CN115044534A - Method for producing pancreatic islet beta cells in vitro by using BMP7 factor, pancreatic islet beta cells obtained by same and application thereof - Google Patents
Method for producing pancreatic islet beta cells in vitro by using BMP7 factor, pancreatic islet beta cells obtained by same and application thereof Download PDFInfo
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- CN115044534A CN115044534A CN202210301602.5A CN202210301602A CN115044534A CN 115044534 A CN115044534 A CN 115044534A CN 202210301602 A CN202210301602 A CN 202210301602A CN 115044534 A CN115044534 A CN 115044534A
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Abstract
The invention discloses a method for producing pancreatic islet beta cells in vitro by using BMP7 factor, the obtained pancreatic islet beta cells and application thereof, belonging to the technical field of biomedicine. To provide a method for using epithelial cells derived from bile duct and pancreatic duct and BMP7 factor and C 17 H 22 N 2 O 4 S method for obtaining islet cells. The invention provides a method for culturing a bile duct organoid or a pancreatic duct organoid in a proliferation culture medium containing BMP7 to obtain pluripotent pancreatic progenitor cells, and then placing the pluripotent pancreatic progenitor cells in a proliferation culture medium containing C 17 H 22 N 2 O 4 And obtaining the islet beta cells from the differentiation medium of the 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 pancreatic beta cells in vitro by using BMP7 factor, the obtained pancreatic beta cells and application.
Background
Cell replacement therapy can maintain a healthy life for diabetic patients with damaged beta cells. However, the lack of pancreatic donors has made islet transplantation into a dilemma in the treatment of diabetes, and some evidence suggests that progenitor cells capable of differentiating into insulin-secreting cells are present in adult pancreatic ducts. However, for patients requiring cell transplantation, immune rejection in the absence of donor tissue and xenogeneic cells still needs to be overcome.
Recent studies have shown that progenitor cells removed from a patient's own bile duct by microsurgery can be expanded to culture organoids and cure biliary disease after bile duct or hepatic portal vein transplantation. These findings highlight a pathway for the repair of damaged gastrointestinal organs using the patient's own progenitor cells. The problem with using catheter progenitor cells is that the efficiency of beta cell differentiation in vivo and in vitro is rather low. A recent report by Hush M showed that only 1% of insulin positive cells were derived from the organoid where Epcam + TSQ-pancreatic ductal progenitor cells grew. Over the past decades, a variety of cytokines and signaling inhibitors have been explored to increase beta cell differentiation, including nicotinamide, noggin, retinoic acid, and Exendin 4, which have been shown to increase insulin expression in ES or iPS derived pancreatic progenitor cells, whereas these molecules promote beta cell differentiation only after ductal cell conversion to endocrine cells.
Chemical methods have proven to be useful tools to promote directed differentiation of stem/progenitor cells. Particularly in the field of embryonic stem cells, small chemical molecules have become a powerful strategy for inducing ES or iPS cells to efficiently differentiate hepatocytes. In addition to those known activators and inhibitors of pancreatic developmental cell signaling pathways, there is still a lack of defined chemical molecules to regulate unknown pathways that enhance beta cell differentiation.
Disclosure of Invention
The invention aims to provide a method for utilizing epithelial cells derived from bile ducts and pancreatic ducts and BMP7 factor and C 17 H 22 N 2 O 4 S method for obtaining islet cells.
The invention provides a method for producing islet beta cells in vitro, which comprises the step of placing organoids in a culture medium containing BMP7 factor to culture to obtain islet beta cells.
Further defined, the step of culturing is: culturing in a proliferation culture medium containing BMP7 factor to obtain pluripotent pancreatic gland progenitor cells, and culturing the pluripotent pancreatic gland progenitor cells in a differentiation culture medium to obtain pancreatic islet beta cells.
Further defined, the organoids are biliary or pancreatic ductal organoids.
Further defined, the concentration of said BMP7 factor is 25-200 ng/mL.
Further defined, the culture medium further comprises a small molecule C17H 22 N 2 O 4 S。
Is further defined as C 17 H 22 N 2 O 4 The concentration of S is 1. mu.M-10. mu.M.
The present invention provides islet beta cells obtained by the above-described method.
The invention provides application of the islet beta cells in preparation of a cell medicament for treating diabetes or a cell medicament for reducing blood sugar.
Further defined, the cellular drug comprises an extracellular matrix compatible with the cells and a pharmaceutical carrier.
Further defined, the cellular drug comprises an extracellular matrix compatible with the cells and a pharmaceutical carrier.
Further defined, the pharmaceutical carrier is a nanomaterial or a microfluidics.
Differentiation medium: mouse pancreas differentiation medium (mDM 9) formulation: including high glucose DMEM medium, 1% penillin-Streptomycin Solution (HyClone; SV30010), 1% glutamine additive, 1% B27 Supplement, 1% ITS (Thermo; 51500056), 50ng/mL VEGF, 50ng/mL Exendin 4, 25ng/mL NOGGIN, 750ng/mL RSPO1, 1. mu. M T3 (TOCRIS; 6666), 10. mu. M A8301, 5. mu.M DAPT, 10. mu.g/mL heparin 100ng/mL Activin A (PEPROTECH, 120-14P), 20ng/mL FGF2, 4.4mM Nicotinamide (Sigma; N0636), and 1. mu.M Retinoic acid.
Human pancreatic differentiation medium (human pancreatic differentiation medium, hDM9) formulation: comprises high-sugar DMEM culture medium, 1% penillin-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 μ M DAPT, 10 μ g/mL heparin, 100ng/mL Activin A, 20ng/mL FGF2, 20ng/mL, 4.4mM Nicotinamide and 1 μ M Retinoic acid.
Proliferation culture medium: human expansion medium (hEM) formulation: 1% Penicilin-Streptomycin Solution, 1% glutamine additive, 1% B27 with out vitamin A (Thermo; 12587010), 50ng/mL EGF, 50ng/mL FGF10, 25ng/mL NOGGIN, 1% N2, 10nM Gastrin, 3 μ M 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 40 mL;
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 make up to 40 mL;
has the advantages that: progenitor cell populations with long-term organoid-forming ability were identified from mouse bile ducts (intrahepatic and extrahepatic) and pancreatic ducts and demonstrated similar self-renewal and multi-differentiation potential. BMP7 is a potent cytokine that converts ductal progenitor cells to a pluripotent state and enhances endocrine differentiation. By screening chemical molecules, TLY142 was found to greatly improve the efficiency of differentiation of mBDO into insulin-secreting cells based on the BMP7 initiation conditions. The human bile duct progenitor cells have long-term organoid forming capability and pluripotency, TLY142 enhances the differentiation of hBDO into insulin-secreting cells by more than 20%, and the transplantation of differentiated mBDO and hBDO in vivo can effectively deal with the glucose stimulation and cure the hyperglycemia of diabetic mice.
The results of the present invention expand the utility of biliary epithelial cells as a potential source of beta cells in diabetic cell replacement therapy. Progenitor cells isolated from autologous bile ducts have been shown to form organoids and enable repair of damaged bile ducts by feedback transplantation. Markers can be used to isolate bile duct progenitor cells from bile duct epithelial cells, which are more readily obtained from autologous bile ducts and donated bile ducts by microsurgery.
The differentiated bile duct organoids have similar endocrine cell lineages to pancreatic ductal organoids, especially insulin secreting cells (fig. 4C and 4D). Transplantation of differentiated human BDO effectively cured model mice for diabetes, demonstrating that biliary progenitor cell-derived organoids are a reliable source of cell transplantation.
The high proportion of glucose-altering-responsive beta cells differentiated from the ductal organoids provides a viable source of beta cells for cell transplantation therapy in diabetes. BMP7 and the chemical small molecule TLY142 are important for enhancing organoid differentiation into beta cells. Supplementation with BMP7 was important for organoid transformation to the Ngn3+ endocrine progenitor state, which is consistent with previous reports that BMP7 significantly increased expression of Ngn 3. Thus, after BMP7 was withdrawn, we defined a DM9 medium that significantly increased the proportion of insulin secreting cells to 7% of total cells over the control (1%) without BMP 7.
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 after renal subconjunctival transplantation.
In summary, epithelial cells in the pancreatic or bile duct are organoids with multiple differentiation potential that can be formed in vitro for long-term culture. They can treat diabetes by differentiating defined media into high proportion of functional beta cells, acting as sugar-lowering agents to replace islets.
Drawings
FIG. 1 is a graph showing the results of organoid preparation using human biliary epithelium, mouse biliary epithelium and pancreatic duct epithelial cells, respectively. The pictures show cytograms of the prepared human bile duct organoid hBDO, mouse bile duct organoid mBDO and mouse pancreatic duct organoid mPDO after 10 passages.
FIG. 2 is a graph of the result of immunostaining to identify organoid pluripotency, pancreatic progenitor cell-specific gene expression, in which A is the expression of PDX1, EPCAM in the immunostaining assay 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 identifying organoid pluripotency, i.e., hepatobiliary progenitor cell-specific gene expression, wherein A is the expression of HNF1B, E-cadherin in hBDO, mBDO and mPDO as immunostaining; b is immunostaining to detect the expression of HNF4A, E-cadherin in hBDO, mBDO and mPDO.
FIG. 4 is a graph of the results of BMP7 combined with mDM9 in promoting secretion and differentiation of mBDO and mPDO into and out of pancreas, wherein A is the expression of NGN3 in mBDO and mPDO after 7 days of induction of BMP7 by quantitative PCR; b, detecting the mRNA expression of pancreas inside and outside secretory cell specificity genes in mBDO and mPDO 21 days after the BMP7 is combined with mDM9 to induce and differentiate by quantitative PCR; c, carrying out immunostaining to detect the expression of specific gene proteins of pancreatic endocrine and exocrine cells in the mBDO 21 days after the induction and differentiation of BMP7 and mDM9 are combined; d is the expression of the pancreatic internal and external secretion cell specific gene protein in mPDO after 21 days of induced differentiation by combining the detection of BMP7 with mDM9 by immunostaining.
FIG. 5 is a graph showing the results of the BMP7 in combination with TLY 142-containing mDM9 promoting the differentiation of mBDO into pancreatic endocrine cells, wherein A is the expression of pancreatic endocrine cell-specific gene mRNA in mBDO after 21 days of differentiation induction by quantitative PCR detection of BMP7 in combination with TLY 142-containing mDM 9; b is the expression of pancreatic endocrine cell specific gene protein in mBDO 21 days after the induction and differentiation of the BMP7 combined with mDM9 containing TLY142 through immunostaining detection.
Fig. 6 is a result of detecting differentiation efficiency and physiological functions of cells after mBDO and mPDO are induced by BMP7 in combination with mDM9, wherein a is a beta cell differentiation ratio in mBDO and mPDO after BMP7 in combination with mDM9 is detected for 21 days by flow cytometry; b is a statistical chart of beta cell differentiation ratio in mBDO and mPDO after 21 days of differentiation induction by combining BMP7 and mDM9 through flow cytometry; c is after 21 days of differentiation induced by BMP7 in combination with mDM9, mBDO and mPDO are stimulated by glucose, and Elisa detects C peptide secretion.
FIG. 7 shows the results of the differentiation efficiency and physiological function of mBDO induced by mDM9 containing TLY142 in combination with BMP7, wherein A is the differentiation ratio of beta cells in mBDO after mDM9 containing TLY142 in combination with BMP7 was induced and differentiated for 21 days by flow cytometry; b is a statistical chart of beta cell differentiation proportion in mBDO after mDM9 induced differentiation of the BMP7 combined with TLY142 for 21 days is detected by flow cytometry; c is BMP7 in combination with TLY142 containing mDM9 induced differentiation after 21 days, mBDO was stimulated by glucose, Elisa detected 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 measurements in vivo in transplanted diabetic mice after induced differentiation of mBDO by BMP7 in combination with mDM9 or mDM9 containing TLY142, wherein A is the change from mBDOs after 21 days of induced differentiation of BMP7 in combination with mDM9 containing TLY142 to the blood sugar of STZ-induced renal cysts in diabetic mice; b and C are the glucose tolerance change of the transplanted mouse; d is the change in the levels of Insulin in the mouse serum; e is the expression of insulin and glucagon detected by immunostaining of transplanted kidney after 8 weeks of transplantation of mBDOs kidney under BMP7 combined with mDM9 induced differentiation containing TLY142 for 21 days.
FIG. 10 is a graph of the results of BMP7 in combination with TLY 142-containing hDM9 promoting hBDO differentiation into pancreatic endocrine cells; wherein, the quantitative PCR detection of A is used for hBDO NGN3 expression 7 days after BMP7 with different concentrations is induced; b is the expression of pancreatic endocrine cell specific gene mRNA in hBDO after 21 days of quantitative PCR detection of BMP7 combined with hDM9 induced differentiation containing 1 mu MTLY 142; c is the expression of pancreatic endocrine cell specific gene protein in hBDO 21 days after the induction and differentiation of the combination of mDM9 containing 1 mu M TLY142 and BMP7 detected by immunostaining; d is the expression of INS gene mRNA in hBDO 21 days after the induction and differentiation of BMP7 combined with hDM9 containing 10 μ M TLY142 by quantitative PCR detection.
FIG. 11 shows the results of the measurement of differentiation efficiency and cell physiological function of hBDO after hDM9 induced by BMP7 combined with TLY142, wherein A is the beta cell differentiation ratio of hBDO after hDM9 induced by BMP7 combined with TLY142 for 21 days by flow cytometry; b is a statistical chart of beta cell differentiation ratio in hBDO after hDM9 combined with TLY142 containing BMP7 is detected to induce differentiation for 21 days by flow cytometry; c is that hBDO is stimulated by glucose after hDM9 induced differentiation of BMP7 combined with TLY142 for 21 days, and Elisa detects C peptide secretion.
FIG. 12 is the results of activity test of hBDO in vivo in transplanted diabetic mice after induction and differentiation by BMP7 in combination with hDM9 containing TLY142, wherein A is the change of hBDOs 21 days after induction and differentiation by BMP7 in combination with mDM9 containing TLY142 to blood sugar after renal cyst induction by STZ in diabetic mice; b and C are the glucose tolerance change of the transplanted mouse; d is the change in the levels of Insulin in the serum of the mice. E is hBDOs kidney cyst transplantation 8 weeks after hDM9 induced differentiation of A BMP7 combined with TLY 142-containing cells for 21 days, and the transplanted kidney is removed and immune staining is carried out to detect the expression of insulin and glucogon.
Detailed Description
The English name of the 1- (3, 4-xylyl) sulfonyl-5- (pyrrolidine-1-carbonyl) pyrrolidine-2-ketone is 1- (3,4-dimethyl phenyl) sulfo-5- (pyrolidine-1-carbonyl) pyrolidin-2-one, the Pubchem CID is 53008166, the molecular formula is C 17 H 22 N 2 O 4 S, named TLY142 in the invention, is shown in the structural diagram of FIG. 8.
Recombinant Human bone morphogenetic protein-7 (BMP7) was purchased under the brand name and from Biyunnan (Recombinant Human BMP-7, P5772-100. mu.g).
Animal and human bile duct tissue
The C57BL/6N and NU/NU immunodeficient mice are purchased from Beijing Wintonli Hua laboratory animal technology Limited company, and are bred in SPF-level laboratory animal laboratories of the institute of Life sciences of northeast forestry university, and the breeding conditions are strictly according to requirements of SPF-level laboratory animal houses.
Intrahepatic and extrahepatic bile ducts were collected from adjacent non-tumor tissues of hepatectomized patients obtained at the second subsidiary hospital of the Harbin medical university. All patients participating in the present invention provided informed consent. The present invention was approved by the research ethics committee.
Differentiation of PDO/BDO into pancreatic cell lineage
mPDO or mBDO with good growth state is taken, cultured for 7 days by mEM (without noggin) containing 100ng/mL BMP7 after passage, and then replaced by fresh mDM9 for 14 days. The medium was changed every other day. And after the differentiation is finished, collecting an RNA sample and an immunostaining sample for identification, and detecting the differentiation efficiency by using a flow cytometer. mDM9 the main components include high glucose 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 M DAPT, 10 μ g/mL heparin, 100ng/mL Activin A, 20ng/mL FGF2, 4.4mM Nicotinamide and 1 μ M retinoic acid. hBDO performed the same pancreatic differentiation procedure as mice using human pancreatic beta cell differentiation medium (hDM9) based on mDM9 and 1% N-2 supplement.
Extraction of Total RNA and cDNA Synthesis
Extracting total RNA of cells by using a Trizol method. According to Vazyme TM 
II QSselect RT SuperMix Kit (product 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 conditions refer to the ChamQ Universal SYBR qPCR Master Mix Kit (Cat. No. Q711-02), each gene is provided with 3 repeats, beta-actin is used as an internal reference gene, and the relative expression level of the target gene is calculated by a Ct method.
Frozen sections and organoid patches
Preparation of frozen sections
Tissues or cultured organoids were fixed with 4% PFA for 15min at room temperature. The tissue or organoids were then dehydrated overnight at 4 ℃ in 30% sucrose and embedded in OCT and approximately 10 μm sections were prepared using a thermostated cryomicrotome.
Preparation of organoid Patch
The cultured organoids were digested with dispase to remove matrigel, then fixed with 4% PFA and washed with cold PBS to ensure clearance of matrigel. During the washing process, the organoids may be allowed to settle naturally overnight at 4 ℃ to ensure organoid recovery. The recovered organoids were resuspended in PBS solution, dropped onto a glass slide, dried at 37 deg.C, and immunofluorescent stained after drying.
Immunofluorescence staining
Punch with 0.3% Triton X-100, incubate 10% horse serum (Zhonghua Jinqiao, ZLI9024) at 37 deg.C for 1h for sealing. Primary antibody (Table 2) was incubated overnight at 4 ℃. The secondary antibody (table 3) was incubated for 2h at room temperature in the dark. Nuclear staining is carried out by adopting Hoechst 33342, and after the blocking piece is blocked by adopting an anti-fluorescence quenching blocking piece, a fluorescence detection result is observed by adopting a high-resolution cell imager Delta vision.
Flow cytometry analysis
And blowing the cultured organoids by using a pre-cooled high-sugar DMEM medium, centrifuging to remove the Matrigel, and repeating the process to ensure the clearance of the Matrigel. Organoids were digested into single cells using 0.25% trypsin. Each experiment was performed at least 5X 10 times 5 And (4) cells. Cells were fixed on ice using 4% PFA, perforated with 0.1% TritonX-100, and blocked with 5% BSA. Primary antibody diluted with 1% BSA was added and incubated on ice for 40min, isotype antibody dilutions served as controls. The secondary antibody (1:100) was diluted in 1% BSA and incubated on ice for 30min protected from light. After washing the cells with PBS, the cells were resuspended with PBS and tested by loading.
Glucose-stimulated C peptide secretion
The matrigel-depleted organoids were resuspended in sugar-free Krebs solution, washed 2-3 times, and then cultured overnight in sugar-free Krebs in low-adsorption plates. The next day, a solution of Krebs containing 2mM glucose was added to resuspend the organoids after removal by sugar-free Krebs centrifugation. After incubation for 10 minutes, the supernatant was collected by centrifugation. The cells were washed with sugar-free Krebs, then resuspended and incubated with 20mM glucose in Krebs for 10 minutes, and the supernatant was collected by centrifugation. The supernatant samples were analyzed for C peptide levels using a mouse/human C peptide ELISA kit (mlbio, ml-1015542) according to standard protocols.
Animal model
Nude mice starved for more than 16 hours 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 down-implantation surgery and then blood glucose was monitored.
Diabetic mouse kidney subcapsular organoid transplantation
After the differentiation culture, matrigel around organoids was removed using a pre-cooled DMEM medium with high glucose. Organoids were collected after centrifugation for subcapsular kidney transplantation. Approximately 10 transplants per mouse 6 Individual cells and transplantation was performed by resuspending organoids using 30% matrix. Eight weeks after transplantation, kidneys of transplanted cells were removed, PFA fixed, sucrose dehydrated, cryosectioned and immunostained for detection.
IPGTT glucose tolerance test
Intraperitoneal glucose tolerance test (IPGTT), mice fasted for 16h and were given free water. A baseline blood sample is collected at the tail of a fully conscious mouse, then D-glucose (2g/kg body weight) is injected into the abdominal cavity, and the blood sugar of a tail vein blood sample is monitored by a Roche glucometer at 15, 30, 60, 90 and 120min after glucose administration. The 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 insulin secretion was measured by ELISA.
Statistical analysis
There were three replicates per experiment. Analysis was performed using one-way analysis of variance (ANOVA), comparing two or more doses, and then Dunnett's test was performed for multiple group comparisons (as applicable) to a single control group. P <0.05, P <0.01, P <0.001 were considered statistically significant.
Preparation of organoids from mouse bile duct and pancreas Main duct
Mouse bile duct (mBD) and mouse pancreatic duct (mPD) were obtained by direct dissection under a dissecting microscope, then digested with collagenase IV at 7 ℃ for 20 minutes, then mechanically minced, washed and centrifuged to resuspend the cells in Matrigel. (Corning,54234) and 600. mu.L of mouse amplification medium (mEM) were added to a 24-well plate. All cells were cultured in a humidified 37 ℃ incubator with 95% air and 5% CO2 and freshly prepared medium was stored at 4 ℃ for 2 weeks. The main components of the mouse amplification medium (mEM) include 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(RSPO1), 25ng/mL Noggin, 100ng/mL FGF10, 10mM Nicotinamide and 10 μ M Y27632. After obtaining luminal-like organoids, the medium was changed to EM without Wnt3A and Y27632.
The cultured Bile Duct Organoid (BDO) and pancreatic duct organoid (mPDO) of the mouse are replaced 1 time every other day according to the proportion of 1:3 was passaged 1 time.
Preparation of human bile duct organoid
Donated human bile duct samples (approximately 1 cm in length) were 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 glass slide. The wash was collected after washing the catheter with sterile PBS, and the pellet was resuspended in Matrigel and allowed to stand at 37 ℃ for 10 minutes. After coagulation, hEM medium containing 30% Wnt3A and 10. mu. M Y-27632 was added and cultured at 37 ℃ under 5% CO 2. When cells proliferate into lumen-like structures, they were switched to normal hEM medium and fresh hEM medium needed to be changed every other day. Passaging 2 times a week at a ratio of 1:3 to 1: 5. Human amplification medium (hEM) was supplemented with 10nm Gastrin, 3. mu.M PGE2, and 5. mu. M A8301.
Human expansion medium (hEM) formulation: 1% Penicillin-Streptomyces Solution (HyClone; SV30010), 1% glutamine additive (gibco; 35050-061), 1% B27 with 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 Gastrin (SIGMA; G9145), 3. mu.M PGE2 (SIGMA; P0409), 5. mu.M 7-01 (MCE; HY-10432), 1mol/L Nicotinamide (Sigma; N0636), 1mol/L N-acetyl-L-cysteine (Sigma; Sigma A9165), 100ng/mL R-Spondin (SH-1), 1 mol/L-715636 (RS; S) and S7168 (RS) in each, 1mL 7126 RS, 3 mL);
mouse expansion medium (mEM) formulation: 1% Penicilin-Streptomyces Solution (HyClone; SV30010), 1% glutamine additive (gibco; 35050-;
pluripotent pancreatic progenitor cell culture medium: the EM (proliferation medium) medium contains BMP7 (Biyunnan; P5772) at final concentrations of 25, 50, 100, and 200ng/mL
Mouse pancreas differentiation medium (mDM 9) formulation: including high-glucose DMEM medium (HyClone; SH30022.01), 1% Penicillin-Streptomyces Solution (HyClone; SV30010), 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; 7150-063RS), 1 μ M T3 (TOS; 6666), 10 μ M A8301 (MCE; MCHY-10432), 5 μ M DAPT (MCE; MCE-13027), 10 μ g/mL heparin (SIGMA; SIGMA 3149), 100 μ g/CRIN (PEACN) 120-2624, Sigma-2624 (Sigma-2624) and Nicement (Sigma-2), 10 μ M).
Human pancreatic differentiation medium (human pancreatic differentiation medium, hDM9) formulation: including high-glucose DMEM medium (HyClone; SH30022.01), 1% Penicillin-Streptomyces Solution (HyClone; SV30010), 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 RSPO 7 (R & D; 50-RS), 1 μ M T3 (TOCRIS; 6666), 10 μ M A HY 8301 (MCE; 5 μ M DAPT (MCE; Action13027), 10 μ g/13027), 10 μ g 7145 (GMA; 10 μ 31 μ g/120 mL), heparin (MCP/31-7331) and heparin (MCP) 3/31-31), 4.4mM Nicotinamide (Sigma; N0636) and 1. mu.M Retinoic acid (SIGMA; R2625).
Example 1 method for preparing epithelial cells
1. Bile duct (mBD) and pancreatic duct (mPD) of mice:
after the mice were sacrificed by cervical dislocation, the mice were immediately immersed in a beaker of 75% alcohol, and the surface of the beaker was sterilized with alcohol cotton, and then quickly placed in a sterilized biosafety cabinet. The mice were soaked and sterilized for about 5 min. The mice were taken out, drained and placed on a sterile dissecting frame, and the skin and peritoneum were sequentially cut along the midline of the abdomen, exposing the abdominal cavity. The mice with the abdominal cavity opened were placed under a dissecting scope to find the liver, pancreas and common bile duct connecting the two (extrahepatic bile duct), and the three were removed together and placed in a 10cm dish containing sterile PBS. Utilize clock and watch tweezers, follow the duct that the common bile duct of bile duct and pancreas in the liver directly peeled off under the microscope, the main duct of pancreas is mainly located pancreas head position, and the head is the widest part of pancreas, is discoid, is located the depressed part of duodenum. When the pancreas head is connected with the duodenum, the pancreas head is cut off gently, so that the pancreas main catheter can be separated better in the later period. For easy observation, the bile duct(s) (mouse bile duct, mBD, extra-hepatic duct and intra-hepatic duct) and pancreatic duct(s) (mPD) were separated using a scalpel after dissection was completed, while maintaining the connection of the chole-pancreatic ducts before dissection was completed. The bile duct and pancreatic duct were placed in separate 15mL centrifuge tubes, respectively, and digested with 2mg/mL collagenase IV37 deg.C for 15min to separate the tissue remaining around the duct, and the centrifuge tubes were shaken vigorously every 5 min. The digested hepatobiliary and pancreatic ducts were replaced in 10cm dishes containing sterile PBS. The tissue remaining around the catheter was dissected using a dissecting scope and rinsed with PBS to ensure removal of excess tissue. Placing the biliopancreatic duct in different sterile 1.5mL centrifuge tubes, respectively cutting the biliopancreatic duct as much as possible with dissecting scissors, adding 1mL of collagenase IV of 2mg/mL, digesting at 37 deg.C for 20min, taking out every 5min, and repeatedly blowing with a 1mL gun head. After passing through a 70 μm sieve, the digested duct cells were centrifuged horizontally for 5min at 300 g. The sediment is the epithelial cells.
2. Human bile duct (mBD)
Human bile duct (hBD) A sample of human bile duct (intrahepatic and extrahepatic) was placed in a 10cm petri dish containing sterile PBS, and the duct was cut open longitudinally with dissecting scissors and unfolded into a square with dissecting forceps. One hand holds the deployed catheter with dissecting forceps (taking care to keep the inside of the catheter-epithelial cell side up) and the other hand scrapes the catheter surface with a sterile slide. The catheter was rinsed on the side of the slide that contacted the catheter and after the procedure with sterile PBS, and the rinse was directly retained in a 10cm petri dish. Collecting PBS containing human common bile duct epithelial cells in a 10cm culture dish, centrifuging for 5min at 300g, and collecting the precipitate. The precipitate is the epithelial cells.
Example 2 method for preparing organoids
The obtained epithelial cells of human/mouse were resuspended in 50. mu.L of Matrigel (Corning,54234), and then dropped into a 24-well plate and allowed to stand at 37 ℃ for 5min to coagulate Matrigel. mu.L of mouse expansion medium (mEM) or human expansion medium (hEM) was added to each well for culture.
The prepared human bile duct organoids (hBDO), mouse bile duct organoids (mBDO) and mouse pancreatic duct organoids (mPDO) can be subcultured continuously for more than 10 generations (fig. 1). Immunostaining results showed that each of hBDO, mBDO and mPDO prepared expressed biliary/pancreatic progenitor marker genes (Sox9, PDX1), hepatobiliary progenitor marker genes (HNF1B, HNF4A), pluripotent stem cell marker gene (EpCAM) and epithelial-specific gene (E-cadherin) (results are shown in fig. 2 and fig. 3), demonstrating that hBDO, mBDO and mPDO are progenitor cells with similar differentiation potential, and the antibodies used are shown in table 1.
TABLE 1
Example 3A method for obtaining islet cells
Culturing mouse bile duct and pancreatic duct organoids: respectively culturing bile duct and pancreatic duct organs of the mouse by using mEM culture medium containing 100ng/mL BMP7, wherein the induction time is 7 days, the quantitative PCR result shows that the specific genes of pancreatic endocrine progenitor cells in mBDOs and mPDos are obviously up-regulated and expressed (figure 4A), so that pancreatic progenitor cells are obtained, replacing mDM9 and continuously culturing for 14 days, and finally, replacing fresh culture medium every other day in the whole process of obtaining the pancreatic beta cells.
Example 4A method for obtaining islet cells
Culturing mouse bile duct and pancreatic duct organoids: respectively culturing bile duct and pancreatic duct organs of the mouse by using mEM culture medium containing 100ng/mL BMP7, wherein the induction time is 7 days, the quantitative PCR result shows that the specific genes of pancreatic endocrine progenitor cells in mBDOs and mPDos are obviously up-regulated and expressed (figure 4A), so that pancreatic progenitor cells are obtained, replacing mDM9 containing TLY142 with 1 mu M at the final concentration for continuous culture for 14 days, and finally replacing fresh culture medium every other day in the whole process for obtaining the pancreatic islet beta cells.
The following experiments were used to verify the effect of the experiment:
(1) fluorescent quantitative PCR: BMP7 was added to mBDOs and mPDOs and cultured for 7 days or after continuous induction culture in stages for 21 days, and then the mixture was collected by centrifugation and lysed using TRIZOL to extract total RNA. Using Primerpcript RT The master kit (Vazyme, R323-01) reverses total RNA to cDNA. The reaction system and conditions were as described in the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) (see Table 2 for primers) and detected using a Roche Light Cycle 480 fluorescent quantitative PCR instrument. 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 amount of mRNA was calculated.
TABLE 2 mouse primer sequences
(2) Immunostaining: after step-by-step induction culture of the mBDOs and the mPDOs for 21 days, respectively washing the mBDOs and the mPDOs by PBS, adding 4% PFA for fixation for 20min, repeatedly washing by cold PBS for 3 times, dripping the cell suspension onto a glass slide, and drying at 37 ℃ until organoids are fixed on the glass slide. The gel was punched at 0.3% Triton X-10037 ℃ for 1h, the antigen retrieval solution (Biyun day, P0090) was retrieved at room temperature for 10min, 10% horse serum was blocked at 37 ℃ for 1h, primary antibody was added and incubated at 4 ℃ overnight, secondary antibody was incubated at room temperature for 2h, Hochestt 33342(1:1000) was stained for 15min, and the blocking piece was quenched with anti-fluorescence (Biyun day, P0126). Fluorescence photography was performed using a high resolution live cell imaging system, DeltaVision. The antibody cargo numbers and the concentrations used are shown in Table 3.
TABLE 3 antibody sources and concentrations used
As a result: after BMP7 was cultured for 7 days, the expression of pluripotent pancreatic progenitor cell marker ngn3 in mBDOs and mPDOs was significantly up-regulated (FIG. 4A), mBDOs and mPDOs induced by BMP7 had multidirectional differentiation potential, and the gene and protein expression of the pancreatic endocrine and exocrine markers of mBDOs and mPDOs were significantly up-regulated after mDM 9-induced differentiation (FIG. 4B-D).
As a result: TLY142 can effectively promote mBDOs to differentiate into pancreatic cells in vitro, and the gene and protein expression of the differentiated mBDOs pancreatic endocrine and exocrine markers are obviously up-regulated (fig. 5A-B).
(3) Flow cytometry: digesting differentiated mBDOs and mPDOs into single cells by using trypsin, fixing on 4% PFA ice for 15min, washing by using PBS, perforating by using 0.1% TritonX-100 at room temperature for 10min, washing by using PBS, sealing by using 5% BSA at room temperature for 15min, placing for 15min at room temperature, diluting primary antibody (C-peptide, 1:200) by using 3% BSA, incubating for 40min on ice, adding homotypic antibody diluent into a control group, washing by using PBS, diluting secondary antibody (donkey anti-rabbit 488, 1:100) by using 3% BSA, incubating for 30min in dark on ice, washing by using PBS, and then resuspending on a machine for detection by using 500 mu L.
(4) Glucose-stimulated C-peptide secretion: and (3) resuspending the differentiated organoids by using a sugar-free Krebs solution, cleaning for 2-3 times, placing the organoids in a low-adsorption culture plate, and incubating overnight. Krebs solution containing 2mM glucose was added thereto, and the mixture was incubated for 10min, and the supernatant was collected by centrifugation. After sugar-free Krebs is used for cleaning cells, Krebs solution with 20mM glucose is added for heavy suspension, incubation is carried out for 10min, and supernatant is collected by centrifugation. The C peptide levels in the supernatant samples were analyzed using a mouse C peptide ELISA kit (mlbio, ml-1015542) according to standard protocols.
As a result: BMP7 was effective in promoting differentiation of mbds and mPDOs into functional β cells, and flow cytometry indicated that C-peptide + cell ratios in mbds and mPDOs after differentiation were approximately 8.49 ± 0.45% and 7.23 ± 0.63% (fig. 6A-B), while C-peptide was secreted in response to glucose stimulation (fig. 6C), TLY142 increased the rate of mbds differentiation into pancreatic β cells by 19.90 ± 0.62% (fig. 7A-B), and the differentiated cells had physiological responses to glucose stimulation of mature β cells (fig. 7C). The structural formula of TLY142 is shown in FIG. 8.
(5) In vivo transplantation experiment: injection of streptozotocin (160mg/kg) intraperitoneally 7 days before Nu/Nu mice transplantation induced diabetes. The glucometer measures non-fasting blood glucose in tail vein samples, and mice with elevated blood glucose levels above 16.8mM were selected as diabetic model mice. Beating 2-stage induced and differentiated mBDOs into single cells with pancreatin, and pressing according to 10 6 One cell/cell was transplanted into the kidney capsule of recipient mice, and regular non-fasting blood glucose was measured every 7 days after transplantation. At week 8 of transplantation, nephrectomy was performed to examine the effect of removing the transplanted organoids or islets on improvement of blood glucose.
(6) Glucose tolerance test: the glucose tolerance test is carried out according to a standard scheme, the mice are hungry overnight, 1g/kg of glucose is injected into the abdominal cavity, the blood glucose levels are detected for 0min, 15min, 30min, 60 min, 90 min and 120min, meanwhile, serum before and after glucose injection is collected, and the change of the insulin content is measured by adopting an ELISA method.
(7) Immunostaining of transplanted kidney cryosection: the kidney of the mouse transplanted with organoid obtained by nephrectomy is fixed by PFA overnight, and after dehydration by 30% sucrose, a tissue section with thickness of 10 μm is obtained by using a frozen section technology, and the differentiation condition of the transplanted organoid is detected by using immunostaining. After the section is dried at room temperature, a wax ring is drawn, PBS is used for washing for 3 times to remove embedding agents, 4% PFA is added for fixing for 20min, cold PBS is used for washing for 3 times, 0.3% Triton X-10037 ℃ is used for punching for 30min, antigen repairing liquid (Biyunshi, P0090) is used for repairing for 5min at room temperature, 10% horse serum is used for sealing for 1h at 37 ℃, primary antibody is added for incubation overnight at 4 ℃, secondary antibody is used for incubation for 2h at room temperature, Hochests 33342(1:1000) is used for dyeing for 15min, and anti-fluorescence quenching sealing pieces (Biyunshi, P0126) are used. Fluorescence photography was performed using the high resolution live cell imaging system DeltaVision.
As a result: after 2-stage induced differentiation, the mBDOs were transplanted into STZ-induced renal cysts of diabetic mice, and a significant decrease in blood glucose was observed in the mice, and the TLY 142-induced differentiation of the mBDOs had a hyperglycemic relief effect more similar to that of the positive control group for islet transplantation (fig. 9A), and glucose tolerance experiments showed that compared to the diabetic control group, the TLY 142-induced differentiation of the mBDOs mice had glucose tolerance similar to that of the positive control group for islet transplantation (fig. 9B-C), while the levels of Insulin in serum were increased (fig. 9D). After nephrectomy for transplantation, the blood glucose levels in mice were significantly elevated (fig. 9A), further demonstrating that indeed the blood glucose was reduced due to the transplanted mBDOs. Immunostaining demonstrated that transplanted TLY 142-induced differentiated mBDOs contained β cells expressing insulin and α cells expressing glucagon (FIG. 9E).
2. Culture of human bile duct organoid
The epithelial cells of human bile duct (intrahepatic and extrahepatic bile ducts) obtained by mechanical scraping were seeded in extracellular matrix, cultured in hEM medium at 37 deg.C and 5% CO 2 Human bile duct organoids (hBDOs) were formed after culturing under incubator conditions.
(1) Subculturing of human biliary organoids (human extrahepatic double product organoids, hbds): organoids were resuspended by blowing using pre-cooled high-sugar DMEM medium (HyClone; SH30022.01), centrifuged to remove supernatant, and the pellet was subcultured using Matrigel and hEM. And (4) carrying out passage once at a ratio of 1:3-1:5 every 7-8 days according to the cell density.
(2) Cryopreservation of human bile duct organoids (hbds): the organoids were crushed and resuspended in precooled high-glucose DMEM medium (HyClone; SH30022.01), after centrifugation to remove the supernatant, 1mL of precooled organoid cryopreservation solution was added, the resuspended organoids were transferred to a cell cryopreservation tube, the cryopreservation tube was placed in a cryopreservation box, then transferred to a refrigerator at-80 ℃ overnight, and the cryopreservation tube was transferred to liquid nitrogen for storage the next day.
The hBDOs obtained could be passaged for a long period of time, had stable proliferation and passaging ability, and could maintain good cell state as it was even when the hBDOs were passed to the 10 th generation, and the results are shown in FIG. 1. The structural formula of TLY142 is shown in FIG. 7A. Culturing the human bile duct organoids in hEM culture medium containing 25, 50, 100 and 200ng/mL BMP7 at final concentration for 7 days, replacing hDM9 containing 1 or 10 μ M TLY142 at final concentration, and culturing for 14 days to obtain pancreatic islet cells. The whole process needs to replace fresh culture medium every other day.
The experimental effect was verified using the following experiment:
(1) fluorescent quantitative PCR: after 21 days of continuous induction culture of hBDOs in stages, the hBDOs are collected by centrifugation and are cracked in TRIZOL to extract total RNA. Using Primerpcript RT The master kit (Vazyme, R323-01) converts total RNA to cDNA. The reaction system and conditions were as described in the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) (see Table 4 for primers) and detected using a Roche Light Cycle 480 fluorescent quantitative PCR instrument. 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 amount of mRNA was calculated.
TABLE 4 human primer sequences
(2) Immunostaining: after hBDOs are subjected to induction culture for 21 days in stages, the hBDOs are washed by PBS, 4% PFA is added for fixation for 20min, cold PBS is repeatedly washed for 3 times, the cell suspension is dripped on a glass slide, and the glass slide is dried at 37 ℃ until organoids are fixed on the glass slide. The membrane is punched at 0.3% Triton X-10037 ℃ for 1h, the antigen retrieval solution (Biyun day, P0090) is retrieved at room temperature for 10min, 10% horse serum is blocked at 37 ℃ for 1h, primary antibody is added for incubation at 4 ℃ overnight, secondary antibody is incubated at room temperature for 2h, Hochestt 33342(1:1000) is stained for 15min, and the blocking piece (Biyun day, P0126) is quenched by anti-fluorescence. Fluorescence photography was performed using the high resolution live cell imaging system DeltaVision. The antibody cargo numbers and the concentrations used are shown in Table 2.
As a result: 25.50, 100 and 200ng/mL BMP7 can respectively up-regulate the expression of pancreatic endocrine progenitor cell gene NGN3 after being cultured for 7 days (figure 10A), 1 mu M TLY142 can effectively promote the differentiation of hBDOs to islet cells in vitro, the mRNA and protein expression of the differentiated gene (INS, GCG and SST) of hBDOs pancreatic endocrine markers are both remarkably up-regulated (figure 10B-C), and meanwhile, 10 mu M TLY142 also has the effect of promoting the up-regulation expression of INS gene expression (figure 10D).
(3) Flow cytometry: digesting differentiated hBDOs into single cells by using trypsin, fixing the hBDOs on 4% PFA ice for 15min, washing by using PBS, punching by using 0.1% TritonX-100 at room temperature for 10min, washing by using PBS, sealing by using 5% BSA at room temperature for 15min, placing the hBDOs at room temperature for 15min, then incubating primary antibody (C-peptide, 1:200) diluted by using 3% BSA on ice for 40min, adding homotypic antibody diluent into a control group, washing by using PBS, incubating secondary antibody (donkey anti-rabbit 488, 1:100) diluted by using 3% BSA in a dark place for 30min, washing by using PBS, and then resuspending the hBDOs on an ice for detection by using a 500 mu L resuspension machine.
(4) Glucose-stimulated C peptide secretion: and (3) resuspending the differentiated organoids by using a sugar-free Krebs solution, cleaning for 2-3 times, placing the organoids in a low adsorption culture plate, and incubating overnight. Krebs solution containing 2mM glucose was added thereto, and the mixture was incubated for 10min, and the supernatant was collected by centrifugation. After sugar-free Krebs is used for cleaning cells, Krebs solution with 20mM glucose is added for heavy suspension, incubation is carried out for 10min, and supernatant is collected by centrifugation. The C peptide levels in the supernatant samples were analyzed using a mouse C peptide ELISA kit (mlbio, ml-1015542) according to standard protocols.
As a result: BMP7 in combination with TLY142 was effective in promoting differentiation of hbds into functional β cells, and flow cytometry indicated that the C-peptide + cell ratio in the differentiated hbds was about 25.50 ± 4.82 (fig. 11A-B), and was also able to secrete C-peptide in response to glucose stimulation (fig. 11C), with physiological response to glucose stimulation of mature β cells.
(5) In vivo transplantation experiment: injection of streptozotocin (160mg/kg) intraperitoneally 7 days before Nu/Nu mice transplantation induced diabetes. The glucometer measures non-fasting blood glucose in tail vein samples, and mice with elevated blood glucose levels above 16.8mM were selected as diabetic model mice. Beating hBDOs induced and differentiated by stages into single cells with pancreatin, pressing according to 10 6 One cell/mouse was transplanted into the kidney capsule of the recipient mouse, and the regular non-fasting blood glucose was measured every 7 days after transplantation. At the 8 th week of transplantation, a nephrectomy was performed to examine the effect of removing the transplanted organoids or islets on improvement of blood glucose.
(6) Glucose tolerance test: the glucose tolerance test is carried out according to a standard scheme, mice are starved overnight, 1g/kg of glucose is injected into the abdominal cavity, the blood glucose levels of 0min, 15min, 30min, 60 min, 90 min and 120min are detected, meanwhile, serum before and after the glucose injection is collected, and the change of the insulin content is measured by adopting an ELISA method.
(7) Immunostaining of transplanted kidney cryosection: the kidney of the mouse transplanted with organoid obtained by nephrectomy is fixed by PFA overnight, and after dehydration by 30% sucrose, a tissue section with thickness of 10 μm is obtained by using a frozen section technology, and the differentiation condition of the transplanted organoid is detected by using immunostaining. After the section is dried at room temperature, a wax ring is drawn, PBS is used for washing for 3 times to remove embedding agents, 4% PFA is added for fixing for 20min, cold PBS is used for washing for 3 times, 0.3% Triton X-10037 ℃ is used for punching for 30min, antigen repairing liquid (Biyunshi, P0090) is used for repairing for 5min at room temperature, 10% horse serum is used for sealing for 1h at 37 ℃, primary antibody is added for incubation overnight at 4 ℃, secondary antibody is used for incubation for 2h at room temperature, Hochests 33342(1:1000) is used for dyeing for 15min, and anti-fluorescence quenching sealing pieces (Biyunshi, P0126) are used. Fluorescence photography was performed using a high resolution live cell imaging system, DeltaVision.
As a result: when the BMP7 and the hBDOs induced by the TLY142 are transplanted into the kidney capsule of the STZ-induced diabetic mouse, the blood sugar of the mouse can be obviously reduced (figure 12A), and a glucose tolerance experiment shows that compared with a control group, the glucose tolerance of the transplanted hBDOs mouse is relieved (figure 12B-C), and the level of Insulin in serum is improved (figure 12D). After nephrectomy for transplantation, the blood glucose levels in mice were significantly elevated (fig. 12A), further demonstrating that the drop in blood glucose was indeed due to the transplanted hBDOs. Immunostaining demonstrated that the transplanted organoids contained β cells expressing insulin as well as α cells expressing glucogon (fig. 12E).
SEQUENCE LISTING
<110> northeast forestry university
<120> a method for producing islet beta cells in vitro using BMP7 factor, islet beta cells obtained thereby, and islet beta cells produced thereby
By using
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<170> PatentIn version 3.5
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<400> 16
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Claims (10)
1. An in vitro method for producing islet beta cells, wherein the islet beta cells are obtained by culturing organoids in a culture medium containing BMP7 factor.
2. The method of claim 1, wherein the step of culturing is: the organoids are cultured in proliferation medium containing BMP7 factor to obtain pluripotent pancreatic progenitor cells, and then the pluripotent pancreatic progenitor cells are cultured in differentiation medium to obtain islet beta cells.
3. The method of claim 1, wherein the organoid is a biliary organoid or a pancreatic ductal organoid.
4. The method of claim 1, wherein the BMP7 factor is present at a concentration of 25-200 ng/mL.
5. The method of claim 1, wherein the medium further comprises small molecule C 17 H 22 N 2 O 4 S。
6. The method of claim 5, wherein C is 17 H 22 N 2 O 4 The concentration of S is 1-10. mu.M.
7. Islet beta cells obtained by the method of any one of claims 1-6.
8. Use of the islet beta cells obtained according to claim 7 for the preparation of a cellular medicament for the treatment of diabetes or for the preparation of a cellular medicament for lowering blood glucose.
9. The use of claim 8, wherein the cellular drug comprises an extracellular matrix compatible with cells and a pharmaceutical carrier.
10. The use of claim 9, wherein the pharmaceutical carrier is a nanomaterial or a microfluidics.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305457A (en) * | 2013-06-06 | 2013-09-18 | 浙江省医学科学院 | Method for expanding pancreatic beta cell in vitro |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305457A (en) * | 2013-06-06 | 2013-09-18 | 浙江省医学科学院 | Method for expanding pancreatic beta cell in vitro |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| GHANI M. W. ET AL.: "Differentiation of rat pancreatic duct stem cells into insulin-secreting islet-like cell clusters through BMP7 inducement", TISSUE CELL, vol. 67 * |
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