CN112980774B

CN112980774B - Culture method for preparing islet beta cells by inducing pluripotent stem cells to differentiate directionally

Info

Publication number: CN112980774B
Application number: CN202110295903.7A
Authority: CN
Inventors: 高歌; 周安宇
Original assignee: Ixcell Biotechnology Co ltd
Current assignee: Ixcell Biotechnology Co ltd
Priority date: 2021-03-19
Filing date: 2021-03-19
Publication date: 2022-04-01
Anticipated expiration: 2041-03-19
Also published as: CN112980774A

Abstract

The invention relates to a culture method for preparing islet beta cells by inducing pluripotent stem cells to differentiate directionally, which comprises the following specific steps: s100, preparing an induced multifunctional cell ball; s200, performing primary differentiation, and performing directional differentiation culture on the induced multifunctional cell balls by using a culture solution A to obtain definitive endoderm cells; s300, performing secondary differentiation, namely inducing and differentiating the definitive endoderm cells by using the culture solution B to obtain pancreatic precursor cells; s400, performing three-time differentiation, and performing induced differentiation on pancreatic precursor cells by using a culture solution C to obtain pancreatic endocrine progenitor cells; and S500, differentiating for four times, and inducing and differentiating the pancreatic endocrine progenitor cells by using the culture solution D to obtain the required pancreatic islet beta cells. The proportion of the culture solution in different stages of cell culture is different, and all components play roles in stages and directionally, and by the method, the induced multifunctional stem cells can be directionally differentiated and cultured in a short time to obtain the islet beta cells.

Description

Culture method for preparing islet beta cells by inducing pluripotent stem cells to differentiate directionally

Technical Field

The invention belongs to the technical field of cell engineering, and particularly relates to a culture method for preparing islet beta cells by inducing pluripotent stem cells to differentiate directionally.

Background

Diabetes Mellitus (DM) is a metabolic disorder characterized clinically by elevated blood sugar, and its main pathogenesis is the reduction of insulin secretion and utilization disorder. At present, diabetes mellitus becomes the third disease which is a serious threat to human health and influences the quality of life of people after tumors and cardiovascular and cerebrovascular diseases. Recent reports from the World Health Organization (WHO) in 2016 show that 4.22 million people worldwide have diabetes, while the number of diabetes patients in china exceeds 1 million people, which is the first of the world.

There are two types of diabetes: type I diabetes (T1D), which accounts for 5-10% of the number of diseases, is an autoimmune disease caused by the selective destruction of islet beta cells; type II diabetes (T2D), accounting for over 90% of the number of diseases, is a disease that results from insulin resistance in peripheral organs, including the liver, fat and muscle. Diabetes patients show loss of insulin-producing cells in vivo, i.e., islet beta cells, or decreased insulin utilization, and the current treatment means is exogenous insulin injection to control blood sugar balance in vivo. Although this method can effectively control the disease progression, the long-term insulin injection can not stably maintain the physiological balance of blood sugar in the body, which results in some high-risk diseases and complications. Vascular disorders caused by complications such as hypoglycemia and hyperglycemia can cause cardiovascular, renal or neurological diseases. Thus, there is a need for a treatment strategy for diabetes that can reduce or even eliminate long-term complications.

One possible approach is to implant human islets into a patient for the treatment of diabetes. The method can well control blood sugar balance in human body, avoid dependence and pain of long-term insulin injection, and further improve the whole life quality. However, the lack of donor islets, donor-recipient islet immune rejection, and variability in islet preparation limit, making islet transplantation regimens for treating diabetes not universally applicable. Therefore, research is currently focused on alternatives that can obtain large numbers of insulin-producing secretory cells.

One such method is the direct differentiation of embryonic stem cells (hESCs) into insulin-secreting cells. However, due to ethical limitations, the currently available embryonic stem cell lines are limited and are not suitable for future clinical cell therapy. Subsequent studies focused primarily on the differentiation of pancreatic endocrine cells using other types of pluripotent stem cells, which appeared to be achieved using a combination of signaling molecules and their associated inhibitors/agonists, typically following 6-7 successive differentiation stages, respectively: definitive Endoderm (DE), Primitive embryonic Gut Tube (Primitive Gut Tube), Posterior Foregut (Posterior forego), Pancreatic Endoderm (PE), endocrine precursor cells (EP), and β -like early cells, and further differentiated into mature pancreatic islet β -like cells. However, these differentiation methods still produce immature beta cells, which express only a limited and unstable variety of hormones and have limited insulin content, and thus cannot be used for transplantation therapy of diabetic patients.

In conclusion, since insulin injection causes various complications, islet transplantation sources are limited, and the existing islet beta cell differentiation scheme is immature, has a small amount or does not have the function of GSIS, so that the treatment of diabetes is greatly limited, and a new islet beta cell differentiation method and a new diabetes treatment strategy are urgently needed to be solved.

Disclosure of Invention

1. Technical problem to be solved by the invention

The purpose of the present invention is to solve the problems of a small number of islet beta cells and an incomplete function of islet beta cells produced by the differentiation of islet beta cells in the prior art.

2. Technical scheme

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the invention relates to a culture method for preparing islet beta cells by inducing pluripotent stem cells to differentiate directionally, which comprises the following steps:

s100, preparing an induced multifunctional cell ball;

s200, performing primary differentiation, and performing directional differentiation culture on the induced multifunctional cell balls by using a culture solution A to obtain definitive endoderm cells;

s300, performing secondary differentiation, namely inducing and differentiating the definitive endoderm cells by using the culture solution B to obtain pancreatic precursor cells;

s400, performing three-time differentiation, and performing induced differentiation on pancreatic precursor cells by using a culture solution C to obtain pancreatic endocrine progenitor cells;

and S500, differentiating for four times, and inducing and differentiating the pancreatic endocrine progenitor cells by using the culture solution D to obtain the required pancreatic islet beta cells.

Preferably, the step S100 of preparing the induced pluripotent stem cell pellet specifically comprises digesting the adherently cultured induced pluripotent stem cells into small cell blocks, then resuspending the cells in a complete mTeSR-1 medium containing ROCK1 inhibitor, and then resuspending the cells in a complete medium of 0.2 × 10⁶/cm²The cell density of the cell is inoculated in an ultra-low adsorption six-hole plate, the cell is cultured for 24 hours under the conditions of 37 ℃ and 5 percent oxygen until the cell becomes a regular pellet, and then 3D suspension culture is carried out for 2-3 days, so as to obtain the induced multifunctional cell pellet.

Preferably, the step S200 is to culture the induced multifunctional cell balls by using the culture solution A at 37 ℃ and 5% CO₂And (3) continuing to culture for 3 days under the condition to obtain definitive endoderm cells, and replacing the culture solution A once every 1 day during the culture period, wherein the main component of the definitive endoderm cell induction culture medium is the culture solution A.

Preferably, in step S300, definitive endoderm cells are cultured in culture medium B at 37 ℃ with 5% CO₂Continuing the cultivation under the conditionsPancreatic progenitor cells were obtained on 5 days, and culture solution B was replaced every 1 day during the culture, and the main component of the pancreatic progenitor cell induction medium was culture solution B.

Preferably, the step S400 is to culture the pancreatic precursor cells in the culture medium C at 37 ℃ and 5% CO₂And (3) continuously culturing for 7 days under the condition to obtain pancreatic endocrine progenitor cells, and replacing the culture solution C every 1 day during the culture period, wherein the main component of the pancreatic endocrine progenitor cell induction culture medium is the culture solution C.

Preferably, the step S500 is to culture the pancreatic endocrine progenitor cells in the culture medium D at 37 ℃ and 5% CO₂Under the condition, 7 days of culture are carried out to obtain the islet beta cells, the culture solution D is replaced every 2-4 days during the continuous culture period, and the main component of the islet beta cell induction culture medium is the culture solution D.

Preferably, the culture solution A comprises Advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, vitamin C, TGF-beta factor, WNT signal pathway activator and JNK signal inhibitor, wherein the concentration of TGF-beta factor is 30-100 ng/mL; the concentration of WNT signal pathway activator is 1-5 μ M; the concentration of JNK signal inhibitor is 0.5-5 μ M.

Preferably, the culture solution B comprises Advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, vitamin C, EGF signal factor, retinoic acid, keratin growth factor, protein kinase C activator and Sirt1 inhibitor, wherein the concentration of EGF signal factor is 2-20 ng/mL; retinoic Acid (Retinoic Acid) concentration is 1-5 μ M; the concentration of the keratin growth factor is 20-40 ng/mL; the concentration of the protein kinase C activator is 100-300 nM; the concentration of Sirt1 inhibitor was 10-40. mu.M.

Preferably, the culture solution C comprises Advanced DMEM/F12 medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, vitamin C, TGF- β inhibitor, hedgehog signaling pathway inhibitor, Notch signaling pathway inhibitor, FGF signaling factor, and thyroid hormone; wherein the concentration of the TGF-beta inhibitor is 5-20 mu M; the concentration of the hedgehog signal pathway inhibitor is 10-50 ng/mL; the concentration of Notch signaling pathway inhibitor is 0.2-1 μ M; the concentration of FGF signal factor is 5-50 ng/ml; the concentration of thyroid hormone is 0.1-1 μ M.

Preferably, the culture solution D comprises Advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, vitamin C, TGF-beta inhibitor, C-Met inhibitor and trace elements, wherein the concentration of the TGF-beta inhibitor is 5-20 mu M; the concentration of the C-Met inhibitor is 20-100 nM.

3. Advantageous effects

Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects:

Drawings

FIG. 1 is a photomicrograph of Induced Pluripotent Stem Cells (iPSCs) in suspension culture; wherein, FIG. 1-A is the picture of normal iPSCs clone (adherent culture), and FIGS. 1-B, C and D are the cell ball morphology after 2h, 24h and 72h of suspension culture (10 Xmirror picture), respectively.

FIG. 2 is a photomicrograph of islet beta cells cultured according to the present invention; wherein FIGS. 2-A, B, C and D are the cell pellet morphology of the four stages (DE stage, PP stage, EN stage and islet beta cell stage) respectively, cultured using the method of example 3; and FIGS. 2-A 'to D' are the cell pellet morphology (4 Xmirror images) at four stages of culture using the method of example 4 (addition of labeled molecules).

FIG. 3 is the identification of characteristic protein expression of DE cells, including immunofluorescence microscopy and flow cytometry, as key stages in the process of the present invention for the directed differentiation of islet beta cells; wherein FIGS. 3-A' are the immunofluorescence pictures of the cell balls at the DE stage, including the combined pictures of DE Marker-FOXA2 and SOX 17; FIGS. 3-B' are photographs of the DE stage single cell immunofluorescence; FIGS. 3-C' are the results of the DE-stage cytometric flow cytometry, including two markers FOXA2 and SOX17 (both immunofluorescence pictures were taken at 20 Xmirror).

FIG. 4 shows the results of immunofluorescence assay of characteristic protein expression of islet precursor cells (PP) at key stages during the process of committed differentiation of islet beta cells according to the present invention; FIGS. 4-A "and FIGS. 4-B" are immunofluorescence staining pictures of the cell balls and single cells at the PP stage, respectively, including PDX1, NKX6.1 and the resultant pictures (all the immunofluorescence pictures are taken with 20 Xlens).

FIG. 5 shows the identification of characteristic protein expression and insulin release function test of the committed differentiated islet beta cells of the present invention; wherein, FIG. 5-A is an immunofluorescence identification picture (20X microscope shoot) of islet beta cell Marker (NKX6.1, C-peptide, insulin and MAFA) and islet alpha cell Marker (glucagon); FIG. 5-B shows the results of Elisa assay of insulin content (or secretion function) of islet beta cells (two experiments Exp-1 and Exp-2); FIG. 5-C shows the results of Elisa assays of insulin secretion function (GSIS) of pancreatic islet beta cells stimulated by different concentrations of glucose.

FIG. 6 is a flowchart of the culturing method of the present invention.

Detailed Description

In order to facilitate an understanding of the invention, the invention will now be described more fully hereinafter with reference to the accompanying drawings, in which several embodiments of the invention are shown, but which may be embodied in many different forms and are not limited to the embodiments described herein, but rather are provided for the purpose of providing a more thorough disclosure of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention; as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1

The culture method for preparing the islet beta cells by inducing the directional differentiation of the pluripotent stem cells comprises the following specific steps:

s100, preparing an induced multifunctional cell ball;

The step S100 of preparing the induced multifunctional cell pellet is to digest the induced multifunctional stem cells cultured by adherence into small cell blocks, then use mTeSR-1 complete culture medium containing ROCK1 inhibitor for heavy suspension, and use the mTeSR-1 complete culture medium for 0.2 multiplied by 10⁶/cm²Inoculating to ultra-low adsorption six-well plate, culturing at 37 deg.C under 5% oxygen for 24 hr until the cells become regular pellets, transferring to 37 deg.C under 5% CO₂And under the condition, performing 3D suspension culture in a spinner flash at 70-120 rpm, preferably 100rpm for 2-3 days to obtain the induced multifunctional cell ball.

Step S200 is to culture the induced multifunctional cell balls by using culture solution A at 37 ℃ and 5% CO₂Culturing for 3 days to obtain definitive endoderm cells, and replacing culture solution A every 1 day during cultureThe main component of the cell induction culture medium is culture solution A.

Step S300 is to culture the definitive endoderm cells with the culture solution B at 37 ℃ and 5% CO₂And continuously culturing for 5 days under the condition to obtain pancreatic precursor cells, and replacing the culture solution B every 1 day during the culture, wherein the main component of the pancreatic precursor cell induction culture medium is the culture solution B.

Step S400 is to culture pancreatic progenitor cells in culture medium C at 37 ℃ with 5% CO₂And (3) continuously culturing for 7 days under the condition to obtain pancreatic endocrine progenitor cells, and replacing the culture solution C every 1 day during the culture period, wherein the main component of the pancreatic endocrine progenitor cell induction culture medium is the culture solution C.

Step S500 is specifically to culture pancreatic endocrine progenitor cells using culture medium D at 37 ℃ with 5% CO₂Under the condition, 7 days of culture are carried out to obtain the islet beta cells, the culture solution D is replaced every 2-4 days during the continuous culture period, and the main component of the islet beta cell induction culture medium is the culture solution D.

Wherein, the advanced DMEM/F12 culture medium is used as a basic culture medium, and the nutrient components comprise glucose (0.4-1g/L), sodium bicarbonate (1.23g/L), HSA (1-5 g/L), N2 (1X), B27 (1X), glutamine (1X) and vitamin C (0.1-0.5 mM). Advanced DMEM/F12 medium, glucose, Human Serum Albumin (HSA) N2, B27 and glutamine are essential components in each differentiation stage, and rich nutrition is provided for long-term survival and proliferation of cells.

The culture solution A is used for inducing the differentiation of the multifunctional cell balls into the stage of definitive endoderm cells, the culture solution B is used for the stage of differentiation of the definitive endoderm cells into pancreatic precursor cells, the culture solution C is used for the stage of differentiation of the pancreatic precursor cells into pancreatic endocrine progenitor cells, and the culture solution D is used for the stage of differentiation of the pancreatic endocrine progenitor cells into islet beta cells.

The culture solution A comprises advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, vitamin C, TGF-beta factor, WNT signal pathway activator and JNK signal inhibitor, wherein the concentration of TGF-beta factor is 30-100ng/mL, preferably 50 ng/mL; the concentration of WNT signaling pathway activator is 1-5. mu.M, preferably 3. mu.M; the concentration of the JNK signal inhibitor is 0.5-5. mu.M, preferably 1. mu.M.

The culture solution B comprises advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, vitamin C, EGF signal factor, retinoic acid, keratin growth factor, protein kinase C activator and Sirt1 inhibitor, wherein the concentration of the EGF signal factor is 2-20ng/mL, preferably 10 ng/mL; retinoic acid (retinoic acid) concentration of 1-5 μ M; the concentration of the keratin growth factor is 20-40ng/mL, preferably 30 ng/mL; the concentration of the protein kinase C activator is 100-300nM, preferably 200 nM; the concentration of the inhibitor of Sirt1 is 10-40. mu.M, preferably 20. mu.M.

The culture solution B also comprises an ALK4/5/7 inhibitor, and the concentration of the ALK4/5/7 inhibitor is 10-50 nM, preferably 20 nM. The ALK4/5/7 inhibitor is A83-01, can selectively inhibit TGF-beta activin receptors ALK4, I-type receptors ALK5 and node receptors ALK7, can also inhibit the transformation of epithelial cells into mesenchymal cells, and has the functions of promoting the specific differentiation of pancreatic lineage cells and the expansion of pancreatic precursor cells.

The culture solution C comprises advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, a vitamin C, TGF-beta inhibitor, a hedgehog signaling pathway inhibitor, a Notch signaling pathway inhibitor, FGF signaling factors and thyroid hormone; wherein the concentration of the TGF-beta inhibitor is 5-20 mu M, preferably 10 mu M; the concentration of the hedgehog signal pathway inhibitor is 10-50ng/mL, preferably 30 ng/mL; the concentration of Notch signaling pathway inhibitor is 0.2-1. mu.M, preferably 0.5. mu.M; the concentration of FGF signal factor is 5-50ng/ml, preferably 10 ng/ml; the concentration of thyroid hormone is 0.1-1. mu.M, preferably 0.5. mu.M.

The culture solution C also comprises a CDK5 inhibitor, and the concentration of the CDK5 inhibitor is 5-30 nM, preferably 15 nM. The CDK5 inhibitor is AT7519, which has dual efficacy: firstly, activators P35 and P39 of CDK5 are expressed in pancreatic cells and play a role in regulating and controlling the maturation of pancreatic islet beta cells, secondly, AT7519 also has a GSK3 beta inhibitory function and can activate a WNT signal pathway to promote cell differentiation, and in addition, the action of CDK5 inhibitors can be enhanced under the premise of the existence of Notch signal inhibitors.

The culture solution D comprises advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, a vitamin C, TGF-beta inhibitor, a C-Met inhibitor and trace elements, wherein the concentration of the TGF-beta inhibitor is 5-20 mu M, preferably 10 nM; the concentration of the C-Met inhibitor is 20-100nM, preferably 50 nM.

Broth D also included an L-type calcium channel (LTCC) activator and a Hippo signaling pathway effector inhibitor; wherein the concentration of the L-type calcium channel (LTCC) activator is 10-30 nM, preferably 20 nM; the concentration of the Hippo signal pathway effector inhibitor is 0.2-0.6. mu.M, preferably 0.35. mu.M. The L-type calcium ion channel (LTCC) activator is BayK8644, can activate Ras signals, enhances the cell cycle and has the function of promoting the proliferation of islet beta cells; the Hippo signal pathway effector YAP inhibitor is Super-TDU1-31, can destroy the interaction between YAP and TEADS transcription factors, and has important effects in promoting the differentiation of endocrine cells and inhibiting the proliferation of precursor cells.

The TGF-beta superfamily factor is Activin A, the WNT signal pathway activator is BML-284 or WNT3a, and the TGF-beta superfamily factor is a key factor for endoderm lineage cell specific differentiation; the JNK signaling pathway inhibitor CC-930 can enhance the effects of Activin A and WNT3a, and can remarkably promote the efficient differentiation of pluripotent stem cells to epiblast; retinoic Acid (RA) can promote migration to PDX1⁺Differentiation of pancreatic precursor cells; the Sirt1 inhibitor is Nicotinamide, and can promote the action on NKX6.1⁺Differentiation of pancreatic precursor cells; the combined use of epidermal growth factor and keratin growth factor can promote PDX1⁺And NKX6.1⁺Differentiation of double positive pancreatic precursor cells; the protein kinase C activator is TPB, can promote the specific differentiation to pancreatic lineage cells, has synergistic effect with RA, and can remarkably enhance PDX1⁺Differentiation of the cells; the hedgehog pathway antagonist is N-acetylcysteine or cyclopamine, and can promote the differentiation of the endocrine precursor cells; FGF10 promotes survival and proliferation of pancreatic endocrine progenitor cells, enhancing differentiation at this stageEfficiency and stability, and has a great synergistic effect on the final yield of pancreatic beta; also, the combination of hedgehog pathway antagonist and FGF10 facilitates delivery to PDX1⁺Differentiation of endocrine precursor cells; the TGF-beta inhibitor is ALK5i II, can promote differentiation and maturation of islet beta cells, and can increase release of insulin in the islet beta cells; the combined use of TGF-beta inhibitors and thyroid hormones may promote NGN3⁺Amplifying the endocrine precursor cells; the Notch signaling pathway inhibitor is FLI-06 or DAPT, and can increase NGN3⁺The number of endocrine precursor cells of (a); the C-Met inhibitor is BMS-777607, and can promote the maturation of pancreatic islet beta cells.

The culture solution of this example is prepared by using Advanced DMEM/F12 medium, and adding some key components necessary for maintaining cell growth, such as glucose, sodium bicarbonate, Human Serum Albumin (HSA), glutamine, vitamin C, and the like, agonists or antagonists including signal pathways of JNK, hedgehog, EGF, FGF, TGF- β, WNT, and the like, and some necessary small molecule compounds, trace elements, and the like, and each component acts in a stepwise and directional manner, and can efficiently induce the directed differentiation of pluripotent stem cells into islet β cells. By the method, the induced multifunctional stem cells can be directionally differentiated and cultured in a short time to obtain the islet beta cells. Firstly, the quantity of raw materials is not limited, the induced pluripotent stem cells can be expanded infinitely, and moral disputes in the aspect of using the embryonic stem cells can be avoided; secondly, the period for culturing the islet beta cells is about three weeks, compared with the existing differentiation technology, the preparation speed is greatly improved, and the production cost is reduced; thirdly, 3D suspension culture is adopted for culturing the islet beta cells, compared with the existing adherent (2D) culture technology, the yield of differentiated cells is greatly increased, and the loss caused by reagent replacement in the culture process is greatly increased; fourthly, a specific signal pathway inhibitor and a small molecular compound are adopted in the process of culturing the islet beta cells, so that the purity and the yield of the islet beta cells can be greatly improved; fifthly, the cultured islet beta cells selectively adopt a cell sorting or biomaterial wrapping technology, so that poor treatment effect caused by low cell purity or potential safety hazard caused by immunological rejection after cell transplantation can be avoided.

Example 2

The content of this example is mainly the preparation of the islet beta cell differentiation culture solution, which is specifically as follows:

the islet beta cell differentiation culture solution is divided into four stages, namely a stage I (DE induction culture medium consisting of culture solution A), a stage II (PP induction culture medium consisting of culture solution B), a stage III (EN induction culture medium consisting of culture solution C) and a stage IV (islet beta cell induction culture medium consisting of culture solution D), wherein the culture solution components consist of basic culture solution components (basal medium) and additive components (supplement), and the components and the ratio are as shown in the following tables 1-4:

TABLE 1 DE Induction Medium composition and proportions

TABLE 2 composition and proportions of PP Induction Medium

Remarking: the addition of A83-01 can obviously improve the cell differentiation efficiency.

TABLE 3 EN Induction Medium composition and proportions

Remarking: the addition of AT7519 can significantly improve the cell differentiation efficiency.

TABLE 4 composition and proportion of islet beta cell induction medium

Remarking: the addition of BayK8644 and Super-TDU1-31 can significantly improve the differentiation efficiency of the cells.

Example 3

The main content of this example is to culture and induce pluripotent stem cells by using a 3D suspension culture method, as follows:

first, required reagent

The culture solution used for inducing the culture of the multifunctional stem cells (iPSCs) is mTeSR-1 complete culture medium, the digestive enzyme used for amplification and passage is EDTA, the reagent used for cell rinsing and balancing is DPBS, and the inhibitor used for promoting the cell survival is Y27632.

Second, culture process

Discarding a culture medium of iPSCs subjected to adherent culture in 100mm-dish, moistening the iPSCs by using 2mL of DPBS, adding 4mL of EDTA digestive enzyme, standing the mixture at 37 ℃ for 3-4 min, discarding the digestive enzyme after intercellular contact separation, adding 12mL of mTeSR-1 complete culture medium containing 10 mu M Y7632 of final concentration, blowing and separating the cells into small cell blocks, wherein each small cell block is aggregated by 10-30 cells.

The separated cell small blocks are averagely transferred to an ultra-low adsorption 6-well plate, cultured for 24h under the condition of 37 ℃ and 5% oxygen until the cells become regular pellets, and then transferred to 75 or 125mL spinner flash for 3D suspension culture at the rotating speed of 70-120 rpm, preferably 100 rpm.

Third, cell ball morphology observation

100mm-dish was placed in a sterile operating table, the suspension cell pellet cultured for 1 or 3 days was transferred to the dish using a 10mL pipette, the dish was gently shaken to uniformly distribute the cell pellet, and observed and photographed under an EVOS microscope.

The results are shown in FIG. 1: the method can obtain large-quantity cell balls with uniform shapes.

Example 4

The main content of this example is directed differentiation culture of islet β cells using induced pluripotent stem cells, as follows:

first, culture of undifferentiated cells

Induction of pluripotent Stem cells 3D suspension cells were obtained as described in example 2, cultured in 125mL of spinner flash, passaged as single cells every 3-4 days using Accutase digestive enzyme, and resuspended in mTeSR-1 complete medium containing 10. mu. M Y7632 inhibitor at 37 ℃ in 5% CO₂The incubator is subjected to suspension culture at the rotating speed of 70-100 rpm.

When preparing islet beta cell differentiation, cells digested by Accutase are treated in a 2-8 x 10 manner⁵cell/mL, preferably 6X 10⁵cell/mL density was plated in spinner flash described above and replaced with differentiation medium after 3 days of culture.

Directed differentiation culture of islet beta cells

Naturally settling the undifferentiated iPSCs cell balls cultured for 3 days for 3-5 min, lightly discarding the upper mTeSR-1 culture medium and dead cells by using a 10mL pipette, sequentially adding the differentiation stage induction culture medium (without marked additives) in example 1 (30mL/Flask, preferably in situ preparation, no more than 7 days if stored at 4 ℃, and taking out 30 minutes before use and balancing to room temperature), standing at 37 ℃, and keeping at 5% CO₂The rotating speed is adjusted to 70rpm above the magnetic stirrer in the incubator, and the corresponding stage of culture time is set. Wherein the culture solution is replaced every 2 days after 3 days of stage one culture, 5 days of stage two culture, 7 days of stage three culture and more than 7 days of stage four culture.

Three, pancreatic islet beta cell ball morphology observation

100mm-dish was placed on a sterile operating table, the islet β cell pellet cultured for about three weeks was transferred to dish using a 10mL pipette, and the dish was gently shaken to uniformly distribute the cell pellets, which were then observed under an EVOS microscope.

FIGS. 2A to D show islet beta-cell pellets (including the morphology of the cell pellets at each stage) cultured by the differentiation method of example 4, and it is understood that islet beta-cell pellets having a good morphology can be obtained by this method.

Example 5

The main content of this example is a method for differentiating islet β cells after adding a small molecule compound (to improve differentiation efficiency), which includes the following steps:

using the islet beta cell differentiation method of example 4, small molecule compounds A83-01, AT7519, BayK8644 and Super-TDU1-31 (marked by the symbol x) were selectively added to the induction medium AT the PP, EN and islet beta cell stages of differentiation, and the morphology and number of the resulting islet beta cells were compared after three weeks of culture.

As shown in FIGS. 2-A 'to D', a larger number of islet beta cells could be obtained in the induction medium after addition of the small molecule compound, and the morphology of the islet beta cell spheres was more uniform.

Example 6

The main content of this example is the identification of the expression of a protein characteristic of DE stage cells by immunofluorescence and flow cytometry, as follows:

the method of example 5 was used to perform the directed differentiation of islet beta cells, during which induction of stage one (DE cells) was the initiation and key to differentiation, and the identification of DE cells was performed using immunofluorescence and flow cytometry as follows.

a. An immunofluorescence method:

placing the cells cultured to the first stage in an ultra-low adsorption 24-pore plate, and standing for 1min to allow the cell balls to sink to the bottom of the pore plate; after carefully aspirating the upper layer of the culture medium, 1 XPBS (pH7.4, the same below, 1 mL/well) was slowly added along the wall of the 24-well plate and washed 2 times; 4% PFA (paraformaldehyde, 0.5 mL/well) pre-warmed at 37 ℃ was then added slowly along the plate walls, the cells were fixed for 18 minutes at room temperature, the PFA was gently aspirated, and the plates were washed 3 times (1 mL/well/time) with 1 XPBS.

0.3% Triton X100 (0.5 mL/well) was added and incubated at 37 ℃ for 30 minutes; then blocked by adding 5% BSA (0.5 mL/well) and incubated at 37 ℃ for 30 min; primary antibody (SOX17 and FOXA2) was then added directly to the blocking solution in an amount of 10. mu.L/well, incubated at 37 ℃ for 1 hour, and washed 3 times with 1 XPBS (1 mL/well/time).

Adding a secondary antibody diluted by 1% BSA (dilution ratio 1:1000), wherein the addition amount of the secondary antibody is 0.5 mL/hole, incubating at 37 ℃ for 60 minutes in the dark, absorbing the secondary antibody, adding 1 XPBS, and washing for 3 times (1 mL/hole/time) for 5 minutes each time; then, 1. mu.g/mL DAPI (0.5 mL/well) prepared with 1 XPBS was added to the cells, stained for 5 minutes, the DAPI was aspirated, and the cells were washed 2 times with 1 XPBS (1 mL/well/time); finally, 1 × PBS (0.5 mL/well) was added to resuspend the cell pellet, and the pellet was observed under a microscope and photographed for processing.

Remarking: in addition to immunofluorescent staining of the cell spheres as per the protocol described above, the spheres were also plated for immunofluorescence in 24-well plates using Accutase digestion into single cells (better to see the expression of each marker in the single cells).

b. Flow cytometry:

placing the cell balls finished in the first stage of culture in a 15mL centrifuge tube, naturally settling for 1-3 min, then discarding the culture solution, and rinsing once by using 1mL DPBS and discarding; adding 1-2 mL of Accutase or TrypLE digestive juice into the cell balls, placing the cell balls at 37 ℃ for 3-5 minutes, and separating the cell balls into single cells; then adding 2-3 times of DPBS to dilute the digestive juice, centrifuging at a rotating speed of 200-300 g for 3 minutes, and discarding the digestive juice and the DPBS.

1mL of 4% Paraformaldehyde (PFA) was added to the cells, fixed at 4 ℃ for 30 minutes and discarded; then adding 0.3% Triton X100 (1mL) or other stabilizing buffer, sealing and permeating for 30 minutes at 4 ℃, and then discarding; primary antibody (SOX17 and FOXA2) in 1mL blocking solution was then added overnight at 4 ℃.

The primary antibody is discarded the next day, 1mL of secondary antibody prepared by confining liquid is added, and the mixture is incubated for 2 hours at 4 ℃ in a dark place; finally, flow cytometry analysis is carried out.

FIGS. 3-A-B 'and FIGS. 3-C' are immunofluorescence and flow cytometric assay results, respectively, of the DE cells cultured in example 5, and it is known that the cultured DE cells all have high marker molecule expression.

Example 7

The main content of this example is the identification of the expression of a protein characteristic to cells in the PP stage by immunofluorescence, as follows:

cells (PP cells) cultured in stage two were removed and the expression of the key proteins (PDX1 and NKX6.1) in the PP stage cells was examined according to the immunofluorescence procedure of example 6.

FIG. 4 shows the results of immunofluorescence assay of Marker markers (PDX1 and NKX6.1) of PP cells cultured in example 5, which indicates that the PP cells cultured have high Marker molecule expression.

Example 8

The main content of this example is the identification of the expression of a protein characteristic to pancreatic islet β cells using immunofluorescence, as follows:

the cells (islet β cells) cultured to stage four were removed, and the expression of islet β cell key proteins (NKX6.1, C-peptide, insulin) was detected according to the immunofluorescence procedure of example 6.

FIG. 5-A shows the immunofluorescence assay results of islet β cells cultured in example 5, indicating that islet β cells cultured have high expression of marker molecules (NKX6.1, C-peptide, insulin, and MAFA).

Example 9

The main content of this example is to use an Elisa kit to detect the insulin secretion function of pancreatic islet β cells, which includes the following details:

collecting the islet beta cell spheres (20-30) cultured for 7 days in the fourth stage in a 15mL centrifuge tube by using a 5mL pipette, naturally settling the cell spheres for 1-3 minutes, and then removing the upper-layer culture solution. Then, 1mL of 70% ethanol containing 1.5% hydrochloric acid (HCl) was added thereto, and the mixture was left at-20 ℃ for 24 hours. After 24 hours the cell pellet was gently shaken and allowed to stand for another 24 hours.

After 48 hours of standing, the tube was placed in a centrifuge and centrifuged at 2100rcf for 15 minutes, and 1mL of the supernatant was collected in a new 15mL centrifuge tube and neutralized with 1mL of 1M TRIS (pH 7.5). The neutralized liquid was tested for insulin release using the human insulin Elisa kit.

FIG. 5-B shows the results of measurement of insulin release from islet β cells cultured in example 5, which indicates that islet β cells cultured in example 5 have a high insulin release amount.

Example 10

The main content of this example is to use Elisa kit to detect the GSIS function of pancreatic islet β cells, which is as follows:

preparation of reagents: krb buffer was made up of 128mM NaCl, 5mM KCl, 2.7mM CaCl2, 1.2mM MgCl2, 1mM Na2HPO4, 1.2mM KH2PO4, 5mM NaHCO3, 10mM HEPES, and 0.1% BSA with deionized water; low concentration glucose (2mM) and high concentration glucose (20mM) were formulated from Krb buffer; the 30mM KCl is prepared from a 1M KCl aqueous solution and a 20mM high-concentration glucose solution; all reagents were filter sterilized through 0.22 μ M filter after formulation.

GSIS functional verification: firstly, islet beta cell spheres (20) cultured for 14 days in stage four are collected in a 15mL centrifuge tube by using a 5mL pipette, and the cell spheres naturally settle for 1-3 minutes and then the supernatant culture solution is discarded. Then, 1mL of Krb buffer was added for rinsing and pre-incubation with 200. mu.L of low concentration (2mM) glucose Krb solution for 1 hour was used to remove residual insulin. After rinsing again with 1mL of Krb buffer 2 times, the cell pellet was incubated with 200. mu.L of low concentration (2mM), high concentration (2mM) glucose Krb solution and KCl solution for 1 hour (each time the solution was changed to a different concentration, the cell pellet was rinsed with Krb equilibration solution, and the supernatant was collected at the end of the incubation). Finally, 200. mu.L of glucose and KCl stimulated supernatant were collected and insulin Elisa kit was used to detect insulin release (GSIS) levels of islet beta cells stimulated with different concentrations of glucose.

FIG. 5-C shows the GSIS measurements of islet beta cells cultured in example 5, indicating that islet beta cells cultured in example 5 have different levels of insulin release in response to different concentrations of glucose stimulation, and that the amount of insulin released from islet beta cells stimulated to increase with increasing glucose levels is highest after KCl stimulation.

From the identification results of examples 4 to 10, it can be seen that the cells obtained by the culture solution and the culture method for inducing pluripotent stem cells to differentiate directionally not only have the expression of proteins characteristic to islet beta cells, such as PDX1, NKX6.1, C-Peptide and Insulin, but also have high yield and purity, and have the glucose-stimulated-Insulin release (GSIS) function, indicating that the cells are mature and functional islet beta cells.

Compared with other differentiation methods, the culture solution enables the induced multifunctional stem cells cultured by 3D suspension to directionally differentiate into the islet beta cells, and has the following advantages: firstly, the quantity of raw materials is not limited, and induced pluripotent stem cells can be expanded infinitely; secondly, the raw material source is not limited, the induced multifunctional stem cells can be obtained by reprogramming adult cells of healthy people such as peripheral blood mononuclear cells, and the use of the embryonic stem cells has ethical limitation; thirdly, the differentiation efficiency is high, and mature and functional islet beta cells can be obtained by using suspension culture and specific signal pathway protein factors and small molecular compounds; fourthly, the obtained yield is high, and mature islet beta cells with large quantity and higher purity can be obtained by adding a specific small molecular compound; fifthly, the time consumption is short, the culture period is 22-30 days, higher cell yield can be obtained by prolonging the amplification time, and the cost loss is reduced; and sixthly, the method is safe and reliable, and the purity of the obtained cells can be improved or the immune rejection of the cells to a host can be reduced or even eliminated by using methods such as cell sorting or biological material wrapping.

In addition, the use of small molecule compounds such as ALK4/5/7 inhibitors (A83-01), CDK5 inhibitors (AT7519), LTCC activators (BayK8644) and Hippo signaling pathway effector inhibitors (Super-TDU1-31) promotes the directed differentiation of islet beta cells, as evidenced by the massive expansion of intermediates (pancreatic precursor cells) during differentiation, and the maturation and high expansion efficiency of the differentiation products (islet beta cells). The islet beta cells obtained by the culture solution and the differentiation method have the advantages of large quantity, high maturity and complete functions, and a new method is provided for the treatment of future diabetes.

The above-mentioned embodiments only express a certain implementation mode of the present invention, and the description thereof is specific and detailed, but not construed as limiting the scope of the present invention; it should be noted that, for those skilled in the art, without departing from the concept of the present invention, several variations and modifications can be made, which are within the protection scope of the present invention; therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A culture method for preparing islet beta cells by inducing pluripotent stem cells to differentiate directionally is characterized by comprising the following steps:

s100, preparing an induced multifunctional cell ball; specifically, after digesting the adherent culture induced multifunctional stem cells into small cell blocks, the adherent culture induced multifunctional stem cells are resuspended in mTeSR-1 complete culture medium containing ROCK1 inhibitor and added with 0.2 × 10⁶/cm²The cell density is inoculated in an ultra-low adsorption six-hole plate, the cell is cultured for 24 hours under the conditions of 37 ℃ and 5 percent oxygen until the cell becomes a regular pellet, and then 3D suspension culture is carried out for 2-3 days to obtain the induced multifunctional cell pellet;

s200, performing primary differentiation, and performing directional differentiation culture on the induced multifunctional cell balls by using a culture solution A to obtain definitive endoderm cells; specifically, induced multifunctional cell balls are cultured by using culture solution A at 37 ℃ and 5% CO₂Culturing for 3 days under the condition to obtain definitive endoderm cells, and replacing the culture solution A every 1 day during the culture period, wherein the main component of the definitive endoderm cell induction culture medium is the culture solution A; the culture solution A comprises Advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, vitamin C, TGF-beta factor, WNT signal pathway activator and JNK signal inhibitor, wherein the concentration of TGF-beta factor is 30-100 ng/mL; the concentration of WNT signal pathway activator is 1-5 μ M; the concentration of the JNK signal inhibitor is 0.5-5 mu M;

s300, performing secondary differentiation, namely inducing and differentiating the definitive endoderm cells by using the culture solution B to obtain pancreatic precursor cells; specifically, definitive endoderm cells were cultured in culture medium B at 37 deg.C with 5% CO₂Continuously culturing for 5 days under the condition to obtain pancreatic precursor cells, and replacing the culture solution B every 1 day during the culture period, wherein the main component of the pancreatic precursor cell induction culture medium is the culture solution B; the culture solution B comprises Advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, vitamin C, EGF signal factor, retinoic acid, keratin growth factor, protein kinase C activator and Sirt1 inhibitor, wherein the concentration of the EGF signal factor is 2-20 ng/mL; retinoic Acid (Retinoic Acid) concentration is 1-5 μ M; the concentration of the keratin growth factor is 20-40 ng/mL;the concentration of the protein kinase C activator is 100-300 nM; the concentration of Sirt1 inhibitor is 10-40 μ M;

s400, performing three-time differentiation, and performing induced differentiation on pancreatic precursor cells by using a culture solution C to obtain pancreatic endocrine progenitor cells; specifically, pancreatic progenitor cells were cultured in culture medium C at 37 ℃ with 5% CO₂Continuously culturing for 7 days under the condition to obtain pancreatic endocrine progenitor cells, and replacing the culture solution C every 1 day during the culture period, wherein the main component of the pancreatic endocrine progenitor cell induction culture medium is the culture solution C; the culture solution C comprises Advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, a vitamin C, TGF-beta inhibitor, a hedgehog signaling pathway inhibitor, a Notch signaling pathway inhibitor, FGF signaling factors and thyroid hormone; wherein the concentration of the TGF-beta inhibitor is 5-20 mu M; the concentration of the hedgehog signal pathway inhibitor is 10-50 ng/mL; the concentration of Notch signaling pathway inhibitor is 0.2-1 μ M; the concentration of FGF signal factor is 5-50 ng/ml; the concentration of thyroid hormone is 0.1-1 μ M;

s500, differentiating for four times, and inducing and differentiating the pancreatic endocrine progenitor cells by using a culture solution D to obtain the needed pancreatic islet beta cells; specifically, pancreatic endocrine progenitor cells were cultured in culture medium D at 37 ℃ with 5% CO₂Under the condition, 7 days of culture are carried out to obtain islet beta cells, a culture solution D is replaced every 2-4 days during the continuous culture period, and the main component of an islet beta cell induction culture medium is the culture solution D; the culture solution D comprises Advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, Human Serum Albumin (HSA), N2, B27, glutamine, a vitamin C, TGF-beta inhibitor, a C-Met inhibitor and trace elements, wherein the concentration of the TGF-beta inhibitor is 5-20 mu M; the concentration of the C-Met inhibitor is 20-100 nM.