CN112961823B - Culture solution for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells - Google Patents

Culture solution for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells Download PDF

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CN112961823B
CN112961823B CN202110297381.4A CN202110297381A CN112961823B CN 112961823 B CN112961823 B CN 112961823B CN 202110297381 A CN202110297381 A CN 202110297381A CN 112961823 B CN112961823 B CN 112961823B
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CN112961823A (en
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高歌
周安宇
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Ixcell Biotechnology Co ltd
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Abstract

The culture solution for preparing the islet beta cells by inducing the directional differentiation of the pluripotent stem cells comprises an advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, human Serum Albumin (HSA), N2, B27, glutamine, a vitamin C, JNK inhibitor, a hedgehog pathway antagonist, an EGF signal factor, an FGF signal factor, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecular compound and trace elements, wherein the culture solution has different proportions at different stages of cell culture, and each component plays a role in a directional manner in stages, so that the pluripotent stem cells can be efficiently induced to be directionally differentiated into the islet beta cells.

Description

Culture solution for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells
Technical Field
The invention belongs to the technical field of cell engineering, and particularly relates to a culture solution for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells.
Background
Diabetes (DiabetesMellitus, DM) is a metabolic disorder characterized clinically by elevated blood glucose, and its main pathogenesis is a decrease in insulin secretion and its utilization disorder. At present, diabetes mellitus has become the third disease threatening human health and affecting people's quality of life after tumor and cardiovascular and cerebrovascular diseases. Recent reports by the World Health Organization (WHO) in 2016 indicate that 4.22 million people worldwide have diabetes, whereas the number of people with diabetes in china exceeds 1 million people, and is the first worldwide.
There are two types of diabetes: type I diabetes (T1D), accounting for 5-10% of the number of diseases, is an autoimmune disease caused by the selective destruction of islet beta cells; type II diabetes mellitus (T2D), which accounts for more than 90% of the number of diseases, is a disease caused by insulin resistance of peripheral organs including liver, fat and muscle. Diabetes patients manifest a loss of insulin-producing cells, islet beta cells, or a decrease in insulin utilization, and current therapies are all exogenous injections of insulin to control blood glucose balance in vivo. Although this method can effectively control disease progression, prolonged insulin injections do not stably maintain blood glucose physiological balance in the body, resulting in the occurrence of high risk diseases and complications. Vascular lesions caused by complications such as hypoglycemia and hyperglycemia may cause cardiovascular, renal or neurological diseases. Thus, there is a need for a therapeutic strategy for the treatment of diabetes that can reduce or even eliminate long-term complications.
One possible approach is to implant human islets into a patient for 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 overall life quality. However, this is limited by the lack of donor islets, immune rejection of islets between donor-recipient, and variability in islet preparation, making islet transplantation approaches not universally applicable to the treatment of diabetes. Thus, current research is focused on alternatives to the availability of large numbers of insulin-producing secreting cells.
One such method is the use of embryonic stem cells (hESCs) to differentiate directly 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 therapies. The latter studies have focused mainly on the use of other types of pluripotent stem cells to differentiate into pancreatic endocrine cells, expressed by the combined use of signaling molecules and their related inhibitors/agonists, generally in accordance with 6-7 successive differentiation stages, respectively: definitive Endoderm (DE), primordial germ tube (PrimitiveGutTube), posterior foregut (postforegut), pancreatic Endoderm (PE), endocrine precursor cells (EP), and beta-like early cells, and further differentiated into mature islet beta-like cells. However, such differentiation methods produce still immature beta cells, whose expressed hormones are limited and unstable, of various types and limited insulin content, and cannot be used for transplantation therapy in diabetics.
In conclusion, due to the fact that insulin injection can cause various complications, islet transplantation sources are limited, and the original islet beta cell differentiation scheme is immature, few in number or not provided with GSIS functions and the like, diabetes treatment 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 invention aims to solve the problems of small quantity and imperfect functions of islet beta cells generated by differentiating the existing islet beta cells.
2. Technical proposal
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
the invention relates to a culture solution for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells, which comprises an advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, human Serum Albumin (HSA), N2, B27, glutamine, a vitamin C, JNK inhibitor, a hedgehog pathway antagonist, an EGF signal factor, an FGF signal factor, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecular compound and microelements, wherein the culture solution has different proportions at different stages of cell culture.
Preferably, the JNK inhibitor is CC-930, the hedgehog pathway antagonist is N-acetylcysteine or cyclopamine, the EGF signal factor is EGF, the FGF signal factor is FGF10, the TGF-beta superfamily factor is ActivinA, the TGF-beta inhibitor is ALK5iII, the WNT signal pathway activator is BML-284 or WNT3a, and the Notch signal pathway inhibitor is FLI-06 or DAPT.
Preferably, the small molecule compounds include keratin growth factors, protein kinase C activators, ROCK1 inhibitors, sirt1 inhibitors, C-Met inhibitors, thyroid hormones, and retinoic acid (Retinoic acid).
Preferably, the keratin growth factor is KGF, the protein kinase C activator is TPB, the ROCK1 inhibitor is Y27632, the Sirt1 inhibitor is Nicotinamide, the C-Met inhibitor is BMS-777607, and the thyroid hormone is T3.
Preferably, the kit further comprises an ALK4/5/7 inhibitor, a CDK5 inhibitor, an L-type calcium channel (LTCC) activator and a Hippo signal pathway effector inhibitor, wherein the ALK4/5/7 inhibitor is A83-01, the CDK5 inhibitor is AT7519, the L-type calcium channel (LTCC) activator is BayK8644, and the Hippo signal pathway effector inhibitor is Super-TDU1-31.
Preferably, the ratio of the culture solution in different stages of cell culture is different, and the culture solution can be specifically divided into a culture solution A, a culture solution B, a culture solution C and a culture solution D, wherein the culture solution A is used for inducing the differentiation of the multifunctional cell pellet into a definitive endoderm cell stage, the culture solution B is used for differentiating the definitive endoderm cell into a pancreatic precursor cell stage, the culture solution C is used for differentiating the pancreatic precursor cell into a pancreatic endocrine progenitor cell stage, and the culture solution D is used for differentiating the pancreatic endocrine progenitor cell into a pancreatic islet beta cell stage.
Preferably, the culture solution A comprises an 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 the TGF-beta factor is 30-100ng/mL; the concentration of WNT signal pathway activator is 1-5. Mu.M; JNK signal inhibitor concentration is 0.5-5. Mu.M.
Preferably, the culture solution B comprises an advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, human Serum Albumin (HSA), N2, B27, glutamine, a vitamin C, EGF signal factor, retinoic acid, a keratin growth factor, a protein kinase C activator and a Sirt1 inhibitor, wherein the concentration of the EGF signal factor is 2-20ng/mL; retinoic acid (Retinoic acid) at a concentration of 1-5 μm; the concentration of the keratin growth factor is 20-40ng/mL; the concentration of the protein kinase C activator is 100-300nM; the concentration of Sirt1 inhibitor is 10-40. Mu.M.
Preferably, the culture solution C comprises an 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 TGF-beta inhibitor is 5-20 mu M; the concentration of the hedgehog signaling pathway inhibitor is 10-50ng/mL; the concentration of Notch signaling pathway inhibitor is 0.2-1. Mu.M; FGF signal factor concentration is 5-50ng/ml; the concentration of thyroid hormone is 0.1-1. Mu.M.
Preferably, the culture solution D comprises an 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 20-100nM.
The concentration of the additive factors in the culture solutions A-D is in a concentration range which can successfully induce and differentiate the cell types in the stage, and factors lower or higher than the concentration range can not successfully obtain final cells with poor quality (such as low induction efficiency, small quantity of final products, incomplete functions and the like).
3. Advantageous effects
Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects:
the culture solution for preparing the islet beta cells by inducing the directional differentiation of the pluripotent stem cells comprises an advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, human Serum Albumin (HSA), N2, B27, glutamine, a vitamin C, JNK inhibitor, a hedgehog pathway antagonist, an EGF signal factor, an FGF signal factor, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecular compound and trace elements, wherein the culture solution has different proportions at different stages of cell culture, and each component plays a role in a staged and directional manner, so that the pluripotent stem cells can be efficiently induced to be directionally differentiated into the islet beta cells.
Drawings
FIG. 1 is a photomicrograph of Induced Pluripotent Stem Cells (iPSCs) suspension culture; wherein FIG. 1-A is a picture of normal iPSCs clone (adherent culture), and FIGS. 1-B, C and D are cell sphere morphology (10 Xmirror picture) after 2h, 24h and 72h of suspension culture, respectively.
FIG. 2 is a photomicrograph of islet beta cells cultured in accordance with the invention; wherein FIGS. 2-A, B, C and D are the morphology of the cell spheres of the four stages (DE stage, PP stage, EN stage and islet beta cell stage) cultivated using the method of example 3, respectively; while figures 2-a '-D' show the morphology of the cell spheres (4 x mirror images) at four stages of culture using the method of example 4 (addition of marker molecules).
FIG. 3 is an identification of protein expression characteristic of DE cells, including immunofluorescence microscopy and flow cytometry, at a key stage in the process of directed differentiation of islet beta cells of the present invention; FIGS. 3-A' are the immunofluorescence pictures of the cell ball in the DE stage, including DE Marker-FOXA2 and SOX17 and the close-up; FIGS. 3-B' are single cell immunofluorescence pictures of DE stage; FIGS. 3-C' show the results of the DE stage cytometry identification, including FOXA2 and SOX17 markers (immunofluorescence images were taken with 20 Xmirror).
FIG. 4 shows immunofluorescence identification of protein expression characteristic of islet precursor cells (PP) at a critical stage in the process of directed differentiation of islet beta cells according to the invention; wherein, the figures 4-A 'and 4-B' are respectively immunofluorescence staining pictures of the cell ball and single cell in the PP stage, including PDX1, NKX6.1 and the combined pictures (the immunofluorescence pictures are all taken by a 20X mirror).
FIG. 5 is an identification of protein expression and insulin release function test characteristic of the directionally differentiated islet beta cells of the invention; wherein FIG. 5-A is an immunofluorescence identification picture (20 Xmirror photograph) of islet beta cell Marker (NKX 6.1, C-peptide, insulin and MAFA) and islet alpha cell Marker (Glucagon); FIG. 5-B shows the results of an Elisa assay for insulin content (or secretion function) of islet beta cells (experiments Exp-1 and Exp-2); FIG. 5-C shows the results of an insulin secretion function (GSIS) Elisa assay after stimulation of islet beta cells with varying concentrations of glucose.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which, however, the invention may be embodied in many different forms and are not limited to the embodiments described herein, but are instead provided for the purpose of providing a more thorough and complete 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; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Example 1
The culture solution for preparing the islet beta cells by inducing the directional differentiation of the pluripotent stem cells comprises an advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, human Serum Albumin (HSA), N2, B27, glutamine, a vitamin C, JNK inhibitor, a hedgehog pathway antagonist, an EGF signal factor, an FGF signal factor, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecular compound and microelements, wherein the culture solution has different proportions at different stages of cell culture. Wherein, the advanced DMEM/F12 culture medium is taken as a basic culture medium, and the added nutrient components comprise glucose (0.4-1 g/L), sodium bicarbonate (1.23 g/L), HSA (1-5 g/L), N2 (1X), B27 (1X), glutamine (1X) and vitamin C (0.1-0.5 mM).
The JNK inhibitor is CC-930, the hedgehog pathway antagonist is N-acetylcysteine or cyclopamine, the EGF signal factor is EGF, the FGF signal factor is FGF10, the TGF-beta superfamily factor is ActivinA, the TGF-beta inhibitor is ALK5iII, the WNT signal pathway activator is BML-284 or WNT3a, and the Notch signal pathway inhibitor is FLI-06 or DAPT.
The small molecule compounds include keratin growth factors, protein kinase C activators, ROCK1 inhibitors, sirt1 inhibitors, C-Met inhibitors, thyroid hormones, and retinoic acid (RetinoicAcid).
The keratin growth factor is KGF, the protein kinase C activator is TPB, the ROCK1 inhibitor is Y27632, the Sirt1 inhibitor is Nicotinamide, the C-Met inhibitor is BMS-777607, and the thyroid hormone is T3.
Also included are ALK4/5/7 inhibitors, CDK5 inhibitors, L-type calcium channel (LTCC) activators, and Hippo signal pathway effector inhibitors, wherein the ALK4/5/7 inhibitor is A83-01, the CDK5 inhibitor is AT7519, the L-type calcium channel (LTCC) activator is BayK8644, and the Hippo signal pathway effector inhibitor is Super-TDU1-31.
The components in the culture solution can synergistically induce the iPSC cells to directionally differentiate into islet beta cells in a staged way. Among them, advanced DMEM/F12 medium, glucose, human Serum Albumin (HSA), N2, B27 and glutamine are essential components for each differentiation stage, improving abundant nutrition for long-term survival and proliferation of cells.
The TGF-beta superfamily factor is Activin A,the WNT signal pathway activator is BML-284 or WNT3a, and is a key factor for the specific differentiation of endodermal lineage cells; the JNK signal pathway inhibitor CC-930 can enhance the actions of the Activin A and the WNT3a and can obviously promote the efficient differentiation of pluripotent stem cells into endoderm; retinoic Acid (RA) may promote the flow to PDX1 + Differentiation of pancreatic precursor cells; sirt1 inhibitor is Nicotinamide and can promote the direction of NKX6.1 + Differentiation of pancreatic precursor cells; the epidermal growth factor and keratin growth factor have synergistic effect, and can promote PDX1 when used in combination + And NKX6.1 + Differentiation of double positive pancreatic precursor cells; the protein kinase C activator is TPB, can promote the specific differentiation to cells of pancreatic lineage, has synergistic effect with RA, and can remarkably enhance PDX1 + Differentiation of cells; the hedgehog pathway antagonist is N-acetylcysteine or cyclopamine, and can promote differentiation of the inwardly secreted precursor cells; FGF10 can promote the survival and proliferation of pancreatic endocrine progenitor cells, improves the differentiation efficiency and stability at the stage, and has great synergy on the final pancreatic beta yield; at the same time, the combination of hedgehog pathway antagonists and FGF10 can promote the promotion of PDX1 + Differentiation of endocrine precursor cells; the TGF-beta inhibitor is ALK5iII, can promote differentiation and maturation of islet beta cells, and can increase the release of insulin in islet beta cells; the combined use of TGF-beta inhibitors and thyroid hormones may promote NGN3 + Is expanded by endocrine precursor cells; the Notch signaling pathway inhibitor is FLI-06 or DAPT, and can increase NGN3 + The number of endocrine precursor cells; the C-Met inhibitor is BMS-777607, and can promote the maturation of islet beta cells.
The ratio of the culture solution in different stages of cell culture is different, and the culture solution can be specifically divided into a culture solution A, a culture solution B, a culture solution C and a culture solution D, wherein the culture solution A is used for inducing the differentiation of multifunctional cell spheres into a definitive endoderm cell stage, the culture solution B is used for differentiating the definitive endoderm cell into a pancreatic precursor cell stage, the culture solution C is used for differentiating the pancreatic precursor cell into a pancreatic endocrine progenitor cell stage, and the culture solution D is used for differentiating the pancreatic endocrine progenitor cell into a pancreatic islet beta cell stage.
The culture solution A comprises an 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 the TGF-beta factor is 30-100ng/mL, preferably 50ng/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 an 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 2-20ng/mL, preferably 10ng/mL; retinoic acid (Retinoic acid) at a concentration of 1-5 μm; the concentration of keratin growth factor is 20-40ng/mL, preferably 30ng/mL; the concentration of the protein kinase C activator is 100-300nM, preferably 200nM; the concentration of Sirt1 inhibitor is 10-40. Mu.M, preferably 20. Mu.M.
Culture broth B also includes an ALK4/5/7 inhibitor, with the concentration of ALK4/5/7 inhibitor being 10-50 nM, preferably 20nM. The ALK4/5/7 inhibitor is A83-01, can selectively inhibit TGF-beta activin receptor ALK4, I type receptor ALK5 and node receptor ALK7, can inhibit transformation of epithelial cells into mesenchymal cells, and has the functions of promoting specific differentiation of pancreatic lineage cells and expansion of pancreatic precursor cells.
The culture solution C comprises an 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 TGF-beta inhibitor is 5-20. Mu.M, preferably 10. Mu.M; the concentration of hedgehog signaling pathway inhibitor is 10-50ng/mL, preferably 30ng/mL; the concentration of Notch signaling pathway inhibitor is 0.2-1. Mu.M, preferably 0.5. Mu.M; FGF signal factor concentration is 5-50ng/ml, preferably 10ng/ml; the concentration of thyroid hormone is 0.1-1. Mu.M, preferably 0.5. Mu.M.
The culture broth C also comprises a CDK5 inhibitor, the CDK5 inhibitor having a concentration of 5 to 30nM, preferably 15nM. The CDK5 inhibitor is AT7519, which has dual efficacy: firstly, the activators P35 and P39 of CDK5 are expressed in pancreas cells and play a role in regulating the maturation of islet beta cells, and secondly, AT7519 also has GSK3 beta inhibition function, can activate WNT signaling pathway to promote cell differentiation, and in addition, the action of CDK5 inhibitor can be enhanced under the premise of the existence of Notch signaling inhibitor.
The culture solution D comprises an 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 10nM; the concentration of the C-Met inhibitor is 20-100nM, preferably 50nM.
Culture broth D also included L-type calcium channel (LTCC) activators and Hippo signaling pathway effector inhibitors; wherein, the concentration of the L-type calcium ion channel (LTCC) activator is 10-30 nM, preferably 20nM; the concentration of the inhibitor of the Hippo signal pathway effector is 0.2 to 0.6. Mu.M, preferably 0.35. Mu.M. The L-type calcium ion channel (LTCC) activator is BayK8644, which can activate Ras signals, strengthen cell cycle and promote proliferation of islet beta cells; the inhibitor of the Hippo signal pathway effector YAP is Super-TDU1-31, which can break the interaction between YAP and TEADS transcription factors and has important roles in promoting the differentiation of endocrine cells and inhibiting proliferative precursor cells.
The culture solution of the embodiment adopts Advanced DMEM/F12 culture medium, and is added with key components such as glucose, sodium bicarbonate, human Serum Albumin (HSA), glutamine, vitamin C and the like which are necessary for maintaining the growth of cells, agonists or antagonists containing JNK, hedgehog, EGF, FGF, TGF-beta, WNT and other signal paths, necessary small molecular compounds, microelements and the like, and the components act in a staged and directional manner, so that the multipotent stem cells can be efficiently induced to be directionally differentiated into islet beta 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 number of raw materials is not limited, the induced multifunctional stem cells can be infinitely amplified, and the moral dispute in the aspect of using embryonic stem cells is avoided; secondly, the period of culturing islet beta cells is about three weeks, and compared with the existing differentiation technology, the preparation speed is greatly improved, and the production cost is reduced; thirdly, the islet beta cells are cultured in a 3D suspension mode, and compared with the existing adherence (2D) culture technology, the yield of differentiated cells and the loss caused by reagent replacement in the culture process are greatly improved; fourth, the specific signal pathway inhibitor and 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; fifth, the cultured islet beta cells selectively adopt cell sorting or biological material wrapping technology, so that the potential safety hazard caused by poor treatment effect due to lower cell purity or immune rejection reaction after cell transplantation can be avoided.
The method for using the culture solution for preparing islet beta cells by inducing the directional differentiation of pluripotent stem cells in the embodiment is as follows:
s100, preparing an induced multifunctional cell sphere;
s200, performing primary differentiation, and performing directional differentiation culture on the induced multifunctional cell spheres by using a culture solution A to obtain definitive endoderm cells;
s300, performing secondary differentiation, and performing induced differentiation on the definitive endoderm cells by using a 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;
s500, performing four differentiation, and performing induced differentiation on pancreatic endocrine progenitor cells by using a culture solution D to obtain required islet beta cells.
The step S100 of preparing the induced multifunctional cytoball comprises digesting the induced multifunctional stem cells subjected to the adherence culture into small cell blocks, and re-suspending with mTESR-1 complete medium containing ROCK1 inhibitor at a ratio of 0.2X10 6 /cm 2 Is inoculated in an ultra-low adsorption six-hole plate, cultured for 24 hours under the condition of 5% oxygen at 37 ℃ until cells become regular pellets, and then transferred to 37 ℃ and 5% CO 2 Under the condition, 3D suspension culture is carried out in a spinner flash at 70-120 rpm, preferably 100rpm, for 2-3 days, and the induction is obtainedMultifunctional cell ball.
Step S200 is specifically performed by using culture solution A for inducing multifunctional cell pellet at 37deg.C and 5% CO 2 And (3) continuing to culture for 3 days under the condition to obtain definitive endoderm cells, wherein the culture solution A is replaced every 1 day during the culture period, and the main component of the definitive endoderm cell induction culture medium is the culture solution A.
Step S300 is specifically performed by culturing definitive endoderm cells in culture medium B at 37deg.C and 5% CO 2 And (3) continuing to culture for 5 days under the condition to obtain pancreatic precursor cells, wherein the culture solution B is replaced every 1 day during the culture period, and the main component of the pancreatic precursor cell induction culture medium is the culture solution B.
Step S400 is specifically performed by culturing pancreatic precursor cells in medium C at 37deg.C and 5% CO 2 Continuously culturing for 7 days under the condition to obtain the 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 performed by culturing pancreatic endocrine progenitor cells with culture medium D at 37deg.C and 5% CO 2 Culturing for 7 days under the condition to obtain islet beta cells, and continuously replacing the culture solution D every 2-4 days during the culture period, wherein the main component of the islet beta cell induction culture medium is the culture solution D.
Example 2
The content of the embodiment is mainly the preparation of islet beta cell differentiation culture solution, and the specific steps are as follows:
the differentiation culture solution of islet beta cells is divided into four stages, namely a first stage (DE induction culture medium composed of culture solution A), a second stage (PP induction culture medium composed of culture solution B), a third stage (EN induction culture medium composed of culture solution C) and a fourth stage (islet beta cell induction culture medium composed of culture solution D), wherein the culture solution components are composed of a basic culture solution component (basal medium) and an additive component (supplement), and the components and the proportions are shown in the following tables 1-4:
TABLE 1 composition and ratio of DE Induction Medium
TABLE 2 composition and ratio of PP Induction Medium
Remarks: the addition of A83-01 can significantly improve the cell differentiation efficiency.
TABLE 3 composition and ratio of EN Induction Medium
Remarks: the addition of AT7519 can significantly increase cell differentiation efficiency.
TABLE 4 composition and ratio of islet beta cell induction Medium
Remarks: the addition of BayK8644 and Super-TDU1-31 can significantly increase the differentiation efficiency of cells.
Example 3
The main content of the embodiment is to culture and induce the multifunctional stem cells by using a 3D suspension culture mode, which is as follows:
1. 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 cell survival is Y27632.
2. Culture process
After the iPSCs which are subjected to the adherence culture in 100mm-dish are discarded and are rinsed by using 2mL of DPBS, 4mL of EDTA digestive enzyme is added and placed at 37 ℃ for 3-4 min, the digestive enzyme is discarded after the intercellular separation, 12mL of mTESR-1 complete medium with the final concentration of 10 mu M Y7632 is added, and the cells are blown and separated into cell small blocks, wherein each cell small block is aggregated by 10-30 cells.
The isolated cell pellet is transferred on average to an ultra-low adsorption 6-well plate, cultured at 37℃under 5% oxygen for 24 hours until the cells become regular pellets, then transferred to 75 or 125mL spinner flash, and subjected to 3D suspension culture at 70-120 rpm, preferably 100 rpm.
3. Cell sphere morphology observation
100mm-dis was placed on a sterile console, suspended cell spheres cultured for 1 day or 3 days were transferred to the above-mentioned dis using a 10mL pipette, the dis was gently shaken to uniformly distribute the cell spheres, and observed under an EVOS microscope and photographed.
The results are shown in FIG. 1: the method can obtain a large number of cell balls with uniform morphology.
Example 4
The main content of the embodiment is to utilize induced multifunctional stem cells to directionally differentiate and culture islet beta cells, and the method is as follows:
1. culture of undifferentiated cells
Induction of multifunctional Stem cells 3D suspension cell pellets were obtained as in example 2, cultured in 125mL of spinner fly, passaged as single cells every 3-4 days with Ackutase digestive enzyme, resuspended in complete medium with mTESR-1 containing 10 μ M Y7632 inhibitor, at 37℃5% CO 2 The incubator of the culture medium is subjected to suspension culture at a rotating speed of 70-100 rpm.
When preparing to differentiate islet beta cells, the cells digested by Ackutase are treated with a method of 2 to 8 multiplied by 10 5 cell/mL, preferably 6X 10 5 cell/mL density seed in the above spinner flash, after 3 days of culture, change to differentiation medium.
2. Directional differentiation culture of islet beta cells
The 3 day old undifferentiated iPSCs pellet was allowed to settle naturally for 3-5 min, the top mTESR-1 medium and dead cells were gently discarded using a 10mL pipette and the induction medium (no addition marked) was added sequentially (30 mL/flash, medium preferably on-the-fly, no more than 7 days if stored at 4℃and removed and equilibrated to room temperature 30 minutes before use) at 37℃at 5% CO as per the differentiation stage induction medium (no addition marked) in example 1 2 Above the magnetic stirrer in the incubator, the rotation speed was adjusted to 70rpm, and the incubation time was adjusted to the corresponding period. Wherein the culture medium is changed every 2 days after the culture of the first stage for 3 days, the culture of the second stage for 5 days, the culture of the third stage for 7 days and the culture of the fourth stage for more than 7 days.
3. Morphology observation of islet beta cell spheres
100mm-dish was placed on a sterile console, the islet beta cell pellet cultured for about three weeks was transferred into dish using a 10mL pipette, and the dish was gently shaken to uniformly distribute the cell pellet, and observed under an EVOS microscope.
FIGS. 2-A-D show islet beta cell pellets (including pellet morphology at each stage) cultured by the differentiation method of example 4, and it is found that islet beta cell pellets having a preferable morphology can be obtained by this method.
Example 5
The main content of this example is the islet β cell differentiation method after adding small molecular compound (to increase differentiation efficiency), specifically as follows:
using the islet beta cell differentiation method of example 4, the morphology and number of islet beta cells obtained after three weeks of culture were compared by selectively adding small molecule compounds A83-01, AT7519, bayK8644, and Super-TDU1-31 (labeled with a sign) to the differentiated PP, EN, and islet beta cell induction media.
As shown in the results of FIG. 2-A '-D', the induction medium after adding the small molecular compound can obtain more islet beta cells, and the morphology of islet beta cell spheres is more uniform.
Example 6
The main content of this example is the identification of the expression of proteins characteristic of cells in the DE phase by immunofluorescence and flow cytometry, as follows:
the directional differentiation of islet beta cells was performed using the procedure of example 5, in which stage one (DE cells) induction was the initiation and key of differentiation, and DE cell identification was performed using immunofluorescence and flow cytometry as follows.
a, immunofluorescence method:
placing the cells cultured to the first stage in an ultra-low adsorption 24-pore plate, and standing for 1min to enable the cell spheres to sink into the bottom of the pore plate; after the upper layer of the culture solution was carefully aspirated, 1 XPBS (pH 7.4, the same shall apply hereinafter, 1 mL/well) was slowly added along the 24-well plate wall and washed 2 times; then, 4% PFA (paraformaldehyde, 0.5 mL/well) preheated at 37℃was slowly added along the plate wall, and the cells were fixed by standing at room temperature for 18 minutes, the PFA was gently aspirated, and 1 XPBS was added for 3 washes (1 mL/well/time).
0.3% Triton X100 (0.5 mL/well) was added and incubated at 37℃for 30 min; then 5% BSA (0.5 mL/well) was added for blocking and incubated at 37℃for 30 min; next, primary antibodies (SOX 17 and FOXA 2) were directly added to the blocking solution in an amount of 10. Mu.L/well, incubated at 37℃for 1 hour, and washed 3 times (1 mL/well/time) with 1 XPBS.
Adding secondary antibody diluted by 1% BSA (dilution ratio 1:1000), wherein the addition amount of the secondary antibody is 0.5 mL/hole, and after incubation at 37 ℃ in dark for 60 minutes, the secondary antibody is sucked, and the secondary antibody is washed 3 times (1 mL/hole/time) by adding 1 XPBS for 5 minutes each time; DAPI (0.5 mL/well) formulated with 1 XPBS at a final concentration of 1 μg/mL was then added, stained for 5 minutes, blotted off, and washed 2 times (1 mL/well/time) with 1 XPBS; finally, 1 XPBS (0.5 mL/well) was added to resuspend the pellet, and the pellet was observed under a microscope and photographed.
Remarks: in addition to performing the immunofluorescent staining of the cell spheres as described above, the cell spheres were also subjected to immunofluorescent staining (better looking at the expression of each marker in the cells of the list) using Accutase to single cell plating in 24-well plates.
b, flow cytometry:
placing the cell balls which are cultured to the first stage in a 15mL centrifuge tube, naturally settling for 1-3 min, discarding the culture solution, and using 1mL DPBS for one time and discarding; adding 1-2 mL of Ackutase or TrypLE digestive juice into the cell ball, placing at 37 ℃ for 3-5 minutes, and separating the cell ball into single cells; then adding 2-3 times of DPBS to dilute the digestion solution, centrifuging at 200-300 g for 3 minutes, and discarding the digestion solution and the DPBS.
1mL of 4% Paraformaldehyde (PFA) was added to the cells, and the cells were fixed at 4℃for 30 minutes and discarded; then 0.3% Triton X100 (1 mL) or other stabilizing buffer was added and blocked and permeabilized at 4deg.C for 30 minutes and discarded; next, 1mL of the primary antibody (SOX 17 and FOXA 2) was added at 4℃overnight.
Discarding the primary antibody in the next day, adding 1mL of secondary antibody prepared by the sealing solution, and incubating at 4 ℃ in a dark place for 2 hours; and finally, performing flow cytometry analysis.
FIGS. 3-A-B "and FIGS. 3-C' show immunofluorescence and flow cytometry identification, respectively, of DE cells cultured in example 5, showing that the cultured DE cells have high expression of marker molecules.
Example 7
The main content of this example is the identification of the expression of proteins characteristic of cells in PP phase using immunofluorescence, in particular as follows:
cells (PP cells) cultured to stage two were removed, and the expression of the critical proteins (PDX 1 and NKX 6.1) in the PP stage was examined according to the immunofluorescence procedure of example 6.
FIG. 4 shows the results of immunofluorescence identification of Marker (PDX 1 and NKX 6.1) of PP cells cultured in example 5, showing that PP cells cultured have high expression of Marker molecules.
Example 8
The main content of the embodiment is the identification of the expression of the islet beta cell characteristic protein by using an immunofluorescence method, which is specifically as follows:
cells cultured to stage four (islet beta cells) were removed, and the expression of islet beta cell key proteins (NKX 6.1, C-peptide, insulin) was examined according to the immunofluorescence procedure of example 6.
FIG. 5-A shows the immunofluorescence assay results of islet beta cells obtained by culturing in example 5, which shows that islet beta cells obtained by culturing have high expression of marker molecules (NKX 6.1, C-peptide, insulin and MAFA).
Example 9
The main content of the embodiment is to use an Elisa kit to detect insulin secretion function of islet beta cells, and the method is as follows:
islet beta cell pellets (20-30) cultured for 7 days in the stage four are collected in a 15mL centrifuge tube by using a 5mL pipette, and the cell pellets naturally settle for 1-3 minutes and then the upper culture solution is discarded. 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 pellet was gently shaken and left for a further 24 hours.
After 48 hours of placement, the tube was centrifuged at 2100rcf for 15 minutes, 1mL of supernatant was collected in a fresh 15mL centrifuge tube, and 1mL of 1M TRIS (pH 7.5) was added for neutralization. The neutralized liquid was tested for insulin release using the human insulin Elisa kit.
FIG. 5-B shows the results of insulin release assays of islet beta cells obtained by culturing in example 5, which shows that the islet beta cells obtained by culturing have a high amount of insulin release.
Example 10
The main content of this example is to use the Elisa kit to detect GSIS function of islet β cells, specifically as follows:
reagent preparation: krb buffer was prepared from 128mM NaCl, 5mM KCl, 2.7mM CaCl2, 1.2mM MgCl2, 1mM Na2HPO4, 1.2mM KH2PO4, 5mM NaHCO3, 10mM HEPES and 0.1% BSA using deionized water; low concentration glucose (2 mM) and high concentration glucose (20 mM) were prepared from Krb buffer; 30mM KCl was formulated from 1M KCl in water and 20mM high concentration glucose solution; all reagents were filter sterilized with a 0.22 μm filter membrane after formulation.
GSIS functional verification: first, islet beta cell pellets (20) cultured for 14 days in stage four were collected in a 15mL centrifuge tube using a 5mL pipette, and the pellets were allowed to settle naturally for 1 to 3 minutes, and then the upper culture solution was discarded. Then, 1mL of Krb buffer was added for rinsing, and pre-incubation was performed for 1 hour using 200 μl of a low concentration (2 mM) glucose Krb solution for removing residual insulin. After washing again with 1mL of Krb buffer 2 times, the cell pellet was incubated with 200 μl of low concentration (2 mM), high concentration (2 mM) glucose Krb solution and KCl solution, respectively, for 1 hour (each time the solutions of different concentrations were changed, krb equilibration solution was used for washing, and supernatants were collected at the end of incubation). Finally, 200 μl of glucose and KCl-stimulated supernatants were collected and insulin release (GSIS) levels of islet β cells stimulated with different concentrations of glucose were detected using the insulin Elisa kit.
FIG. 5-C shows the GSIS test results of islet beta cells obtained by the culture of example 5, showing that the islet beta cells obtained by the culture have different degrees of insulin release for different concentrations of glucose stimulation, the insulin content released by the stimulated beta cells increases with increasing glucose content, and the content reaches the highest after KCl stimulation.
From the results of the identification in examples 4 to 10, it can be seen that the cells obtained by the induced directional differentiation culture of the multifunctional stem cells have not only the expression of proteins characteristic of islet beta cells such as PDX1, NKX6.1, C-Peptide and Insulin, but also very high yield and purity, and have glucose stimulation-Insulin release (GSIS) function, indicating that they are mature and functional islet beta cells, using the culture solution and the culture method of the present invention.
The culture solution of the invention enables the induced multifunctional stem cells subjected to 3D suspension culture to be directionally differentiated into islet beta cells, and has the following advantages compared with other differentiation methods: firstly, the number of raw materials is not limited, and induced multifunctional stem cells can be infinitely amplified; secondly, the source of the raw materials 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 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 channel protein factors and small molecular compounds; fourthly, the obtained yield is high, and mature islet beta cells with huge quantity and higher purity can be obtained by adding specific small molecular compounds; fifthly, the time consumption is short, the culture period is 22-30 days, and the higher cell yield can be obtained by prolonging the amplification time, so that the cost loss is reduced; sixth, safe and reliable, use methods such as cell sorting or biomaterial encapsulation, etc., can improve the purity of the cell obtained or reduce even have no immune rejection reaction of the cell to host.
In addition, the use of small molecule compounds such as ALK4/5/7 inhibitors (A83-01), CDK5 inhibitors (AT 7519), LTCC activators (BayK 8644) and Hippo signal pathway effector inhibitors (Super-TDU 1-31) promotes the directed differentiation of islet beta cells, which is manifested by a massive expansion of intermediates (pancreatic precursor cells) during differentiation, as well as maturation and high expansion efficiency of 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 perfect functions, and a new method is provided for treating diabetes in the future.
The foregoing examples merely illustrate certain embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention; it should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the concept of the invention, all of which fall within the scope of protection of the invention; accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (3)

1. A culture solution for preparing islet beta cells by inducing multipotent stem cells to directionally differentiate is characterized in that: the culture solution is culture solution A, culture solution B, culture solution C and culture solution D, wherein the culture solution A is used for inducing the differentiation of the multifunctional cell spheroids into a definitive endoderm cell stage, the culture solution B is used for differentiating the definitive endoderm cells into pancreatic precursor cell stages, the culture solution C is used for differentiating the pancreatic precursor cells into pancreatic endocrine progenitor cells, and the culture solution D is used for differentiating the pancreatic endocrine progenitor cells into pancreatic islet beta cell stages;
before using the culture solution A, re-suspending the induced multifunctional stem cells by using a complete mTESR-1 culture medium containing a ROCK1 inhibitor Y27632, and culturing the induced multifunctional cell spheres in a 3D suspension mode;
the culture solution A is an 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 TGF-beta superfamily factor is ActivinA; the WNT signaling pathway activator is WNT3 a; the JNK inhibitor is CC-930; wherein the concentration of TGF-beta factor is 30-100ng/mL; the concentration of WNT signal pathway activator is 1-5. Mu.M; JNK signal inhibitor concentration is 0.5-5. Mu.M;
the culture solution B is an Advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, human serum albumin HSA, N2, B27, glutamine, vitamin C, EGF signal factors, retinoic acid, keratin growth factors, protein kinase C activators and Sirt1 inhibitors, wherein the EGF signal factors are EGF, the keratin growth factors are KGF, the protein kinase C activators are TPB, the Sirt1 inhibitors are Nicotinamide, and the concentration of the EGF signal factors is 2-20ng/mL; retinoic acid concentration is 1-5 μm; the concentration of the keratin growth factor is 20-40ng/mL; the concentration of the protein kinase C activator is 100-300nM; the concentration of the Sirt1 inhibitor is 10-40 mu M;
the culture solution C is an Advanced DMEM/F12 culture medium, glucose, sodium bicarbonate, human serum albumin HSA, N2, B27, glutamine, a vitamin C, TGF-beta inhibitor, a hedgehog signal pathway inhibitor, a Notch signal pathway inhibitor, an FGF signal factor and thyroid hormone, wherein the TGF-beta inhibitor is ALK5iII, the hedgehog pathway antagonist is cyclopamine, the Notch signal pathway inhibitor is FLI-06, the FGF signal factor is FGF10, the thyroid hormone is T3, and the concentration of the TGF-beta inhibitor is 5-20 mu M; the concentration of the hedgehog signaling pathway inhibitor is 10-50ng/mL; the concentration of Notch signaling pathway inhibitor is 0.2-1. Mu.M; FGF signal factor concentration is 5-50ng/ml; the concentration of thyroid hormone is 0.1-1 μm;
the culture solution D is an 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 microelements; wherein the C-Met inhibitor is BMS-777607 and the TGF-beta inhibitor is ALK5iII, and the concentration of the TGF-beta inhibitor is 5-20 mu M; the concentration of the C-Met inhibitor is 20-100nM.
2. The culture solution for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells according to claim 1, wherein the culture solution is characterized by: the culture solution B is also added with an ALK4/5/7 inhibitor, wherein the ALK4/5/7 inhibitor is A83-01, and the ALK4/5/7 inhibitor is 10-50 nM.
3. The culture solution for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells according to claim 1, wherein the culture solution is characterized by: the culture solution C is also added with a CDK5 inhibitor which is AT7519, and the concentration of the CDK5 inhibitor is 5-30 nM; the culture solution D is also added with an L-type calcium ion channel LTCC activator which is BayK8644 and a Hippo signal pathway effector inhibitor which is Super-TDU1-31; the concentration of the L-type calcium ion channel LTCC activator is 10-30 nM; the concentration of the inhibitor of the Hippo signal pathway effector is 0.2 to 0.6. Mu.M.
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