CN116144584B - Pancreatic progenitor cells differentiated from human induced pluripotent stem cells, and preparation method and application thereof - Google Patents
Pancreatic progenitor cells differentiated from human induced pluripotent stem cells, and preparation method and application thereof Download PDFInfo
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- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
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Abstract
The application provides a preparation method of pancreatic progenitor cells differentiated from human induced pluripotent stem cells, comprising the following steps: providing hiPSCs, and inoculating the hiPSCs on a culture plate coated by matrigel for culture; performing endodermal induced differentiation on the hiPSCs to obtain endodermal cells, wherein the endodermal induced differentiation process comprises: firstly, culturing for 1 day by using a definitive endoderm induction culture medium A, and then replacing the definitive endoderm induction culture medium B for continuous culture for 2-3 days; differentiating the endoderm cells into a primitive gut tube; differentiating the raw intestinal tube into a hind foregut; differentiation of the hind foregut into pancreatic progenitor cells. The preparation method can efficiently utilize human induced pluripotent stem cells to differentiate to obtain pancreatic progenitor cells with good purity, high activity and perfect functions, and is expected to be used for clinical research and application.
Description
Technical Field
The application relates to the field of cell biology, in particular to pancreatic progenitor cells differentiated from human induced pluripotent stem cells, and a preparation method and application thereof.
Background
Diabetes is a group of metabolic diseases characterized by hyperglycemia, and is largely classified as type I diabetes, type II diabetes, gestational diabetes, and other specific types of diabetes. In recent years, the incidence of diabetes is on the rise and tends to be younger, and diabetes has become a common endocrine and metabolic disease that seriously jeopardizes human health. Type I diabetics, whose insulin secretion is insufficient due to damaged islet cells, need to rely entirely on exogenous insulin therapy, account for nearly 10% of the total number of diabetics. Islet transplantation is the most effective method at present for curing diabetes, however, the wide application of the therapy is seriously hindered by the relative shortage of donors, immune rejection after transplantation and the like, so that a need exists for searching for new sources of islet cells.
Human induced pluripotent stem cells (Human induced pluripotent stem cells, hiPSC) are a cell type that are reprogrammed by somatic cells to give characteristics similar to embryonic stem cells. Studies have shown that hiPSCs can be induced in vitro by small molecules into different types of somatic cells, including pancreatic progenitor cells and pancreatic beta cells. However, due to the complexity of molecular networks in pancreatic organogenesis, a unified induction scheme for in vitro differentiation of hiPSCs into islet progenitor cells has not been formed at present, and the problems of large dependence on cell lines, low operational repeatability and the like generally exist in the operation process of the existing induction scheme.
Disclosure of Invention
In view of the above, the present application provides a method for preparing pancreatic progenitor cells differentiated from human induced pluripotent stem cells, which can efficiently utilize human induced pluripotent stem cell differentiation to obtain pancreatic progenitor cells with good purity, high survival rate and perfect function, and is expected to be used in clinical research and application.
In a first aspect, the present application provides a method for preparing pancreatic progenitor cells differentiated from human induced pluripotent stem cells, comprising the steps of:
(1) Providing human induced pluripotent stem cells, and inoculating the cells on a culture plate coated with matrigel for culture;
(2) Performing endodermal induction differentiation on the human induced pluripotent stem cells to obtain endodermal cells; the endodermal induced differentiation process includes: firstly culturing for 1 day by using a definitive endoderm induction culture medium A, and then replacing the definitive endoderm induction culture medium A with a definitive endoderm induction culture medium B for further culturing for 2 to 3 days, wherein the definitive endoderm induction culture medium A is an RPMI1640 basal medium containing 100 to 110ng/mL of Activin A and 2 to 4 mu mol/L of CHIR99021, and the definitive endoderm induction culture medium B is an RPMI1640 complete medium containing 100 to 110ng/mL of Activin A;
(3) Transferring the endoderm cells into a first differentiation medium for culturing for 2-3 days, and changing liquid every day to differentiate the endoderm cells into a primitive intestinal canal;
(4) Discarding the first differentiation medium in the step (3), replacing the first differentiation medium with a second differentiation medium for 2-3 days, and replacing the liquid every day to differentiate the raw intestine into the hind foregut;
(5) Discarding the second differentiation medium in the step (4), changing to a third differentiation medium for culturing for 5-6 days, changing liquid every day to induce the metaintestinal differentiation, and collecting to obtain pancreatic progenitor cells.
Optionally, in the step (2), the process of replacing the definitive endoderm induction medium B with the definitive endoderm induction medium B for 2-3 days comprises: cell culture was performed with daily replacement of fresh definitive endoderm induction medium B, and serum replacement was added to the daily replacement of fresh definitive endoderm induction medium B. Optionally, the serum replacement is 0.8% -10% by volume.
Further, optionally, in the step (2), the process of replacing the definitive endoderm induction medium B with the culture medium B for 2-3 days further comprises: cell culture is carried out by replacing fresh definitive endoderm induction medium B every day, and serum substitutes with different volume percentages are added into the definitive endoderm induction medium B which is replaced every day, the serum substitutes with the volume percentages of 0.8-1.0% are added into the definitive endoderm induction medium B in the first day, and the serum substitutes with the volume percentages of 8-10% are added into the definitive endoderm induction medium B in the next and subsequent days.
For example, in step (2), the replacement of definitive endoderm induction medium B for 2 days, comprising: the method comprises the steps of replacing fresh definitive endoderm induction medium B every day for cell culture, wherein 0.8-1.0% of serum replacement is added into the definitive endoderm induction medium B in the first day, and 8-10% of serum replacement is added into the definitive endoderm induction medium B in the second day.
Alternatively, for example, in step (2), the replacement of definitive endoderm induction medium B for 3 days, comprising: the fresh definitive endoderm induction medium B is replaced every day for cell culture, wherein 0.8-1.0% of serum replacement is added into the definitive endoderm induction medium B in the first day, and 8-10% of serum replacement is added into the definitive endoderm induction medium B in the second and third days.
Alternatively, the serum replacement is a well-defined, non-Fetal Bovine Serum (FBS) -containing cell culture supplement, which may be, but is not limited to, commercial serum replacement. Further alternatively, the serum replacement is KnockOut TM Serum substitutes.
Wherein, the Activin A is Activin A, is a multifunctional cytokine and is a member of TGF-beta superfamily. CHIR99021 is a potent and selective GSK-3 alpha/beta inhibitor and is also a potent Wnt/beta-catenin signaling pathway activator.
Alternatively, in step (1), the hiPSCs may be directly a single-cell suspension of hiPSCs, or a single-cell suspension of hiPSCs obtained by dissociation treatment with a cell dissociation agent. The hiPSCs single cell suspension is beneficial to the follow-up accurate cell inoculation work.
Alternatively, in step (1), the hiPSCs are cultured in a hiPSCs-specific medium that is a mTeSR containing 10 μ M Y27632 (or Y-27632) TM 1 culture medium. Wherein the Y27632 is a selective inhibitor of ROCK, a potent, cell permeable, reversible, selective Rho-associated protein kinase inhibitor.
Optionally, the first differentiation medium is based on MCDB131 medium and comprises 48-52 ng/mL FGF7, 0.15-0.35 mmol/L ascorbic acid, 2-4% defatted BSA, 1.5-2.5 mmol/L glutamine, 5-15 mmol/L glucose and 2.5-3.5 g/L sodium bicarbonate.
Wherein the MCDB131 culture medium is a low-protein and serum-free culture medium designed for specific cells. The MCDB131 culture medium can be a culture medium which is artificially configured or a commercial culture medium.
Alternatively, the FGF7 is fibroblast growth factor 7. The ascorbic acid is vitamin C, is chemically named as L- (+) -threose type 2,3,4,5, 6-pentahydroxy-2-hexenoic acid-4-lactone, and is also named as L-ascorbic acid, and is an antioxidant. The defatted BSA is fatty acid-free bovine serum albumin and does not contain IgG.
Optionally, the mTeSR TM The culture medium 1 is a serum-free culture medium with definite chemical components, is used for maintaining and culturing and expanding undifferentiated human embryonic stem cells without feeder cells, and can also be used for inducing and maintaining human induced pluripotent stem cells. In the present application, the mTESR TM The medium 1 may be, but is not limited to, commercial medium.
Optionally, the second differentiation medium is based on MCDB131 medium and comprises 50-55 ng/mL FGF7, 0.15-0.35 mmol/L ascorbic acid, 0.2-0.3 mu mol/L SANT-1, 1-1.2 mu mol/L retinoic acid RA, ITS-X in a dilution ratio of 1:100-1:200, 90-110 nmol/LLDN193189, 190-210 nmol/L TPB, 2% -4% defatted BSA, 1.5-2.5 mmol/L glutamine, 5-15 mmol/L glucose and 2.5-3.5 g/L sodium bicarbonate.
Optionally, the third differentiation medium is based on MCDB131 medium and comprises 1.5-2.5 ng/mL FGF7, 0.15-0.35 mmol/L ascorbic acid, 0.2-0.3 mu mol/L SANT-1, 0.05-0.15 mu mol/L retinoic acid RA, ITS-X with a dilution ratio of 1:100-1:200, 190-210 nmol/LLDN193189, 90-110 nmol/L TPB, 2% -4% defatted BSA, 1.5-2.5 mmol/L glutamine, 5-15 mmol/L glucose and 2.5-3.5 g/L sodium bicarbonate.
Wherein the SANT-1 has a molecular formula of C 23 H 27 N 5 Is a potent Smoothened (Smo) antagonist. The retinoic acid RA is a natural oxidative metabolite of vitamin a. The ITS-X is insulin-transferrin-selenium-ethanolamine, a medium supplement. The LDN193189 (DM 3189) is a selective BMP signaling inhibitor, inhibiting ALK1, ALK2, ALK3 and ALK6. The TPB is a benzolactam-derived cell-permeable Protein Kinase C (PKC) activator.
Optionally, in step (2), the human induced pluripotent stem cells have a degree of fusion of 90% or more when induced to differentiate. In the application, when the fusion degree of the human induced pluripotent stem cells reaches more than 90%, endodermal induction differentiation is started, so that the conversion efficiency of the human induced pluripotent stem cells to endodermal cells can be greatly improved.
Alternatively, the human induced pluripotent stem cells have a degree of fusion of greater than or equal to 90% and less than 100%.
Optionally, the Matrigel is Matrigel. In other embodiments, the matrigel may also be selected from other matrigel or equivalents thereof, such as fibronectin, polylysine.
Optionally, in step (1), the human induced pluripotent stem cells are present at 2.0X10 × 5 ～2.6×10 5 Individual cells/cm 2 Is inoculated on a culture plate coated with matrigel. The matrigel can be polymerized to form a three-dimensional matrix with biological activity after being coated at room temperature, and simulates the structure, composition, physical characteristics and functions of a basement membrane of cells in vivo, thereby being beneficial to the culture and differentiation of cells in vitro and the research on cell morphology, biochemical functions, migration, infection and gene expression.
In the present application, the cell plate may include, but is not limited to, a cell culture dish. For example, the cell plate may be, but is not limited to, one or more of an orifice plate, a culture flask, and a culture dish.
The concentrations marked in the application are all final concentrations, wherein the defatted BSA is mass and volume percent, and ITS-X is prepared into the culture medium by commercial ITS-X (100X) according to the dilution ratio of 1:100-1:200.
The cell culture in the preparation process of the application is all cultured under the conventional environment, such as 37 ℃ and 5% CO 2 Is cultured in a constant temperature incubator environment.
According to the preparation method of pancreatic progenitor cells differentiated from human induced pluripotent stem cells, high-efficiency differentiation from human induced pluripotent stem cells to endoderm cells can be achieved in a very short time, the whole preparation method can obtain pancreatic progenitor cells with good purity, high activity rate and perfect functions only by five steps for about 15 days, and the preparation method is good in repeatability, strong in operability and wide in application prospect.
In a second aspect, the present application also provides a pancreatic progenitor cell differentiated from a human induced pluripotent stem cell, prepared by the method of the first aspect of the application, having a double positive expression level of PDX1 and nkx6.1 of 67-71%.
Wherein, both PDX1 and nkx6.1 are key markers for pancreatic progenitor cells. Meanwhile, pancreatic progenitor cells are characterized by co-expression of the transcription factors PDX1 and NKX 6-1. Determination of the biscationic expression levels of PDX1 and Nkx6.1 based on pancreatic progenitor cells can strongly indicate the level of activity and the level of pancreatic progenitor cells.
The pancreatic progenitor cells differentiated from the human induced pluripotent stem cells provided by the second aspect of the application have the characteristics of good purity, high activity rate and perfect functions, and the double positive expression level of PDX1 and Nkx6.1 can reach 67-71%, so that the pancreatic progenitor cells can be widely applied to various clinical tests of in-vitro pancreatic progenitor cells, and have profound research significance. For example, a well-functioning pancreatic progenitor cell with good purity, high viability may be used as an important cell source for further induced differentiation into islet beta cells.
In a third aspect, the present application also provides the use of pancreatic progenitor cells differentiated from human induced pluripotent stem cells prepared by the preparation method according to the first aspect of the application or provided by the second aspect for the preparation of a medicament for the prevention, diagnosis and treatment of diabetes.
In the present application, the application may be, but is not limited to, a detection kit, a targeted drug carrier or an application in a drug screening model. For example, the use of the third aspect of the application may be as an application associated with islet beta cells.
In the application of the third aspect of the present application, the pancreatic progenitor cells prepared by the preparation method of the first aspect of the present application or differentiated from the human induced pluripotent stem cells provided by the second aspect of the present application can greatly enrich the application range of the pancreatic progenitor cells differentiated from the human induced pluripotent stem cells provided by the present application due to the high efficiency of the preparation method and the characteristics of good purity, high activity rate and perfect functions of the prepared pancreatic progenitor cells, and has a wide and abundant research value.
Drawings
For a clearer description of the present application, reference will be made to the following detailed description of embodiments taken in conjunction with the accompanying drawings.
FIG. 1 is a flow chart of a method for preparing pancreatic progenitor cells from human induced pluripotent stem cells according to an embodiment of the application;
FIG. 2 is a graph showing the growth status of endoderm cells (100X) in each experimental group according to the example of the present application;
FIG. 3 shows the immunofluorescence of the test groups of the first embodiment of the present application for detecting the expression of SOX17 and FOXA2 markers at the endodermal differentiation stage (400X);
FIG. 4 shows the quantitative fluorescent detection of the expression of c-Kit, CXCR4, SOX17 markers at the endoderm differentiation stage of each experimental group in example I of the effect of the present application;
FIG. 5 shows the state of cell growth (100X) at various stages of pancreatic progenitor cells induced by different inducers according to example II of the effect of the application;
FIG. 6 shows the effect of the application in example II (400X) of fluorescence detection of the expression of PDX1 and Nkx6.1 markers at the differentiation stage of pancreatic progenitor cells;
FIG. 7 shows the second embodiment of the present application, in which the expression of PDX1 and Nkx6.1 markers is detected in the differentiation stage of pancreatic progenitor cells of the first strain;
FIG. 8 shows the second embodiment of the present application for detecting the expression of PDX1 and Nkx6.1 markers at the differentiation stage of pancreatic progenitor cells;
FIG. 9 shows the expression of pancreatic islet beta cell markers induced by pancreatic progenitor cells in example III, and an insulin secretion assay under in vitro glucose stimulation.
Detailed Description
The following description is of the preferred embodiments of the present application, and it should be noted that it will be apparent to those skilled in the art that modifications and variations can be made without departing from the principle of the application, and these modifications and variations are also regarded as the scope of the application.
In one embodiment of the present application, as shown in FIG. 1, there is provided pancreatic progenitor cells differentiated from human induced pluripotent stem cells and a method for preparing the same, comprising the steps of:
s01, providing human induced pluripotent stem cells, and inoculating the cells on a culture plate coated with matrigel for culture;
s02, performing endoderm induction differentiation on the human induced pluripotent stem cells to obtain endoderm cells; the endodermal induced differentiation process includes: firstly culturing for 1 day by using a definitive endoderm induction culture medium A, and then replacing the definitive endoderm induction culture medium A with a definitive endoderm induction culture medium B for further culturing for 2 to 3 days, wherein the definitive endoderm induction culture medium A is an RPMI1640 basal medium containing 100 to 110ng/mL of Activin A and 2 to 4 mu mol/L of CHIR99021, and the definitive endoderm induction culture medium B is an RPMI1640 complete medium containing 100 to 110ng/mL of Activin A;
s03, transferring the endoderm cells into a first differentiation medium for culturing for 2-3 days, and changing the liquid every day to differentiate the endoderm cells into a primitive gut tube;
s04, discarding the first differentiation medium in the step (3), replacing the first differentiation medium with a second differentiation medium for 2-3 days, and replacing the liquid every day to differentiate the raw intestinal canal into a hind foregut;
s05, discarding the second differentiation medium in the step (4), replacing the second differentiation medium with a third differentiation medium for culturing for 5-6 days, replacing liquid every day to induce the metaintestinal differentiation, and collecting to obtain pancreatic progenitor cells.
Wherein, in the S01, the hiPSCs may be a commercial cell line or may be induced by donor cells, and the donor cells may include, but are not limited to, one or more of villus cells, fibroblasts, neural stem cells, liver cells, mesenchymal stem cells, peripheral blood cells, mammary epithelial cells, and adipose stem cells.
In some embodiments, hiPSCs may be derived from somatic cells or other progenitor cells of individuals with a unique genetic background. For example, hiPSCs may be produced by cells from individuals with disease conditions, individuals with high risk disease conditions, and/or individuals with low risk disease conditions. Cells produced by individuals with different genetic backgrounds or cells differentiated in vitro therefrom as described herein may be used to study the mechanism of disease conditions (such as diabetes) and to identify therapeutic targets.
The following examples are given to further illustrate the present application, and unless otherwise indicated, the reagents used in the various examples, comparative examples and effect examples of the present application are commercially available.
Example 1A method for the preparation of pancreatic progenitor cells differentiated from human induced pluripotent stem cells
Resuspension of hiPSCs after resuscitating in mTeSR containing 10 μ M Y27632 TM 1, counting, placing proper amount of cells into a culture dish coated with Matrigel matrix gel for culturing, changing the culture medium every day to maintain the undifferentiated state, dissociating the cells into single cells with mild cell dissociation reagent (GCDR) when the cells are fused to 70% -80%, counting, and adding 2.6X10 g 5 cells/cm 2 Is inoculated in a six-hole plate coated by Matrigel matrix glue, the growth state of cells is observed after overnight culture, and when the cell fusion degree reaches 90% -100%, induced differentiation is started. At the stage of differentiation of hiPSCs into endoderm cells at S02, after 1 day of culture with RPMI1640 basal medium containing 100ng/mL of Activin A and 3. Mu. Mol/L of CHIR99021, the culture medium was replaced with 100ng/mL of Activin A and 0.8% of KnockOut TM The complete medium of RPMI1640 for serum replacement was continued for 1 day, and the culture was changed to 100ng/mL of Activin A and 8% of KnockOut by 3 rd day TM RPMI1640 complete medium of serum replacement. The growth state of the endoderm cells induced by the induction factors was observed and photographed. At the stage of differentiation of endodermal cells into the gastrula of S03: the differentiated MCDB131 medium was supplemented with 2% BSA, 2mM glutamine (100X), 10mM glucose, 3g/L sodium bicarbonate, 50ng/mL FGF7, 0.25mM ascorbic acid (Sigma), and incubated for 3 days with daily replacement of new oneFresh medium. At the hind foregut differentiation stage of S04: MCDB131 medium added with 2%BSA, 2mM glutamine (100X), 10mM glucose, 3g/L sodium bicarbonate, 50ng/mL FGF7, 0.25mM ascorbic acid, 0.25 u M SANT-1, 1 u M RA, 1:200ITS-X, 100nM LDN193189, 200nM TPB, with the medium culture for 3 days, every day replacement of fresh medium. At the metaintestinal differentiation stage of S05 towards pancreatic progenitor cells: MCDB131 medium added with 2% BSA, 2mM glutamine (100X), 10mM glucose, 3g/L sodium bicarbonate, 2ng/mL FGF7, 0.25mM ascorbic acid, 0.25 u MSANT-1, 0.1 u M RA, 1:200ITS-X, 200nM LDN193189 and 100nM TPB, with this medium differentiated for 5 days, cells were changed every day for fresh culture, and then harvested to give pancreatic progenitor cells, and example 1 group was named SD group.
Comparative example 1A method for preparing pancreatic progenitor cells differentiated from human induced pluripotent stem cells
Consistent with the experimental procedure of example 1 above, the only difference was in the stages of differentiation of hiPSCs into endodermal cells of S02: during S02, on day 1 of differentiation, MCDB131 medium containing 3. Mu.M CHIR99021 and 115ng/mL Activin A was added to the cell culture dish; on day 2 of differentiation, the cell culture broth was replaced with MCDB131 medium containing 0.3. Mu.M CHIR99021 and 110ng/mL Activin A; day 3 of differentiation was replaced with MCDB131 medium containing 100ng/mL Activin A. Then, pancreatic progenitor cells were obtained by differentiating stage of endoderm cells into protointestinal tract, differentiating stage of protointestinal tract into hind foregut of S04, differentiating stage of hind foregut into pancreatic progenitor cells of S05, and designated as Activin a group of comparative example 1.
Comparative example 2A method for preparing pancreatic progenitor cells differentiated from human induced pluripotent stem cells
Consistent with the experimental procedure of example 1 above, the only difference was in the stages of differentiation of hiPSCs into endodermal cells of S02: GDF8 is used in combination with CHIR-99021 (where GDF8 belongs to the TGF-beta superfamily and is a secreted growth and differentiation factor that regulates cell growth and differentiation in embryonic and adult tissues). On day 1 of differentiation, MCDB131 medium containing 3. Mu.M CHIR99021 and 100ng/mL GDF8 was added to the cell culture dish; on days 2 and 3, the medium was replaced with MCDB131 medium containing 100ng/mL GDF 8. Then, pancreatic progenitor cells were obtained by differentiating stage of endoderm cells into protointestinal tract, differentiating stage of protointestinal tract into hind foregut of S04, differentiating stage of hind foregut into pancreatic progenitor cells of S05, and designated as GDF8 group of comparative example 2.
In order to evaluate the effect of pancreatic progenitor cells differentiated from human induced pluripotent stem cells prepared by the above-described method of the application, the following effect examples were carried out.
Effect example one: validation of differences in endodermal cells induced by different inducers
Immunofluorescence assays were performed on the differentiation stages of hiPSCs of S02 of example 1 (SD group), comparative example 1 (Activin a group), and comparative example 2 (GDF 8 group) into endodermal cells to examine the expression of endodermal cell markers SOX17 and FOXA 2. Taking 4×10 5 After overnight incubation on a 20mm diameter glass-bottomed cell culture dish, the individual cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 15min at room temperature, washed 3 times with PBS, and incubated with cell-permeabilizing solution for 5min at room temperature. Subsequently, the mixture was blocked with a serum sealant at room temperature for 1h. After pipetting off the blocking solution, the primary antibodies SOX17 and FOXA2 were added and incubated overnight at 4 ℃. PBS was used for 3 times, the cell nuclei were stained with a species-specific fluorescent conjugated antibody (Alexa Fluor-488 conjugated anti-rabbit IgG (H+L) antibody, alexa Fluor-555 conjugated anti-mouse IgG (H+L)) incubated for 1H at room temperature in the dark, PBS was used for 3 times, 4', 6-diamino-2-phenylindole (DAPI) was used for staining, and photographs were taken using an inverted fluorescent microscope.
The expression of c-Kit, CXCR4 and SOX17 markers at the differentiation stage of S1 endoderm was quantitatively detected by Q-PCR, enough cells were harvested and total RNA of each group of cells was extracted according to QIAzol Lysis Reagent instructions, and the concentration and purity of RNA were detected by a micro-spectrophotometer, followed by reverse transcription of RNA into cDNA according to a reverse transcription Kit. The mRNA expression levels of hSOX17, hCHCR 4 and hKIT were detected by Q-PCR using cDNA as a template and GAPDH as an internal reference. Forward and reverse primers (or upstream and downstream primers) were each designed based on the mRNA sequence provided by NCBI and were finally synthesized by Shanghai Biotechnology, inc. using Primer 3Plus software. The PCR primer sequences are shown in Table 1.
TABLE 1 primers for real-time quantitative PCR analysis
The morphology of endodermal cells grown in the S02 stage of example 1 (SD group), comparative example 1 (Activin a group), and comparative example 2 (GDF 8 group) is shown in fig. 2, the morphology of the cells undergoing induced differentiation is changed, the cells become dense and there is a clear limit in the contact between endodermal cells, and the gaps between cells of GDF8 group begin to take on the bulge-like morphology. As shown in FIG. 3, the marker immunofluorescence detection results show that the double positive rates of SOX17 and FOXA2 of SD group and Activin A group are not greatly different, the expression rate is 85-90%, and the double positive rate of GDF8 group is the lowest. As shown in FIG. 4, the results of the fluorescent quantitative determination are shown in FIG. 4, wherein (a), (b) and (c) in FIG. 4 are the fluorescent quantitative determination data of the control group, the active A group, the GDF8 group and the SD group, respectively, and the relative expression levels of the endodermal cell markers c-Kit, CXCR4 and SOX17 of the active A group are the highest, while the relative expression level of the GDF8 group is the least.
Effect example two: verification of the Effect of different inducer-induced endodermal cells in the preparation of pancreatic progenitor cells
During the induction of pancreatic progenitor cells in each of example 1 (SD group), comparative example 1 (Activin A group) and comparative example 2 (GDF 8 group), cells were taken for immunofluorescence detection as described in the procedure of effect example one, and the expression of PDX1 and Nkx6.1 markers at the differentiation stage of S05 pancreatic progenitor cells was examined. Cell antigen detection: cells were collected and washed with DPBS, centrifuged, 1mL of DPBS was added to resuspend cell pellet, incubated at room temperature for 10min in 1. Mu. L Fixable Viability Stain 700 dye, centrifuged to discard supernatant and washed 1 time with BD stain buffer. 1mL BD 1 XFix/Perm Buffer was added, incubated at 4deg.C for 50 minutes in the dark, and then washed 2 times with 1 XPerm/Wash Buffer. Cells were resuspended using 100. Mu.L of stand buffer and incubated at 4℃for 50min in the dark after addition of antibody NKX6.1-APC and PDX 1-FITC. After the antibody incubation was completed, the antibody was washed 2 times with BD stand buffer. Cells were resuspended by addition of 350. Mu. L BD stain buffer and analyzed mechanically on a flow cytometer.
The cell growth states of the pancreatic progenitor cells induced by different induction factors in example 1 (SD group), comparative example 1 (Activin a group) and comparative example 2 (GDF 8 group) are shown in fig. 5, the cell stack growth of the Activin a group and the GDF8 group is obvious from the S03 stage, the bottom layer tiling cells are gradually thinned, the spherical and ellipsoidal structures are gradually increased, the GDF8 group has the most of the linear, spherical and ellipsoidal structures after the induction to the S05 stage, the Activin a group has relatively less cells, and the cells of the SD group have the least of the dense growth, spherical and ellipsoidal structures. After digestion, the cells of each group were re-plated for immunofluorescent staining as shown in FIG. 6, the pancreatic progenitor cells of SD group had the highest double positive rates of PDX-1 and NKx6.1, while the GDF 8-induced cells had lower double positive rates of PDX-1 and NKx6.1 than the cells induced by Activin A, and the cells of each group at S05 stage were taken for flow detection of the expression levels of PDX1 and Nkx6.1, with significant differences in the double positive expression levels of the cells of the different induction systems. The flow data of the first and second pancreatic progenitor differentiation stages PDX1 and Nkx6.1 markers in FIGS. 7 and 8, respectively, show that the expression rate of actin A was about 20% and that of GDF8 was about 7% lower. The method of comparative example 1 was referred to "culture medium and use thereof and method of inducing differentiation of pluripotent stem cells into islets" (application publication No. CN 112251396A), the method of comparative example 2 was referred to (Biomedicines, 2020,8,179; doi:10.3390/Biomedicines 8070179), but the purity of the induced pancreatic progenitor cells was far lower than that described in the text, and the factors of the induction failure were likely to be different among the cell lines used, whereas the SD group cell line induced by the same cell line was apparent in the present application, PDX-1 was expressed about 90% and double positive was expressed about 67 to 71%. Meanwhile, the two detection results of the first strain and the second strain are basically consistent, so that the preparation method provided by the application has the advantages of good repeatability, strong operability and wide application prospect. Existing pancreatic progenitor cell culture kit (e.g., STEMdiff TM Brand) the purity of the obtained pancreatic progenitor cells (PDX1+NKX6.1+) is generally in the range of 66.5% -74.5%, which means thatThe method provided by the application can achieve the same induction effect as the finished product kit, but the components in the finished product kit are kept secret, the price is high, and the number of induced cells is limited, while the pancreatic progenitor cells obtained by the preparation method disclosed by the application have the advantages of good purity, high activity rate and complete functions, and the reagent has simple components, is simple and convenient to prepare and operate, and greatly reduces the preparation cost.
Effect example three: verification of insulin secretion levels of pancreatic progenitor cells induced by different induction factors into islet beta cells
Pancreatic progenitor cells obtained by induction in the S05 phase of each of example 1 (SD group), comparative example 1 (Activin A group), and comparative example 2 (GDF 8 group) were digested and resuspended in induction medium to a density of 3X 10 6 Transferring the cells to an ultralow adsorption pore plate for continuous culture, and continuously inducing the S4 pancreatic progenitor cells for 33 days by referring to the culture medium and the method for differentiating induced pluripotent stem cells into pancreatic islets (application publication No. CN 112251396A) disclosed by the application, wherein the used induction factors comprise: retinoic acid RA, T3, LDN193189, ALK5 inhibitor II, zinc sulfate, heparin sodium, gamma-secretase inhibitors, N-acetylcysteine, trolox, R428, induced into islet beta cells, and immunofluorescence tested for expression of insulin markers, and validated whether the induced cells were able to secrete insulin under glucose stimulation at concentrations of 3mM and 20 mM.
As a result, as shown in FIGS. 9 (a) and (b), the immunofluorescence was examined for insulin expression, and positive expression was found in each group, but the positive expression regions were smaller in the Activin A group and the GDF8 group than in the SD group. Cells were cultured simultaneously in glucose concentrations of 3mM and 20mM for 1.5 hours, insulin was detected in the culture supernatant, and insulin secretion increased with increasing glucose concentration, with the highest insulin secretion in SD group and very little secretion in Activin A group. In-vitro insulin secretion detection experiments show that the pancreatic islet cells differentiated by the pancreatic progenitor cells prepared by the application can better respond to the stimulation of high-concentration glucose to secrete insulin, which proves that the pancreatic progenitor cells prepared by the application have perfect functions and can better differentiate into functional mature pancreatic islets.
The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (4)
1. A method for preparing pancreatic progenitor cells differentiated from human induced pluripotent stem cells, comprising the steps of:
(1) Providing human induced pluripotent stem cells, and inoculating the cells on a culture plate coated with matrigel for culture;
(2) Performing endodermal induction differentiation on the human induced pluripotent stem cells to obtain endodermal cells; the endodermal induced differentiation process includes: culturing for 1 day by using a definitive endoderm induction culture medium A; then, replacing fresh definitive endoderm induction medium B every day for continuous culture for 2 days, wherein 0.8-1.0% of serum substitute in volume percentage is added into the definitive endoderm induction medium B in the first day, and 8-10% of serum substitute in volume percentage is added into the definitive endoderm induction medium B in the second day; wherein, the definitive endoderm induction culture medium A is an RPMI1640 basal culture medium containing 100-110 ng/mL of Activin A and 2-4 mu mol/L of CHIR99021, and the definitive endoderm induction culture medium B is an RPMI1640 complete culture medium containing 100-110 ng/mL of Activin A;
(3) Transferring the endoderm cells into a first differentiation medium for culturing for 2-3 days, and changing liquid every day to differentiate the endoderm cells into a primitive intestinal canal; the first differentiation culture medium takes MCDB131 culture medium as a basic culture medium and comprises 48-52 ng/mL FGF7, 0.15-0.35 mmol/L ascorbic acid, 2-4% defatted BSA, 1.5-2.5 mmol/L glutamine, 5-15 mmol/L glucose and 2.5-3.5 g/L sodium bicarbonate;
(4) Discarding the first differentiation medium in the step (3), replacing the first differentiation medium with a second differentiation medium for 2-3 days, and replacing the liquid every day to differentiate the raw intestine into the hind foregut; wherein the second differentiation culture medium takes MCDB131 culture medium as basic culture medium, contains 50-55 ng/mL FGF7, 0.15-0.35 mmol/L ascorbic acid, 0.2-0.3 mu mol/L SANT-1, 1-1.2 mu mol/L retinoic acid RA, ITS-X with a dilution ratio of 1:100-1:200, 90-110 nmol/L LDN193189, 190-210 nmol/L TPB, 2% -4% defatted BSA, 1.5-2.5 mmol/L glutamine, 5-15 mmol/L glucose and 2.5-3.5 g/L sodium bicarbonate;
(5) Discarding the second differentiation medium in the step (4), changing to a third differentiation medium for culturing for 5-6 days, changing liquid every day to induce the metaintestinal differentiation, and collecting to obtain pancreatic progenitor cells; wherein the third differentiation culture medium takes MCDB131 culture medium as basic culture medium, contains 1.5-2.5 ng/mL FGF7, 0.15-0.35 mmol/L ascorbic acid, 0.2-0.3 mu mol/L SANT-1, 0.05-0.15 mu mol/L retinoic acid RA, ITS-X with a dilution ratio of 1:100-1:200, 190-210 nmol/L LDN193189, 90-110 nmol/L TPB, 2% -4% defatted BSA, 1.5-2.5 mmol/L glutamine, 5-15 mmol/L glucose and 2.5-3.5 g/L sodium bicarbonate; the prepared double positive expression level of PDX1 and Nkx6.1 of the pancreatic progenitor cells reaches 67-71%.
2. The method for producing pancreatic progenitor cells differentiated from human induced pluripotent stem cells according to claim 1, wherein the degree of fusion of the human induced pluripotent stem cells reaches 90% or more when the human induced pluripotent stem cells are induced to differentiate in step (2).
3. The method of preparing pancreatic progenitor cells differentiated from human-induced pluripotent stem cells according to claim 1, wherein the Matrigel is Matrigel.
4. The method for producing pancreatic progenitor cells differentiated from human-induced pluripotent stem cells according to claim 1, wherein in the step (1), the human-induced pluripotent stem cells are present at a ratio of 2.0×10 5 ～2.6×10 5 Individual cells/cm 2 Is inoculated into the warpOn a matrigel coated culture plate.
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| Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells;Jamie Trott等;《Stem Cell Reports》;第8卷(第6期);第1675-1688页全文，尤其是第1683页左栏第3段，图5E。 * |
| Optimization of activin-A: a breakthrough in differentiation of human induced pluripotent stem cell into definitive endoderm;Sadegh Ghorbani-Dalini等;《Biotech》;第10卷(第5期);文献号215第1-7页全文，尤其是摘要，第3页右栏第1段，第5页左栏第1、2段，图1、3，第6页左栏第4段、右栏第1、2、4段。 * |
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