CN115028730B - Preparation method of adult stem cell-derived organoid - Google Patents

Abstract

The invention relates to a preparation method of an adult stem cell-derived organoid. The invention generates the monoclonal antibody of anti-GSK-3 beta by screening specific antigen epitope and through a proprietary hybridoma technology. The antibodies reported in the present invention have high binding affinity; the protein is specifically combined with GSK-3 beta human protein, and has no cross reaction; can effectively inhibit the expression of GSK-3 beta, can promote bone marrow mesenchymal stem cells to construct kidney-like organs, can also promote iPSC to be differentiated into neural stem cells, and has good application prospect.

Description

Preparation method of adult stem cell-derived organoid

Technical Field

The invention relates to the field of biology, in particular to a preparation method of an adult stem cell-derived organoid.

Background

Organoids (Organoids), meaning that it resembles an organ of tissue. In fact, it is a model that is built based on a 3D in vitro cell culture system and is highly similar to the source tissue or organ in vivo. These 3D in vitro culture systems replicate the complex spatial morphology of differentiated tissues and are capable of exhibiting cell-to-cell interactions and spatial position morphology between cells and their surrounding matrix. But also can achieve the physiological reaction similar to the tissue and organ differentiated in the body, and has extremely high similarity to the source tissue. Compared with the traditional 2D cell culture mode, the organoids cultured in 3D comprise various cell types, can form functional 'micro-organs', and can be better used for simulating the generation process and the physiological and pathological states of organ tissues, so that the organoids have wide application prospects in the aspects of basic research and clinical diagnosis and treatment.

Organoids are used for physiological studies, and the material can be derived from pluripotent stem cells (hPSCs) or adult stem cells. The pluripotent stem cells can be derived from Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs), while the somatic stem cells are generally derived from separation and purification of tissue samples obtained by surgical operation. In addition to physiological studies such as embryonic development, normal tissue organoids can also be used in tumor studies, including the study of mechanisms of carcinogenic risk factors and the establishment of tumor organoids under gene editing conditions.

Organoid research has focused on disease models such as developmental related problems, genetic diseases, tumor cancers, and the like. By using the iPSCs of the patient, a valuable disease model can be established, and the disease model of the patient can be simulated and reproduced in vitro; meanwhile, the establishment of the organoids can realize more effective and more real detection on the drug effect and toxicity of the drug. Because the organoids can be directly cultured and generated by the human iPSCs, the inconsistency of detection results caused by the difference between the animal cells and the human cells is avoided to a great extent compared with an animal model.

It has been shown that stem cells can be induced to differentiate into collecting ducts and nephrons by directed differentiation. Ureteric buds also originate in the mesoderm and interact with an adjacent cell population (i.e., metanephric mesenchyme), which will give rise to the ureteric tree, forming the ureter, renal pelvis, and collecting ducts, while metanephric mesenchymal progenitor cells give rise to nephrons. Ureteral epithelium is derived from one side branch of the mesonephron duct, which itself forms in the anterior mesoderm, while the posterior mesenchyme is derived from the posterior mesoderm. Cells migrate from the primitive streak (somite mesoderm) cephalically to form the mesoderm. Early migrating primitive streak cells form ureteral epithelium under the stimulation of fibroblast growth factor9 (FGF 9), while a selective inhibitor of hepatic glycogen synthesis kinase3 β receptor (CHIR 99021) stimulates late migrating primitive streak cells by altering the duration of Wnt signaling, causing an increase in postrenal mesenchyme. According to this principle, in 2015 foreign scholars obtained kidney-like organs by inducing the directed induced differentiation of pluripotent stem cells. The Xiaofenglin and the like successfully construct kidney-like organs by adopting BM-MSCs, utilizing selective inhibitors of CHIR99021 hepatic glycogen synthetic kinase3 beta receptors and controlling the differentiation of the BM-MSCs through cytokines. It follows that liver glycogen synthesis kinase3 β is a key step in organoid formation.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase which is well conserved in evolution, is commonly found in eukaryotic cells of mammals, and can act on a plurality of signal proteins, structural proteins and transcription factors to regulate differentiation, proliferation, survival and apoptosis of cells besides the activity of the Glycogen Synthase (GS) which is originally discovered and regulated. In the research of various serious diseases such as cancer, neurodegenerative diseases, neuropsychiatric diseases and the like, the selection of the compound as a therapeutic target is receiving attention from more and more researchers. Liver glycogen synthesis kinase3 beta pathway inhibitors currently exist in a few classes, and a few classes are available for selection.

Disclosure of Invention

The invention provides a monoclonal antibody specifically aiming at GSK-3 beta. The antibody is obtained by screening specific dominant antigen epitope.

In one aspect, the invention provides a GSK-3 beta-C1 monoclonal antibody having an affinity dissociation constant (Kd) of 7.23X 10 ^－10 And belongs to high affinity antibodies.

The light and heavy chain sequences of the monoclonal antibodies are respectively as follows:

light chain variable region (SEQ ID NO: 2)

DIVITQRPALMAASPGEKVTITCMLYLRDMKAKSKWYQQKSGISPKPWIYFNTKMSCGVPARFSGSGSGTSYSLTITSMEAEDAATYYCQSDYREVDVFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 3)

EVQLEESATELARPGASVKLSCKASGYIFSTFYQVWIKQRPGQGLEWIGVICLEDTYDLLHKELGGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGSIYYSPSWGLGTTLAVSS

Preferably, the amino acid sequence of the heavy chain variable region is an analogous sequence having at least 50% homology to sequence 3, preferably, the amino acid sequence of the heavy chain variable region is an analogous sequence having at least 60% homology to sequence 3, preferably, the amino acid sequence of the heavy chain variable region is an analogous sequence having at least 70% homology to sequence 3, preferably, the amino acid sequence of the heavy chain variable region is an analogous sequence having at least 80% homology to sequence 3, preferably, the amino acid sequence of the heavy chain variable region is an analogous sequence having at least 90% homology to sequence 3, preferably, the amino acid sequence of the heavy chain variable region is an analogous sequence having at least 99% homology to sequence 3.

Preferably, the amino acid sequence of the light chain variable region is an analogous sequence having at least 50% homology to sequence 2, preferably, the amino acid sequence of the light chain variable region is an analogous sequence having at least 60% homology to sequence 2, preferably, the amino acid sequence of the light chain variable region is an analogous sequence having at least 70% homology to sequence 2, preferably, the amino acid sequence of the light chain variable region is an analogous sequence having at least 80% homology to sequence 2, preferably, the amino acid sequence of the light chain variable region is an analogous sequence having at least 90% homology to sequence 2, preferably, the amino acid sequence of the light chain variable region is an analogous sequence having at least 100% homology to sequence 2.

The present disclosure includes nucleic acid molecules encoding the immunoglobulin light and heavy chain genes of GSK-3 β monoclonal antibodies, vectors comprising such nucleic acids, and host cells capable of producing the anti-GSK-3 β monoclonal antibodies of the present disclosure.

The GSK-3 β monoclonal antibodies of the invention may be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For recombinant expression of the antibody, the host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cell, optionally secreted into the medium in which the host cell is cultured, from which medium the antibody can be recovered. Antibody heavy and light chain genes are obtained using standard recombinant DNA methods, integrated into recombinant expression vectors, and the vectors introduced into host cells.

GSK-3 β monoclonal antibodies of the present disclosure include intact molecules and antibody fragments (e.g., fragments of Fab and F (ab ') 2 that specifically bind hpg. Fab and F (ab')) 2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of an animal or plant, and may have less non-specific tissue binding than an intact antibody. Thus, the antibody fragments may be used for therapeutic applications among other applications.

The term "antibody fragment" refers to a portion of a full-length antibody, typically the target binding or variable region. Examples of antibody fragments include Fab, fab ', F (ab') 2 and Fv fragments. An "Fv" fragment is the smallest antibody fragment that contains an intact target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain (V1, V2) in close, non-covalent association. H-VL in this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of V. H-VL dimer. Typically, six CDRs confer antibody target binding specificity. However, in some cases, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) can have the ability to recognize and bind a target, albeit with a lower affinity than the entire binding site. "Single chain Fv" or "scFv" antibody fragments comprise the domains of VH and VL antibodies, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between Vv and Vb. H and VL enable scFv to form domains that target the desired structure. A "single domain antibody" is a domain that displays sufficient affinity for HPG by a single VH or VL.

The GSK-3 β monoclonal antibody may be formulated in a composition. Optionally, the compositions provided herein are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the desired method of administration to the patient).

In another aspect of the invention, the use of a GSK-3 β monoclonal antibody in the preparation of a pharmaceutical composition for inhibiting GSK-3 β activity is provided.

The GSK-3 β monoclonal antibodies of the invention may be administered to a patient by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, topical, intrathecal and intracerebroventricular. In any given case, the most suitable route of administration will depend on the particular antibody, the subject, the nature and severity of the disease and the physical condition of the subject. The antibody can be formulated as an aqueous solution for administration by subcutaneous injection.

The pharmaceutical compositions may conveniently be presented in unit dosage form comprising a predetermined amount of an anti-GSK-3 β monoclonal antibody of the present disclosure per dose. Such units may comprise, for example, but are not limited to, 5mg to 5g, such as 10mg to 1g, or 20mg to 50mg. The pharmaceutically acceptable carriers for use in the present disclosure may take a variety of forms, for example, depending on the condition to be treated or the route of administration.

By mixing the antibody having a desired purity with an optional pharmaceutically acceptable carrier, excipients or stabilizers (all of which are referred to herein as "carriers"), which are commonly used in the art, i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants and other various additives, are used. These additives must be non-toxic to recipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in a range close to physiological conditions. They may be present at a concentration of about 2mM to about 50 mM. Suitable buffers for use in the present disclosure include organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffer (e.g., lactic acid-sodium lactate mixture, lactic acid-potassium hydroxide mixture, acetic acid-potassium acetate mixture, etc.), and salts thereof (e.g., sodium acetate-acetic acid-sodium acetate mixture, etc.). In addition, phosphate buffers, histidine buffers and trimethylamine salts such as Tris can be used.

In another aspect of the invention, the invention provides an application of the GSK-3 beta monoclonal antibody in promoting bone marrow mesenchymal stem cells to construct kidney-like organs.

Further, the concentration of the GSK-3 beta monoclonal antibody in the application is 10 mu mol/L.

Furthermore, the monoclonal antibody of the invention can also promote iPSC to differentiate into neural stem cells, thereby being beneficial to the subsequent preparation of neural-related organoids.

Advantageous effects

The invention has the advantages that the monoclonal antibody of anti-GSK-3 beta is generated by screening specific antigen epitope and a special hybridoma technology. The antibodies reported in the present invention have high binding affinity; the protein is specifically combined with GSK-3 beta human protein, and no cross reaction exists; can effectively inhibit the expression of GSK-3 beta, can promote bone marrow mesenchymal stem cells to construct kidney-like organs, can also promote iPSC to be differentiated into neural stem cells, and has good application prospect.

Drawings

FIG. 1GSK-3 beta antigen dominant epitope analysis result chart

FIG. 2 is a graph showing the effect of the antibody on GSK-3. Beta. Protein expression

FIG. 3 is a graph showing the effect of the antibody on the differentiation of iPSC into neural stem cell density

Detailed Description

The present invention will be further described below by way of specific embodiments and experimental data. Although specific terms are used below for the sake of clarity, these terms are not meant to define or limit the scope of the invention.

EXAMPLE 1 preparation of GSK-3 beta monoclonal antibody

According to the amino acid sequence of human Glycogen Synthase Kinase-3 Beta, the epitope analysis was performed, and the dominant epitope was selected as follows by computer Parker software analysis (as shown in FIG. 1):

kvsrdkgsk vttvvatpgq gpdrpqevsy tdtkvigngs fgvvyqaklc dsgelvaikk vlqdkrfknnr elqimrkldh cnivrryff ysgekkdev (SEQ ID NO: 1). The antigen was artificially synthesized and used for the following immunization.

(1) Primary immunization: taking 4 healthy female BALB/c mice of 6 weeks old, diluting 800 mu g of GSK-3 beta antigen fragment with 100 mu l of sterile PBS, mixing with 100 mu l of complete Freund's adjuvant, fully emulsifying, and injecting into the abdominal cavity of each mouse;

(2) And (3) second immunization: after three weeks, diluting 80 μ g of GSK-3 β antigen fragment with 100 μ l of sterile PBS, and mixing with 100 μ l of incomplete Freund's adjuvant by emulsifying, and injecting into abdominal cavity of each mouse;

(3) Blood is taken from orbital venous plexus of mice, the serum titer is determined by an indirect ELISA method, and the selected serum titer is 1 ⁵ The mice were subjected to intraperitoneal injection and impact immunization 1 time after being diluted with 200. Mu.g of GSK-3 beta antigen fragment without adjuvant in 100. Mu.l of PBS 3 days before cell fusion;

(4) After three days, BALB/c mouse spleen cells were taken for cell fusion.

(5) Myeloma cells Sp2/0 at logarithmic growth phase, centrifuging at 1000rpm for 5min, discarding supernatant, resuspending cells in 10ml serum-free RPMI-1640 complete medium, counting, and adjusting cell number to 1 × 10 ⁷ Washing with serum-free RPMI-1640 complete medium for 2 times;

(6) Sp2/0 myeloma cells were mixed with spleen cells in a ratio of 1 (V/V) in 50ml plastic conical-bottom centrifuge tubes. Filling a centrifugal tube with serum-free RPMI-1640 complete culture medium, and centrifuging at 1000rpm for 5mim at room temperature;

(7) Preheating 50% PEG solution and serum-free RPMI-1640 complete medium;

(8) Sucking the centrifuged mixed cell supernatant, adding 1ml of hot PEG into the mixed cell sediment, slowly adding dropwise at a constant speed within 1min, gently stirring the cells by using a pipettor every time one drop is added, and gently stirring the cells for 1min after the dropwise addition is finished;

(9) Dropwise adding preheated serum-free RPMI-1640 complete culture medium into the mixed cell liquid, slightly stirring while adding, stopping the action of PEG, respectively adding lml,2ml,3ml,4ml,5ml and 10ml every 2 minutes, and finally supplementing the serum-free RPMI-1640 complete culture medium to 50ml;

(10) Centrifuging at 1000rpm for 5 mm, discarding the supernatant, adding 5ml HAT culture medium, slightly blowing and sucking the precipitated cells, and cutting to prevent the cells fused together from scattering;

(11) Supplementation with HAT and RPMI-1640 complete medium and 1% magic ^TM Cloning factor of hybridoma cell, adding fused cell suspension into 5 96-well plates, 1 × 10 ⁵ Individual cells/(150. Mu.l/well), put in a 37 ℃ cell culture chamber, 5% by volume of CO ₂ And culturing under saturated humidity condition.

(12) On 14 th day after fusion, collecting the supernatant with the growth of the fused cells for detection, screening positive clones by an indirect ELISA method, and taking the culture supernatant of myeloma cell Sp2/0 as a negative control; and (3) co-screening 17 hybridoma cells with better positive, further cloning by using a limiting dilution method, and selecting two hybridoma cell strains with strongest positive, namely GSK-3 beta-C1 and GSK-3 beta-D8 which are stably expressed for later use.

The monoclonal cell line obtained by screening was designated as 10 ⁶ Ascites was prepared by injecting each cell/mouse abdominal cavity. When the ascites of the mouse is generated to the maximum, the neck of the mouse is removed to kill the mouse, the ascites is collected, and the ascites antibody titer is detected by an indirect ELISA method. The antibody in the ascites was purified by an ammonium octylate method and a protein A column.

Detecting the purity and concentration of the antibody by SDS-PAGE and ultraviolet spectrophotometry; the results of subtype identification of the monoclonal antibody using the mouse antibody subtype identification kit are shown in Table 1.

TABLE 1 antibody concentrations and subtypes

Hybridoma cell	Antibody concentration (mg/mL)	Cell subsets
			GSK-3β-C1	12.11±0.55	IgG1
GSK-3β-D8	15.32±0.67	IgG2a

To verify the specificity of the obtained 2 monoclonal antibodies, the two antibodies were combined with SEQ ID NO: 1. comparison of recombinant human GSK-3 beta (Biovision, cat number: 7004-100) and GSK-3 beta additional antigen fragments sidvwsagcv laelllgqpipfgdgsvdvqlveikvllgptreqirernp, HCG and BSA after indirect ELISA detection shows that the cross-reaction rate of 2 monoclonal antibodies obtained by screening and other various substances is less than 0.1 percent, and the monoclonal antibodies can specifically react with GSK-3 beta holoprotein and the immunogen fragment of the invention, which shows that the specificity is better (Table 2).

TABLE 2 Cross-reactivity of antibodies

Example 2 identification of affinity and sequence characteristics of GSK-3 beta-C1 monoclonal antibody

Measuring the affinity of the monoclonal antibody by using an indirect ELISA method, namely coating an ELISA plate with 2 mu g/mL GSK-3 beta protein, sealing, adding the diluted and purified monoclonal antibody in a multiple proportion, taking goat anti-mouse IgG marked by HRP as a secondary antibody, and reading OD (optical density) by using an ELISA reader ₄₅₀ The absorbance at nm, calculated according to the conventional data processing method in the field (A practical guide to monoclonal antibodies), gave GSK-3 beta-C1 monoclonal antibody affinity dissociation constant (Kd) of 7.23X 10 ^－10 And belongs to high affinity antibodies.

The light and heavy chain sequences of the monoclonal antibody obtained by amplifying the light and heavy chain sequences of the antibody by using a PCR method and identifying through sequencing are respectively as follows:

light chain variable region (SEQ ID NO: 2)

DIVITQRPALMAASPGEKVTITCMLYLRDMKAKSKWYQQKSGISPKPWIYFNTKMSCGVPARFSGSGSGTSYSLTITSMEAEDAATYYCQSDYREVDVFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 3)

EVQLEESATELARPGASVKLSCKASGYIFSTFYQVWIKQRPGQGLEWIGVICLEDTYDLLHKELGGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGSIYYSPSWGLGTTLAVSS

Example 3 Effect of GSK-3 beta-C1 monoclonal antibodies on GSK-3 beta

Human colon cancer cells HT-29 (HT 29), cells in logarithmic growth phase digested with 0.25% pancreatin, 2X10 ⁸ cells/L are inoculated in a 6-well plate at the density, monoclonal antibodies (0, 1, 10 and 100 mu mol/L) with different concentrations are added after 2d, BIO (1 mu mol/L, CAS number: 667463-62-9, sigma) is taken as a positive control, the culture is continued for 24h, cells of each group are collected after 0.25 percent of pancreatin without EDTA, and PBS is centrifuged and resuspended for 1 time. Detecting GSK-3 beta protein expression by Western blotting: extracting total cell protein according to the operation steps of a protein extraction kit and determining the protein concentration, adding 20 mu g of total protein per lane for SDS-PAGE electrophoresis, transferring the protein to an NC membrane by wet transfer, sealing with 5% skimmed milk powder at room temperature for 1h, adding mouse anti-GSK-3 beta (1. Scanning a film, analyzing a gray value by QuantityOne software, and calculating the relative protein content of the GSK-3 beta by taking the expression quantity of the beta-actin as the basis of the relative expression quantity. The experiment was repeated 3 times. The results are shown in FIG. 2.

As can be seen from the Western blotting detection results in FIG. 2, the differences between the C1 monoclonal antibody and the positive control group are statistically significant (P < 0.05), and after 100. Mu. Mol/L C1 monoclonal antibody is used for treatment, the relative expression level of GSK-3 beta is only (0.03 + -0.01), which is significantly improved compared with the positive control (0.36 + -0.06). From the results, the GSK-3 beta-C1 monoclonal antibody provided by the invention can obviously inhibit the activity of the GSK-3 beta protein.

Example 4 Effect of GSK-3. Beta. -C1 monoclonal antibody on the differentiation of iPSC into neural stem cells

iPSC cell (A)

iPSM0041, triple starter) were inoculated into Matrigel-coated 6-well plates and mTeSR was added ^TM 1 pluripotent stem cell culture medium for 1 Day, and the following Day (Day 1) was changed to neural differentiation medium (DMEM/F12 and Neurobasal Medium mixed at 1:1, followed by 0.5 XN 2, 0.5 XB 27, 0.1mmol/Lascorbic, 1 XGlutamax, 1 XAntibi-otic-antibacterial). At this stage, the Smad inhibitors SB and DMH1 that favour the differentiation of ipscs towards the ectoderm were added. Meanwhile, in order to study the influence of the GSK-3 beta-C1 monoclonal antibody on the differentiation of iPSC, the GSK-3 beta-C1 monoclonal antibody with different concentrations is added, 4 experimental groups with the concentration of 0, 1, 10 and 100 mu mol/L (mu M) are divided according to different concentrations, the 0 mu mol/L group is taken as a control group, the positive control is BIO (1 mu mol/L, CAS number: 667463-62-9, sigma), the culture is carried out for 6d, and the liquid is changed every other day. Measurement of cell concentration "cell count in units of 1 well of 6-well plate (unit 10) ⁶ One/well) "and NSC markers SOX1, nestin. The results are shown in FIG. 3.

As can be seen from the results in FIG. 3, the higher the concentration of the monoclonal antibody added to the experimental group, the more the cells are aggregated, the greater the number of cells, and the number of cells in the C1 monoclonal antibody group at 1. Mu. Mol/L is (0.70. + -. 0.02). Times.10 ⁶ The number of cells in the positive control group was (0.65. + -. 0.03). Times.10 ⁶ The differentiation and proliferation promoting effect is obvious; the number of the C1 monoclonal antibody group cells at 100. Mu. Mol/L is (1.61 +/-0.05). Times.10 ⁶ The proliferation was most pronounced. The difference between the 0. Mu. Mol/L group and the experimental group is statistically significant (P)<0.05). In addition, immunofluorescence results showed that cells in the positive control group and each experimental group expressed NSC markers SOX1 and Nestin. Indicating that each set of ipscs had differentiated into NSCs. Wherein the average positive rate of the 1 mu mol/L C1 monoclonal antibody group is 92.14 percent, is slightly improved than 90.53 percent of the positive control group, and is also higher than the positive rate of the high-concentration C1 monoclonal antibody. This also indicates that ipscs differentiated into NSCs, with higher concentrations of mab giving greater cell densities, but the higher concentration group had lower expression of SOX1 than the other groups. The C1 monoclonal antibody is suggested to enable the cell proliferation to be more vigorous, the differentiation is not influenced by a certain concentration of the inhibitor (1 mu mol/L), but the differentiation is not influenced by the high concentration of the C1 monoclonal antibody.

Example 5 bone marrow mesenchymal stem cell construction of Kidney-like organ experiment

Bone marrow mesenchymal stem cell (CP-H166, wuhan Punuo sai)Life science and technology limited) using DMEM/F-12 liquid medium containing 10% fetal calf serum, after passage, the degree of confluency is 40% -50% in 2-3d, removing DMEM/F-12 liquid medium containing 10% fetal calf serum from the culture dish, adding APEL liquid medium containing 10 μmol/L C1 monoclonal antibody, culturing at 37 ℃ for 4d, and replacing the medium every 2 d. After 4 days, the medium was changed to APEL medium containing 200ng/ml FGF9+ 1. Mu.g/ml heparin. The culture was carried out at 37 ℃ to day 7, and the medium was changed every 2 days. On day 7, after digesting the cells with trypsin, the cells were centrifuged 400g,3min and resuspended in 3ml of pure APEL liquid medium. About 5X 10 per kidney-like organ is required ⁵ For each cell, the desired amount of cell suspension was pipetted into a 1.5ml Eppendorf tube. 400g was centrifuged for 2min and the supernatant discarded, leaving only 1 cell pellet. APEL liquid medium containing 10. Mu. Mol/L of the mAb was added to a Transwell plate. The Transwell filter was attached to the surface of the medium. The cell pellet was placed on a Transwell filter. Incubate 1h at 37 ℃ and change to 1.2ml APEL broth containing 200ng/ml FGF9+ 1. Mu.g/ml heparin for 5 days, change media every 2 days, and change to pure APEL broth on day 12. Medium was changed every 2d and kidney-like organs were cultured in pure APEL liquid medium to day 25. In the control group, CHIR99021 was used in place of the monoclonal antibody of the present invention. The results show that, in the kidney-like organs, fluorescence markers of distal tubule (ECAD +), proximal tubule (LTL +), glomerulus (WT 1 +) and blood vessel (CD 31) can be detected no matter in the experimental group or the control group, which indicates that the kidney-like organs are primarily formed, and the antibody of the present invention can control the formation ratio of the metanephric mesenchyme by regulating Wnt signals, so as to finally control the formation of nephrons. The results show that the C1 monoclonal antibody of the invention has basically similar action with CHIR99021, and can be used for constructing kidney-like organs by using mesenchymal stem cells.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred embodiments of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the appended claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Sequence listing

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Claims (3)

1. A monoclonal antibody specific to GSK-3 beta, wherein the light chain sequence and the heavy chain sequence of the monoclonal antibody are respectively as follows:

the sequence of the light chain variable region is shown as SEQ ID NO:2, the sequence of the heavy chain variable region is shown as SEQ ID NO:3, respectively.

2. Use of the monoclonal antibody of claim 1 in the preparation of a medicament for promoting differentiation of ipscs into neural stem cells.

3. Use of the monoclonal antibody of claim 1 in the preparation of a medicament for promoting bone marrow mesenchymal stem cells to construct a kidney-like organ.

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