Method for constructing Bardet-Biedl syndrome kidney cell model

Abstract

The invention discloses a method for constructing a Bardet-Biedl syndrome kidney cell model, which comprises the following steps of: constructing a Bardet-Biedl syndrome kidney cell model; the invention relates to the technical field of biology, and provides a method for constructing a Bardet-Biedl syndrome kidney cell model, which comprises the following steps: directionally inducing and differentiating a specific induced pluripotent stem cell into a kidney cell population, and obtaining the kidney cell population, namely the Bardet-Biedl syndrome kidney cell model, the specific induced pluripotent stem cells are derived from BBS7 gene carrying c.849 + 1Ggt; c, and c.754Ggt; the in-vitro peripheral blood mononuclear cells of a Bardet-Biedl syndrome patient with A and A composite heterozygous mutation are reprogrammed to obtain the in-vitro peripheral blood mononuclear cells of the Bardet-Biedl syndrome. The cell model disclosed by the invention can reflect clinical manifestations of kidney dysplasia and dysplasia of cilia of a Bardet-Biedl syndrome patient, so that a platform is provided for a pathogenic mechanism of the disease, drug screening and even gene therapy.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for constructing a Bardet-Biedl syndrome kidney cell model. Background Art

[0002] Bardet-Biedl syndrome (BBS) is a clinical syndrome characterized by six major features, including renal dysplasia, polydactyly, retinitis pigmentosa, obesity, mental retardation, and gonadal dysgenesis. It is a non-motor ciliary dysfunction disease caused by functional mutations in BBS-related genes. More than 90% of BBS patients have renal abnormalities with latent clinical symptoms. As the disease progresses, renal failure occurs, which is the leading cause of death in BBS patients. However, the cause of kidney disease caused by BBS mutations is still unclear, and there is no practical treatment plan.

[0003] Although animal models such as mice and zebrafish have played an important role in studying the pathogenic mechanism of BBS, these models often fail to fully reproduce the pathological characteristics of human diseases. For example, BBS7 knockout mice exhibit phenotypes such as retinal degeneration and obesity, but no significant renal cysts or polydactyly. In addition, there are significant differences between mice and humans in genome structure, renal development patterns, and ciliary function, which makes it difficult for animal models to fully reflect the pathological mechanisms of BBS-related renal abnormalities. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a Bardet-Biedl syndrome kidney cell model.

[0005] In a first aspect, the present invention claims a method for constructing a Bardet-Biedl syndrome kidney cell model.

[0006] The method for constructing a Bardet-Biedl syndrome kidney cell model claimed in the present invention may include the following steps: directing and inducing differentiation of specific induced pluripotent stem cells into a kidney cell population, wherein the obtained kidney cell population is a Bardet-Biedl syndrome kidney cell model; The specific induced pluripotent stem cells are obtained by reprogramming ex vivo peripheral blood mononuclear cells from patients with Bardet-Biedl syndrome carrying compound heterozygous mutations of c.849+1G>C and c.754G>A in the BBS7 gene.

[0007] Among them, the c.849+1G>C refers to a base substitution variation from guanine (G) to cytosine (C) at the intron boundary after the 849th base (donor splicing site) in the cDNA sequence of the BBS7 gene. This variation may affect the subsequent splicing process after gene transcription, and thus affect the protein product or related biological function finally expressed by the gene. The c.754G>A means that compared with the cDNA sequence of the wild-type BBS7 gene, the 754th nucleotide has undergone a substitution from G to A. The compound heterozygous mutation refers to the BBS7 gene having both the c.849+1G>C and the c.754G>A mutations, and both mutations are heterozygous mutations. Among them, the Genebank accession number of the cDNA sequence (wild type) of the BBS7 gene is NM_176824.3 (Update: PRI 20-OCT-2024).

[0008] Exemplarily, the reprogramming may be OCT4 Gene, SOX2 Gene, KLF4 Genes and c-MYC The genes are co-introduced into the peripheral blood mononuclear cells, and cultured until the induced pluripotent stem cells are obtained.

[0009] Furthermore, the directed induction and differentiation of the specific induced pluripotent stem cells into a kidney cell population can be carried out according to a method comprising the following steps: (a1) culturing the induced pluripotent stem cells in an induction differentiation medium 1 to obtain primitive streak cells (corresponding to the primitive streak stage of inducing hiPSCs to differentiate into kidneys, i.e., stage C in the embodiment); the induction differentiation medium 1 is composed of CHIR99021 and a basal medium; (a2) culturing the primitive streak cells in a differentiation induction medium 2 to obtain a kidney cell population (corresponding to the stage of further differentiating the primitive streak cells into a kidney cell population having multiple types, i.e., stage D in the embodiment); the differentiation induction medium 2 comprises FGF9, Heparin and a basal medium; (a3) Maintaining and culturing the renal cell population in a culture medium without differentiation inducing factors to obtain a more mature renal cell population (corresponding to the further maturation stage in the embodiment, i.e., stage E).

[0010] In an embodiment of the present invention, in the differentiation induction medium 1, the basal medium is APEL2 medium. In the differentiation induction medium 2, the basal medium is APEL2 medium. In step (a3), the medium without differentiation induction factors is APEL2 medium.

[0011] Furthermore, in the induction differentiation medium 1, the final concentration of CHIR99021 can be 10 μM (the final concentration of CHIR99021 was tried to be 8 μM, and the results showed that no nephron-like tubular structure could be formed at the 2D level). In the induction differentiation medium 2, the final concentration of FGF9 can be 200 ng / ml, and the final concentration of Heparin can be 1 μg / ml. Further, in step (a1), the induced pluripotent stem cells can be cultured in the induction differentiation medium 1 for 4 days (3 days were tried, and no nephron-like tubular structure could be formed at the 2D level). During this period, the induction differentiation medium 1 was replaced every day. On the 4th day, the expression of primitive streak stage marker genes, T gene (Gene ID: 6862) and TBX6 gene (Gene ID: 6911), will appear.

[0012] Furthermore, in step (a2), the primitive streak cells may be cultured in the differentiation induction medium 2 for 10 days, during which the medium is changed every 1-2 days.

[0013] Furthermore, in step (a3), the kidney cell population may be cultured in the culture medium without differentiation inducing factors for 10-14 days, during which the medium is changed every 1-2 days.

[0014] Furthermore, in the method, the following step may be included before step (a1): the induced pluripotent stem cells are cultured in a culture plate pre-plated with matrix gel in mTeSR plus medium supplemented with Y27632 (whose concentration may be 10 μM) for 1 day, and then cultured in mTeSR plus medium without Y27632 for 2 days, so that the cell confluence is 30-40% (corresponding to the pre-modeling stage in the embodiment, i.e., stage B). The culture time requirement at this stage is relatively loose, and the purpose is to prepare for subsequent differentiation (3 days is recommended, and the state of the cultured cells for 3 days is more stable. After trying to culture for only 1 day at this stage, the cell differentiation is not very good, which is manifested in: more cell death during differentiation is easy, and differentiation for more than 20 days cannot be completed). Furthermore, before this, the step of amplifying and culturing the induced pluripotent stem cells to ensure that the cells are in a good state (corresponding to stage A in the embodiment) may be included, specifically, the induced pluripotent stem cells are cultured for 24 hours in a culture plate pre-coated with matrix gel using mTeSR plus medium supplemented with Y27632 (whose concentration may be 10 μM), and then replaced with mTeSR plus medium without Y27632 for continued culture, and stably passaged for at least 3 times to ensure that the cells are in a good growth state.

[0015] In a second aspect, the present invention claims protection for the kidney cell population prepared by the method described in the first aspect above.

[0016] In a third aspect, the present invention claims the use of the kidney cell population described in the second aspect as or in the preparation of a Bardet-Biedl syndrome kidney cell model.

[0017] In a fourth aspect, the present invention claims the use of the method described in the first aspect above or the kidney cell population described in the second aspect above in any of the following: (B1) Drug screening and / or drug evaluation; (B2) Preparation of products for drug screening and / or drug evaluation; (B3) Preparation of products for studying the pathogenesis of Bardet-Biedl syndrome; (B4) Preparation of products for evaluation of gene therapy strategies for Bardet-Biedl syndrome; In (B1) and (B2), the drug is a drug that delays or improves kidney damage in Bardet-Biedl syndrome.

[0018] In a fifth aspect, the present invention claims a method for preparing a target kidney cell population.

[0019] The method for preparing the target kidney cell population claimed in the present invention may include the steps described in the first aspect above; the kidney cell population formed by the directed induction and differentiation of the induced pluripotent stem cells is the target kidney cell population; The target renal cell population is a renal cell population having all or part of the following characteristics: (b1) a reduced proportion of podocytes and / or renal tubular epithelial cells; (b2) a reduced cilia rate and / or shortened cilia length; (b3) abnormal expression (increased and / or decreased) of cilia-related genes.

[0020] Further, the cilia-related genes may be all or part of the following: GLI1, INVS, AXIN2, PDGFRA, PTCH1, IFT80, IFT74, TMEM67, NPHP1, IFT88, PRKCA, FUZ, CDK5RAP2, GLI3, AHI1, MKKS, CEP41, IFT172, RPGRIP1L, NEK1, CEP290, MKS1, TTC8, KRAS, DYNC2LI1, BBS7, BBS2, HNF1B, CDKN1A, CCND1, FJX1, PTPN5 and / or FOS.

[0021] In one embodiment of the present invention, the podocytes are Nephrin-positive podocytes; and the renal tubular epithelial cells are AQP1-positive renal tubular epithelial cells.

[0022] In a sixth aspect, the present invention claims protection for the use of the target kidney cell population prepared by the method described in the fifth aspect above in screening drugs with functions of: (c1) increasing the proportion of podocytes and / or renal tubular epithelial cells; (c2) increasing the cilia rate and / or increasing the cilia length; (c3) restoring abnormally expressed cilia-related genes to normal.

[0023] Furthermore, the abnormally expressed cilia-related genes include: (d1) genes whose expression is decreased by 2 times or more, including GLI1, INVS, AXIN2, PDGFRA, PTCH1, IFT80, IFT74, TMEM67, NPHP1, IFT88, PRKCA, FUZ, CDK5RAP2, GLI3, AHI1, MKKS, CEP41, IFT172, RPGRIP1L, NEK1, CEP290, MKS1, TTC8, KRAS, DYNC2LI1, BBS7 and / or BBS2; (d2) genes whose expression is increased by 2 times or more, including HNF1B, CDKN1A, CCND1, FJX1, PTPN5 and / or FOS.

[0024] In one embodiment of the present invention, the podocytes are Nephrin-positive podocytes; and the renal tubular epithelial cells are AQP1-positive renal tubular epithelial cells.

[0025] Experiments have shown that the process of inducing the differentiation of hiPSCs carrying the BBS7 mutation into kidney cells in the present invention simulates the development process of the kidney in vivo, and the formed kidney cells and nephron-like structures reproduce the clinical phenotype and disease characteristics of kidney damage in Bardet-Biedl syndrome, including: (1) Compared with wild-type kidney cells, the number of podocytes (NEPHRIN) and renal tubular epithelial cells (AQP1) in the kidney cells induced and differentiated by hiPSCs carrying the BBS7 mutation in the present invention is reduced. (2) Compared with the wild type, the length of cilia in the kidney cells induced and differentiated by hiPSCs carrying the BBS7 mutation in the present invention is shortened, the cilia rate is reduced, and multiple genes related to cilia are differentially expressed, which suggests that the function of cilia as a "biological antenna" is abnormal. In summary, it can be seen that the hiPSCs carrying the BBS7 mutation in the present invention can reflect the clinical manifestations and disease characteristics of kidney dysplasia and cilia abnormalities in patients with Bardet-Biedl syndrome, which provides a good platform for the pathogenic mechanism, drug screening and even gene therapy of Bardet-Biedl syndrome in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1This is a flowchart for the construction of a kidney cell model for Bardet-Biedl syndrome. Stage A: hiPSC (human induced pluripotent stem cells) can enter the next stage after three stable passages. Stage B: Pre-modeling stage. Stage C: Primitive streak induction stage. Stage D: Differentiation stage of kidney cell population. Stage E: Further maturation stage. In the figure, "D+Arabic numerals" represent the day.

[0027] Figure 2 The cells in the primitive streak differentiation stage during the process of hiPSCs from healthy people and hiPSCs from patients with Bardet-Biedl syndrome being induced to differentiate into kidney cells (A, scale bar is 200μm) and the PCR identification results of marker genes in the primitive streak stage (B). In the figure, "wild type" corresponds to the directed differentiation of hiPSCs from healthy people, "mutant type" corresponds to the directed differentiation of hiPSCs from patients with Bardet-Biedl syndrome, and "D+Arabic numerals" corresponds to Figure 1 is consistent in.

[0028] Figure 3 The tubular structures that begin to appear in the late differentiation stage of the kidney cell population differentiated from hiPSCs derived from BBS patients.

[0029] Figure 4 This is the detection of the expression of podocytes and renal tubular epithelial cells in the kidney cell population differentiated by hiPSC directed induction from healthy people (i.e., the "wild type" in the figure) and the kidney cell population differentiated by hiPSC directed induction from BBS patients (i.e., the "BBS model" in the figure). In the figure, the first column is a blank tube.

[0030] Figure 5 The cilia rate and cilia length of the kidney cell population differentiated by hiPSC derived from healthy people (i.e., "wild type" in the figure) and the kidney cell population differentiated by hiPSC derived from BBS patients (i.e., "BBS" in the figure) were detected.

[0031] Figure 6 This is a chip detection heat map of the expression of cilia-related genes in the kidney cell population differentiated by hiPSCs derived from healthy people (i.e., "wild type" in the figure) and the kidney cell population differentiated by hiPSCs derived from BBS patients (i.e., "BBS" in the figure). DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0033] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0034] Example 1. Construction and identification of a Bardet-Biedl syndrome renal cell model 1. Experimental Materials 1. Cell lines BBS patient-derived hiPSCs: Reprogramming of explanted peripheral blood mononuclear cells from Bardet-Biedl syndrome patients (BBS patients) carrying compound heterozygous mutations of c.849+1G>C and c.754G>A in the BBS7 gene OCT4 Gene, SOX2 Gene, KLF4 Genes and c-MYC Human induced pluripotent stem cells obtained by co-introducing the gene into the peripheral blood mononuclear cells. The cells and their specific construction process are recorded in the article "Qian Fu, et al. Generation of a human iPSC line from a Bardet-Biedl syndrome patient compound heterozygousfor the BBS7 variants c.849+1G>C / c.754G>A. Stem Cell Research 54 (2021)102428". The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0035] Among them, the c.849+1G>C refers to a base substitution variation from guanine (G) to cytosine (C) at the intron boundary after the 849th base (donor splicing site) in the cDNA sequence of the BBS7 gene. This variation may affect the subsequent splicing process after gene transcription, and thus affect the protein product or related biological function finally expressed by the gene. The c.754G>A means that compared with the cDNA sequence of the wild-type BBS7 gene, the 754th nucleotide has undergone a substitution from G to A. The compound heterozygous mutation refers to the BBS7 gene having both the c.849+1G>C and the c.754G>A mutations, and both mutations are heterozygous mutations. Among them, the Genebank accession number of the cDNA sequence (wild type) of the BBS7 gene is NM_176824.3 (Update: PRI 20-OCT-2024).

[0036] Healthy human hiPSC: iPS cells DYR0100 were established by reprogramming the foreskin fibroblasts of a healthy newborn to induce OCT4, SOX2, KLF4, and MYC. The cells are products of the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences Typical Culture Collection Committee, with the catalog number SCSP-1301.

[0037] 2. Consumables and reagents Ordinary 24-well cell culture plate: Nest product, item number 702002. Glass-bottom 24-well cell culture plate: Nest product, item number 801006. 6-well cell culture plate: Nest product, item number 703002. Matrigel gel: Corning product, item number 356231. mTeSR plus medium: stemcell product, item number 100-0274. APEL2 medium: stemcell product, item number 05275. Y27632: selleck product, item number S1049. CHIR99021: selleck product, item number S1263. FGF9: sinobiological product, item number 10262-H01H. Heparin: MCE product, item number HY-17567A.

[0038] 2. Construction of Bardet-Biedl Syndrome Cell Model The flowchart of the construction of Bardet-Biedl syndrome cell model is shown below. Figure 1 As shown in the figure, the construction process includes the following five stages: Phase A: hiPSC (human induced pluripotent stem cells) can enter the next phase only after three stable passages. This is to ensure the stability and consistency of the cells and provide a reliable cell source for subsequent modeling.

[0039] Phase B: Pre-modeling phase, during which cells are seeded into 24-well plates and stably cultured for 3 days to prepare for subsequent differentiation.

[0040] Phase C: Primitive streak induction phase, inducing cells to develop towards a specific developmental path.

[0041] Stage D: The stage of differentiation of renal cell populations, which causes the cells to differentiate towards renal cells.

[0042] Stage E: Further maturation stage, allowing differentiated kidney cells to further develop and mature.

[0043] The five stages are described in detail below: 1. Expansion and culture of hiPSCs to ensure good cell condition (Phase A) The hiPSCs from healthy individuals and BBS patients in step 1 above were expanded and maintained in 6-well plates covered with 0.5% matrix gel. The hiPSCs were cultured in mTeSR plus medium. The medium was changed every 1-2 days depending on the number of cells. When the cell confluence reached 80%, the cells were passaged. The passage steps of the two hiPSC lines are as follows: One day in advance, 1 ml of 0.5% matrix gel was spread into a 6-well plate and placed in a cell incubator.

[0044] The next day, take out the 6-well plate with matrix gel laid the day before, aspirate the matrix gel, and add 2 ml of mTeSR plus medium containing Y27632 (10 μM) (liquid medium obtained by adding Y27632 to mTeSR plus medium, the final concentration of Y27632 in the liquid medium is 10 μM) for later use; take out another cell well plate to be subcultured, wash the cells twice with DPBS, then add TrypLE digestion enzyme, incubate at 37°C for 1 minute, discard the digestion enzyme, return to the 37°C incubator and continue digestion for 8 minutes, add 1 ml of the above mTeSR plus medium containing Y27632 (10 μM) to resuspend the cells, aspirate 100 μl of the cell suspension, add it to the new well plate, shake to mix, and return to the incubator for continued culture; after 24 hours, discard the supernatant, replace with mTeSR plus medium without Y27632 to continue cell culture, and subculture when the confluence is about 80%. Stable subculture is performed 3 times (each subculture repeats the previous steps, mainly to stabilize the cell state) to ensure that the cells are in good growth condition before subsequent nephron differentiation can be carried out.

[0045] 2. Pre-modeling stage (Phase B) Subsequent differentiation modeling was performed in 24-well cell culture plates (glass bottom / ordinary plastic bottom).

[0046] Glue laying: 0.5% Matrigel was spread on a 24-well plate one day in advance and placed in a cell culture incubator overnight.

[0047] The next day: Remove the 24-well plate, discard the matrix gel, and add mTeSR plus medium containing Y27632 (10μM) (liquid medium obtained by adding Y27632 to mTeSR plus medium, the final concentration of Y27632 in the liquid medium is 10μM) for later use. Remove the hiPSCs cultured in the 6-well plate, aspirate the mTeSR plus medium and add DPBS to wash away the remaining medium, and wash at least twice. Aspirate DPBS and add 1 ml of accutase (cell digestion solution). Place the cells in a 37℃ incubator for 10 minutes. Use a 1ml pipette to gently blow twice to make a single cell suspension, and transfer the cells to a 15ml test tube containing 1ml of mTeSR plus medium, mix by inverting, and centrifuge at 300g for 3min. Remove the supernatant, wash once with DPBS, and resuspend into a single cell suspension with 2 ml of mTeSR plus medium containing Y27632 (10 μM) (liquid medium obtained by adding Y27632 to mTeSR plus medium, the final concentration of Y27632 in the liquid medium is 10 μM), and aspirate 20 μl of the suspension for cell counting. According to the counting results, inoculate about 20,000 cells in each well of a 24-well plate, and tap the well plate to evenly spread the cells in the well plate. After 24 hours, replace the medium with mTeSR plus medium, continue culturing for 48 hours, and observe the confluence of the cells under a microscope ( Figure 2 A, D0).

[0048] 3. Inducing hiPSCs to differentiate into the primitive streak stage (PS) of the kidney (Stage C) Wells with a confluence of about 30-40% were selected for 4-day PS differentiation induction. During these 4 days, the medium was replaced every day with APEL2 differentiation medium containing CHIR99021 (10 μM) (liquid medium obtained by adding CHIR99021 to APEL2 differentiation medium. In this liquid medium, the final concentration of CHIR99021 is 10 μM). On the 4th day (cell status is as follows Figure 2 (A, D4) The expression of the marker genes of the PS stage, T gene (Gene ID: 6862) and TBX6 gene (Gene ID: 6911), was detected by PCR.

[0049] Among them, the primer sequences used to detect the T gene are as follows: Forward primer: 5′-TATGAGCCTCGAATCCACATAGT-3′ (SEQ ID No. 1); Reverse primer: 5'-CCTCGTTCTGATAAGCAGTCAC-3' (SEQ ID No. 2).

[0050] Primers used to detect TBX6 gene: Forward primer: 5′-CATCCACGAGAATTGTACCCG-3′ (SEQ ID No. 3); Reverse primer: 5'-AGCAATCCAGTTTAGGGGTGT-3' (SEQ ID No. 4).

[0051] The results showed that on the 4th day, the expression of the marker genes of the PS stage, T gene and TBX6 gene, appeared ( Figure 2 Middle B). The cells obtained after this stage of culture are primitive streak cells (PS cells for short).

[0052] 4. Further differentiate the above PS cells into various types of kidney cell populations (D stage) Use APEL2 differentiation medium containing FGF9 (200ng / ml) and Heparin (1μg / ml) (liquid culture medium obtained by adding FGF9 and Heparin to APEL2 differentiation medium. In this liquid culture medium, the final concentration of FGF9 is 200ng / ml, and the final concentration of Heparin is 1μg / ml) for 10 days of culture. Depending on the growth of cells and the color change of the culture medium, the liquid is changed every 1-2 days. In the later stage of this stage, the formation of tubular structures can be seen around 14 days (in the experiment, the earliest was the 11th day). Tubular structures will appear in both healthy people and BBS patients. Figure 3 The image shows the tubular structures that begin to appear in the late differentiation stage of a kidney cell population differentiated from hiPSCs derived from BBS patients.

[0053] 5. Further maturity stage (stage E) At this stage, all factors that induce differentiation are removed, and the cells are maintained in culture with APEL2 medium. The medium is changed every 1-2 days, and the specific amount can be adjusted according to the color change of the medium. Subsequent experiments can be carried out after 10-14 days.

[0054] Finally, the hiPSCs from healthy individuals and BBS patients in the above step 1 were induced to differentiate into more mature kidney cell populations.

[0055] 3. Identification of Bardet-Biedl Syndrome Cell Model The kidney cell populations differentiated from hiPSCs derived from healthy individuals and hiPSCs derived from BBS patients obtained in step 2 above were identified in the following aspects: 1. Detection of the number and proportion of podocytes and renal tubular epithelial cells in the kidney cell population differentiated from hiPSC Flow cytometry was used to detect the proportion of podocytes (Nephrin) and tubular epithelial cells (AQP1) in the kidney cell population differentiated from hiPSC derived from healthy subjects and the kidney cell population differentiated from hiPSC derived from BBS patients obtained in step 2 above.

[0056] Antibodies and reagents used: Nephrin-PE: Santa Cruz product, item number sc-376522; AQP1-PE: Santa Cruz product, catalog number sc-32737; Fixation / Permeabilization Solution Kit: BD Pharmingen product, catalog number 554714.

[0057] Prepare three 1.5 ml EP tubes for collecting cells from two sources of kidney cell populations.

[0058] The specific operations are as follows: (1) Use accutase to fully digest the hiPSC-differentiated population from healthy individuals and the hiPSC-differentiated kidney cell population from BBS patients obtained in step 2 above. After terminating the digestion with serum-containing culture medium, collect the cells from 2-3 wells into a 1.5 ml EP tube to ensure a sufficient number of cells for flow cytometry experiments.

[0059] (2) Add 1 ml of pre-chilled DPBS to wash once, centrifuge, and discard the supernatant.

[0060] (3) Add 250 μl of fixation / permeabilization solution (from the Fixation / Permeabilization Solution Kit) to each tube and immediately use a 200 μl gun to disperse the cell clumps to form a single-cell suspension at 4°C for 20 min.

[0061] (4) Add 1 ml of washing buffer, centrifuge, and discard the supernatant. (5) Repeat the above steps.

[0062] (6) Add 50 μl of washing buffer to each tube to resuspend the cell pellet.

[0063] (7) Incubation with primary antibody: Leave one tube of cells in each group as a blank control group. Add 2.5 μl of antibodies AQP1-PE and Nephrin-PE into an EP tube respectively, resuspend and incubate at 4°C for 30 min.

[0064] (8) Wash twice with washing buffer, centrifuge, and discard the supernatant.

[0065] (9) Add 200 μl of staining buffer (2% bovine serum + DPBS) to resuspend the cells.

[0066] (10) After filtering, the product is put on the machine for inspection.

[0067] The results are as follows Figure 4 As shown, it can be seen that compared with the wild type (i.e., the kidney cell population differentiated by hiPSC derived from a healthy person obtained in step 2 above), the proportion of podocytes (Nephrin) and tubular epithelial cells (AQP1) in the BBS cell population (i.e., the kidney cell population differentiated by hiPSC derived from a BBS patient obtained in step 2 above) was significantly decreased.

[0068] 2. Detection of cilia rate and cilia length of kidney cells differentiated from hiPSCs Consumables: Glass-bottom cell culture dishes.

[0069] DMEM: product of GIBCO, catalog number 11966025.

[0070] FBS: vivocell product, product number C04001-500.

[0071] ProLong™ Gold Antifade Mounting Medium (with DAPI): ThermoFish product, catalog number P36941.

[0072] Primary Antibody: Rootletin: abcam product, catalog number ab121653, marks the root of cilia.

[0073] Acetyl tubulin: a proteintech product, catalog number 66200-1-Ig, used to label ciliary axonemes.

[0074] Secondary Antibody: Goat anti-mouse 488: Invitrogen product, catalog number A-11001.

[0075] Goat anti-rabbit 647: Invitrogen product, catalog number A32733.

[0076] The specific operations are as follows: The hiPSC-derived kidney cell populations obtained in step 2 above and the hiPSC-derived kidney cell populations obtained in step 2 were fully digested with accutase, inoculated into glass-bottomed cell culture dishes, and cultured with DMEM containing 10% serum. After the cells covered the bottom of the culture dish, the serum was removed, and the cells were starved for 24 hours before cilia staining. The staining steps are as follows: (1) Discard the culture medium, wash twice with DPBS, and fix with 4% paraformaldehyde at room temperature for 20 minutes.

[0077] (2) Incubate with 0.3% Triton-100 at room temperature for 10 min.

[0078] (3) Block with 5% goat serum at room temperature for 1 h.

[0079] (4) Dilute the primary antibody in antibody diluent at a ratio of 1:200 and incubate at 4°C overnight.

[0080] (5) The next day, recover the primary antibody.

[0081] (6) Wash with DPBS, 10 min, 3 times.

[0082] (7) Incubate with secondary antibody: dilute the secondary antibody at a ratio of 1:800 using antibody diluent and incubate at room temperature for 1 h.

[0083] (8) Wash three times with DPBS.

[0084] (9) Add 100 μl of anti-fluorescence quenching mounting medium (containing DAPI) and solidify the mounting medium at room temperature overnight.

[0085] (10) The next day, fluorescence images were taken using a Leica inverted microscope to calculate the ciliary rate and ciliary length.

[0086] The results are as follows Figure 5 As shown, it can be seen that compared with wild-type cells (i.e., the kidney cell population differentiated by hiPSC derived from healthy people obtained in step 2 above), BBS cells (i.e., the kidney cell population differentiated by hiPSC derived from BBS patients obtained in step 2 above) have significantly reduced cilia rate and significantly shortened cilia length.

[0087] 3. Detection of cilia-related gene expression in kidney cells differentiated from hiPSCs Using cilia PCR array, we found that cilia-related genes in the BBS model showed significant differential expression.

[0088] Primary cilia PCR array: Woji Gene products, catalog number WC-MRNA0127-H.

[0089] TRIzol reagent: Invitrogen product, catalog number 15596018.

[0090] Reverse transcription kit: Vazyme product, product number R232-01.

[0091] SYBR Green: Applied Biosystems, catalog number A57156.

[0092] (1) RNA extraction The kidney cell populations differentiated from hiPSCs derived from healthy people and the kidney cell populations differentiated from hiPSCs derived from BBS patients obtained in the above step 2 were collected respectively, 1 ml of Trizol solution was added, pipetted and mixed, 200 μl of chloroform was added, oscillated and mixed for 15 s, left at room temperature for 3 min, centrifuged at 12000 g at 4°C for 10 min, the upper aqueous phase was aspirated into another centrifuge tube, and the same volume of isopropanol was added, inverted and mixed, left at room temperature for 10 min, centrifuged at 12000 g at 4°C for 10 min, a white precipitate was visible at the bottom of the centrifuge tube, the supernatant was discarded, 1 ml of freshly prepared 75% ethanol was added to wash the precipitate, the precipitate was gently blown up, centrifuged at 12000 g for 5 min, the supernatant was discarded, the lid was opened and left at room temperature for 10 min to evaporate the ethanol, 10-20 μl of enzyme-free water was added to dissolve the precipitate, and the RNA concentration was measured.

[0093] (2) cDNA synthesis: cDNA was synthesized using the Novagen reverse transcription kit.

[0094] (3) Mix cDNA with SYBR-Green and add it to a 6-well plate with a PCR chip according to the PCR array operating instructions. Then perform quantitative PCR. After the data is exported, calculate the relative expression multiple.

[0095] The results are as follows Figure 6As shown, it can be seen that compared with wild-type cells (i.e., the kidney cell population differentiated by hiPSC derived from healthy subjects obtained in step 2 above), the expression of cilia-related genes in the BBS cell population (i.e., the kidney cell population differentiated by hiPSC derived from BBS patients obtained in step 2 above) was significantly decreased, among which the genes whose expression decreased by 2 times or more included GLI1, INVS, AXIN2, PDGFRA, PTCH1, IFT80, IFT74, TMEM67, NPHP1, IFT88, PRKCA, FUZ, CDK5RAP2, GLI3, AHI1, MKKS, CEP41, IFT172, RPGRIP1L, NEK1, CEP290, MKS1, TTC8, KRAS, DYNC2LI1, BBS7 and BBS2; the genes whose expression increased by 2 times or more included HNF1B, CDKN1A, CCND1, FJX1, PTPN5 and FOS.

[0096] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for constructing a Bardet-Biedl syndrome kidney cell model, comprising the following steps: directing and inducing differentiation of specific induced pluripotent stem cells into a kidney cell population, wherein the obtained kidney cell population is a Bardet-Biedl syndrome kidney cell model; The specific induced pluripotent stem cells are obtained by reprogramming ex vivo peripheral blood mononuclear cells from patients with Bardet-Biedl syndrome carrying compound heterozygous mutations of c.849+1G>C and c.754G>A in the BBS7 gene.

2. The method according to claim 1, characterized in that: Directed induction and differentiation of the specific induced pluripotent stem cells into a kidney cell population is carried out according to a method comprising the following steps: (a1) culturing the induced pluripotent stem cells in an induction differentiation medium 1 to obtain primitive streak cells; the induction differentiation medium 1 consists of CHIR99021 and a basal medium; (a2) culturing the primitive streak cells in an induction differentiation medium 2 to obtain a kidney cell population; the induction differentiation medium 2 comprises FGF9, Heparin and a basal medium; (a3) Maintaining and culturing the kidney cell population in a culture medium that does not contain differentiation inducing factors to obtain a more mature kidney cell population.

3. The method according to claim 2, characterized in that: In the differentiation induction medium 1, the basal medium is APEL2 medium; and / or In the differentiation induction medium 2, the basal medium is APEL2 medium; and / or In step (a3), the culture medium without differentiation inducing factors is APEL2 culture medium.

4. The method according to claim 2, characterized in that: In the differentiation induction medium 1, the final concentration of CHIR99021 is 10 μM; and / or In the differentiation induction medium 2, the final concentration of FGF9 is 200 ng / ml, and the final concentration of Heparin is 1 μg / ml.

5. The method according to claim 2, characterized in that: In step (a1), the induced pluripotent stem cells are cultured in the differentiation induction medium 1 for 4 days; and / or In step (a2), the primitive streak cells are cultured in the differentiation induction medium 2 for 10 days; and / or In step (a3), the kidney cell population is maintained in the culture medium without differentiation inducing factors for 10-14 days.

6. The method according to claim 2, characterized in that: In the method, the following step is further included before step (a1): culturing the induced pluripotent stem cells in mTeSR plus medium supplemented with Y27632 for 1 day, and then culturing in mTeSR plus medium without Y27632 for 2 days.

7. A kidney cell population prepared by the method according to any one of claims 1 to 6.

8. Use of the kidney cell population according to claim 7 as or in the preparation of a Bardet-Biedl syndrome kidney cell model.

9. Use of the method according to any one of claims 1 to 6 or the renal cell population according to claim 7 in any of the following: (B1) Drug screening and / or drug evaluation; (B2) Preparation of products for drug screening and / or drug evaluation; (B3) Preparation of products for studying the pathogenesis of Bardet-Biedl syndrome; (B4) Preparation of products for evaluation of gene therapy strategies for Bardet-Biedl syndrome; In (B1) and (B2), the drug is a drug that delays or improves kidney damage in Bardet-Biedl syndrome.

10. A method for preparing a target kidney cell population, comprising the steps described in any one of claims 1 to 6; in, The kidney cell population formed by the directed differentiation of the induced pluripotent stem cells is the target kidney cell population; The target kidney cell population has all or part of the following characteristics: (b1) a reduced proportion of podocytes and / or renal tubular epithelial cells; (b2) a reduced cilia rate and / or shortened cilia length; (b3) abnormal expression of cilia-related genes.

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