, a decrease in blood glucose in mice was clearly observed (fig. 12A), and glucose tolerance experiments showed that the transplanted hbos mice had an improved glucose tolerance compared to the control group (fig. 12B-C), while the levels of instrin in serum were increased (fig. 12D). The blood glucose levels in mice were significantly elevated after the transplanted nephrectomy (fig. 12A), further demonstrating that blood glucose was indeed reduced by transplanted hbdus. Immunostaining demonstrated that the transplanted organoids contained β cells expressing insulin and α cells expressing glucagon (fig. 12E).
SEQUENCE LISTING
<110> university of northeast forestry
<120> a method for producing islet beta cells in vitro using BMP7 factor and islet beta cells obtained and the use thereof
By using
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<170> PatentIn version 3.5
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Claims (1)
1. A method for producing islet beta cells in vitro, which is characterized in that organoids are placed in a culture medium containing BMP7 factor for culture, and islet beta cells are obtained; the organoids are bile duct organoids or pancreatic duct organoids; culturing organoids in a proliferation medium containing BMP7 factor to obtain pluripotent pancreatic progenitor cells, and culturing the pluripotent pancreatic progenitor cells in a differentiation medium to obtain islet beta cells; the concentration of the BMP7 factor is 25-200 ng/mL; the differentiation medium also comprises small molecule C 17 H 22 N 2 O 4 S, S; the C is 17 H 22 N 2 O 4 The concentration of S is 1 mu M-10 mu M.
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| CN112522181A (en) * | 2020-12-29 | 2021-03-19 | 苏州方舟生物科技有限公司 | Culture medium system and method for inducing and generating human islet beta cells in vitro |
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| CN112522181A (en) * | 2020-12-29 | 2021-03-19 | 苏州方舟生物科技有限公司 | Culture medium system and method for inducing and generating human islet beta cells in vitro |
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| Differentiation of rat pancreatic duct stem cells into insulin-secreting islet-like cell clusters through BMP7 inducement;Ghani M. W. et al.;Tissue Cell;第67卷;摘要 * |
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