Multi-lineage human kidney organ in-vitro fusion technology and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an in-vitro fusion technology for multi-lineage human kidney organs and application thereof. The invention provides a method for realizing culture of multilineage kidney organs by fusing kidney units and ureteral bud organs in vitro. According to the in-vitro fusion technology of the kidney organoid reported by the invention, two organoid with different pedigree sources, namely the kidney unit and the ureteral bud, can be efficiently fused, a UB tubular structure is integrated into the kidney unit organoid, and the UB tubular structure starts from a single ureteral bud and is wrapped by kidney unit precursor cells in a similar in-vivo development process, and finally develops into a kidney with glomerulus and a plurality of kidney cells. The multi-lineage renal organ comprises a catheter, a proximal tube, a marrow tab thin section, a distal tube, a connecting tube and a ureteral bud with a single outlet. A framework is provided for an engineered renal unit and a smooth collection system, and the method is an important step for generating the functional renal tissue from the beginning.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a multi-lineage human kidney organoid in vitro fusion technology and its application. Background Art

[0002] As an important tool for studying normal kidney function and pathological mechanisms, kidney organoids have important application values in the fields of disease model construction, drug screening, and nephrotoxicity assessment. In the future, they may even provide potential solutions for human kidney transplantation. In the past decade or so, significant progress has been made in the technologies of directed differentiation of renal lineage cells and three-dimensional kidney structure reconstruction based on human induced pluripotent stem cells (hiPSCs).

[0003] The embryonic development of the kidney depends on the synergistic action of the ureteric bud (UB) and the metanephric mesenchyme (MM): MM cells induce the branching morphogenesis of the UB through paracrine signals, and then form nephrons containing glomerular podocytes, proximal tubules, thin segments of the loop of Henle, distal tubules, and vascular endothelium. However, there are still major challenges in reconstructing this complex developmental process in vitro.

[0004] Currently, the differentiation protocols of hiPSC-based kidney organoids mainly focus on the directed differentiation of nephrogenic mesenchyme (NM), and form nephron-like structures by inducing the epithelialization of NPCs. However, this strategy has significant limitations - the lack of a collecting system such as a functional ureteric bud. Since the UB is not only the developmental origin of the collecting duct system, but also provides the structural basis for the fluid drainage of the distal part of the nephron, its absence results in the formation of only isolated distal tubule structures in the existing organoids, and a complete lumen network cannot be established. It is worth noting that all nephrons are connected to the UB collecting system at the distal end under physiological conditions, forming a lumen axial arrangement with a unified polarity. Therefore, achieving the spatial coupling of NPCs and UBs and constructing a functional collecting system will be the key step to break through the bottleneck of organoid vascularization and obtain human renal tissue with physiological functions.

[0005] The ureteric bud (UB) generates the collecting system of the kidney. Mature UB develops from ureteric bud progenitor cells (UBPCs), which originate from a different lineage of embryonic cell populations in the metanephric mesenchyme from the nephron progenitors (NPCs). Therefore, UBPCs are not induced in kidney organoids. In recent years, differentiation strategies for UB have emerged. Studies using mouse embryonic stem cells have shown that induced UB and NPC can recapitulate the developmental interactions of the nephrogenic lineage in vitro and generate a very regular collecting system. However, currently, combining the two lineages of human UB and NPC does not produce the same results as mouse cells. After dissociating and mixing single cells of the NPC and UB lineages, organized NPC-UB fusion has not been achieved (related literature: Min Shi, Brittney Crouse, Nambirajan Sundaram, Naomi Pode Shakked, Lioba Ester, Weitao Zhang, Vinothini Janakiram, Raphael Kopan, Michael A Helmrath, Joseph V Bonventre, Kyle W McCracken. Integrating collecting systems in kidney organoids through fusion of distal nephron to ureteric bud. bioRxiv [Preprint]. 2024 Sep 20:2024.09.19.613645.).

[0006] Therefore, it is necessary to develop an organoid construction method for in vitro fusion of UB and NPC organoids. Summary of the Invention

[0007] Current strategies for constructing kidney organoids based on human pluripotent stem cells (hiPSCs) mainly achieve multi-lineage fusion through in vitro co-culture of nephron progenitors (NPCs) and ureteric bud progenitor cells (UBPCs). However, this mechanical mixing strategy has significant limitations: in a three-dimensional culture system, UBPCs show a disordered spatial distribution and cannot recapitulate the directed branching pattern of the ureteric bud (UB) during embryonic development. Therefore, the present invention provides the following technical solutions:

[0008] In the first aspect of the present invention, a method for constructing multi-lineage human kidney organoids is provided, comprising the following steps:

[0009] 1) Construct nephron organoids and ureteric bud organoids separately;

[0010] 2) Use a micro tool to create a notch on the surface of the renal unit organoid, and place the ureteric bud organoid fragments into the notch;

[0011] 3) Fusion, differentiation.

[0012] In some embodiments of the present invention, the method for constructing the renal unit organoid includes the following steps:

[0013] 1) Culture human urine cell-derived induced pluripotent stem cells in BPEL medium until embryoid bodies are formed;

[0014] 2) Culture the embryoid bodies with Stage II medium until 3D-shaped renal unit organoids are formed.

[0015] In some embodiments of the present invention, the BPEL medium includes a basal medium: IMDM: 1X, F12: 1X, PFHMII: 1X, additives: BSA: 0.1-0.4% w / v, ITS-X: 0.7-1.3X, αMTG: 0.002%-0.005%, AA2P: 0.03-0.07 mg / mL, Glutamax: 0.7-1.3X, CD Lipid concentrate: 0.7-1.3X, PVA: 1.7-3.3 mg / mL, CHIR99021: 1-10 μM, Y-27632: 2-10 μM, and β-Mercaptoethanol: 0.7-1.3 mM.

[0016] In some embodiments of the present invention, the Stage II medium includes a basal medium: DMEM: 1X, additives: KOSR: 10-20%, NEAA: 0.7-1.3X, Glutamax: 0.7-1.3X, HEPES: 0.7-1.3X, and PVA: 1.7-3.3 mg / mL.

[0017] In some embodiments of the present invention, the method for constructing the ureteric bud organoid includes the following steps:

[0018] 1) Induce the differentiation of human embryonic stem cell lines using basal differentiation medium, ME medium, UB-I medium, and UB-II medium respectively, and perform flow sorting to obtain KIT + ureteric bud progenitor cells;

[0019] 2) Culture the ureteric bud progenitor cells with hUBCM medium to obtain ureteric bud organoids with a diameter of 0.5-2 mm, and divide them into 8-10 fragments.

[0020] In some embodiments of the present invention, the diameter of the ureteric bud organoid is 1 mm.

[0021] In some embodiments of the present invention, the basic differentiation medium comprises F12: 1X, Glutamax: 0.7 - 1.3X, NEAA: 0.7 - 1.3X, β-Mercaptoethanol: 0.05 - 0.15 mM, B-27 supplement minus vitamin A: 0.7 - 1.3X, ITS: 0.7 - 1.3X.

[0022] In some embodiments of the present invention, the ME medium is added with CHIR99021: 3 - 6 μM and LDN-193189: 7 - 13 nM on the basis of the basic differentiation medium.

[0023] In some embodiments of the present invention, the UB-I medium is added with FGF2: 140 - 260 ng / mL, TTNPB: 0.07 - 0.13 μM, LDN-193189: 21 - 39 nM, and A83-01: 0.1 - 0.3 μM on the basis of the basic differentiation medium.

[0024] In some embodiments of the present invention, the UB-II medium is added with FGF2: 140 - 260 ng / mL, TTNPB: 0.07 - 0.13 μM, and LDN-193189: 21 - 39 nM on the basis of the basic differentiation medium.

[0025] In some embodiments of the present invention, the hUBCM medium is added with LDN-193189: 140 - 260 nM, TTNPB: 0.07 - 0.13 μM, CHIR99021: 2 - 4 μM, JAK inhibitor: 70 - 130 nM, GDNF: 35 - 65 ng / mL, A83-01: 0.1 - 0.3 μM, R-Spondin 1: 70 - 130 ng / mL, FGF7: 35 - 65 ng / mL, SB202190: 3 - 7 μM, and EGF: 35 - 65 ng / mL on the basis of the basic differentiation medium.

[0026] The above equivalents are all final concentration equivalents.

[0027] In some embodiments of the present invention, the above X represents the final standard concentration of a conventional system formulation. For example, "DMEM: 1X" means that the medium finally contains the standard system components of DMEM at a concentration of 1 times, and "HEPES: 0.7 - 1.3X" means that the medium finally contains the standard system components of HEPES at a concentration of 0.7 to 1.3 times. X is a conventional expression of a conventional formulation in the art and is widely recognized in the art.

[0028] In some embodiments of the present invention, the above-mentioned culture medium further comprises an antibiotic, such as Penicillin-Streptomycin.

[0029] In some embodiments of the present invention, the differentiation time of each of the ME medium, UB-I medium, and UB-II medium is 1 to 2 days.

[0030] In some embodiments of the present invention, in step 3), the fusion time is 7 to 9 days; the differentiation time is 5 to 10 days; preferably, the fusion time is 8 days, and the time for redifferentiation after fusion is 8 days.

[0031] In some embodiments of the present invention, the culture medium used for fusion is an hUBCM medium containing Y-27632;

[0032] In some embodiments of the present invention, the culture medium used for differentiation is a mixed medium with a volume ratio of StageII to hUBCM of (2.5 to 3.5):1; preferably, it is 3:1.

[0033] The second aspect of the present invention provides a multi-lineage human kidney organoid prepared by the method described in the first aspect of the present invention.

[0034] The third aspect of the present invention provides the application of the method described in the first aspect of the present invention and the multi-lineage human kidney organoid described in the second aspect of the present invention in disease model construction, therapeutic drug screening, and drug nephrotoxicity screening.

[0035] The beneficial effects of the present invention are:

[0036] The present invention provides a method for culturing multi-lineage kidney organoids by fusing renal units and ureteric bud organoids in vitro. The renal organoid in vitro fusion technology reported in the present invention can efficiently fuse organoids derived from two different lineages of renal units and ureteric buds, integrate the UB tubular structure into the renal unit organoids, and is similar to the in vivo development process starting from a single ureteric bud, being surrounded by renal unit precursor cells, and finally developing into a multi-lineage kidney organoid with glomeruli, proximal tubules, thin segments of the loop of Henle, distal tubules, connecting tubules, and a single outlet ureteric bud. It provides a framework for engineered renal units and a patent collecting system and is an important step in the de novo generation of functional renal tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The following further describes the present invention in conjunction with the drawings and embodiments, wherein:

[0038] Figure 1 It is the immunofluorescence staining result of renal unit organoids obtained by differentiating hiPSCs.

[0039] Figure 2 qPCR results of ureteric bud organoids obtained by H1 differentiation.

[0040] Figure 3 Results of the expression of glomerular podocyte NPHS1, thin descending limb of Henle loop SLC12A1, and distal tubule CDH1 after the fusion of ureteric bud organoids and renal unit organoids differentiated to day 5, day 8, day 11, day 14, and day 17 for qPCR analysis.

[0041] Figure 4 Results of the percentage of podocyte area after the fusion of ureteric bud organoids and renal unit organoids differentiated to day 5, day 8, day 11, day 14, and day 17 by immunofluorescence quantitative analysis.

[0042] Figure 5 Results of qPCR comparison of the expression of cell types developed from two lineages obtained at different ratios, with the medium ratio for co-induced differentiation of fused renal organoids ranging from StageII: hUBCM = 1:3 to StageII: hUBCM = 4:1, and whether FBS was added.

[0043] Figure 6 Results of immunofluorescence comparison of the area of renal unit podocytes obtained at different ratios, with the medium ratio for co-induced differentiation of fused renal organoids ranging from StageII: hUBCM = 1:3 to StageII: hUBCM = 4:1, and whether FBS was added.

[0044] Figure 7 Results of immunofluorescence comparison of the number of fusions between ureteric buds and distal tubules of renal units obtained at different ratios, with the medium ratio for co-induced differentiation of fused renal organoids ranging from StageII: hUBCM = 1:3 to StageII: hUBCM = 4:1, and whether FBS was added.

[0045] Figure 8 Results of immunofluorescence staining for the comparison of the continued differentiation time of fused renal organoids. The renal unit organoids were fused at day 8 and then continued to differentiate for 5 days, 8 days, 10 days, and 13 days. Glomerular podocytes PODXL and vascular endothelium CD31 of the renal unit.

[0046] Figure 9 Results of immunofluorescence of the main cell types of fused renal organoids. Glomerular podocytes PODXL, vascular endothelium CD31, distal tubule ECAD, and ureter GATA3 of the renal unit.

[0047] Figure 10 Results of single-cell sequencing analysis of all cell types developed from two lineages, renal units and ureteric buds, of fused renal organoids. Detailed implementation methods

[0048] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work all fall within the scope of protection of the present invention.

[0049] Experimental cells:

[0050] Human urine cell-derived induced pluripotent stem cells hiPSC (hUC-iPSC): a cell line constructed, purified and identified by the laboratory of Zhang Xiao at the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (has been publicly reported in the literature: Fan K, Zhang S, Zhang Y, et al. A Machine Learning Assisted, Label-Free, Non-Invasive Approach for Somatic Reprogramming in Induced Pluripotent Stem Cell Colony Formation Detection and Prediction. Scientific Reports. 2017, 7(1): 13496.).

[0051] Male standard human embryonic stem cell line H1 (WA01): donated by the WiCell Research Institute cell bank in the United States.

[0052] The experimental reagents are shown in Table 1:

[0053] Table 1 Experimental reagents

[0054]

[0055]

[0056] The experimental consumables are shown in Table 2:

[0057] Table 2 Experimental consumables

[0058]

[0059]

[0060] The experimental instruments and equipment are shown in Table 3.

[0061] Table 3 Experimental instruments and equipment

[0062]

[0063]

[0064] The primers adopted in the present invention are shown in Table 4.

[0065] Table 4 Primer Information

[0066]

[0067] The antibodies adopted in the present invention are shown in Table 5.

[0068] Table 5 Immunofluorescence Staining Antibody Information

[0069]

[0070] Example 1 Method for Differentiating, Constructing and Culturing Renal Organoids

[0071] The hiPS cells are used to differentiate renal organoids in this example, and human renal organoids are constructed in vitro through a two-stage culture mode. The method for differentiation, construction and culture is operated according to the following steps.

[0072] 1. Preparation of culture medium

[0073] The BPEL (Bovine serum albumin polyvinylalcohol essential lipids) and Stage II culture medium required for the differentiation and culture process of renal organoids are prepared according to Table 6 and Table 7 below respectively, and are filtered and sterilized through a 0.22 μm filter membrane and stored in a 4°C refrigerator.

[0074] Table 6 BPEL Culture Medium Formula

[0075]

[0076] Table 7 StageII Culture Medium Formula

[0077]

[0078] 2. Culture method

[0079] Taking the culture system where the cells in 1 10-cm cell culture dish correspond to 1 ultra-low attachment 6-well plate as an example:

[0080] (1) Cell preparation. Culture hiPS cells in a 10-cm dish, and start the subsequent operations after the cells reach ~80% confluence.

[0081] (2) Preparation of BPEL complete medium. Add 14.4 μL of CHIR99021 (10.0 mM) to 18 mL of BPEL medium to a final concentration of 8.0 μM, add 6.0 μL of Y-27632 (10.0 mM) to a final concentration of 3.3 μM, and add 32.7 μL of β-mercaptoethanol (55 mM) to a final concentration of 100 μM. After mixing the medium, add it to an ultra-low attachment 6-well plate at a volume of 2.0 mL / well for standby.

[0082] (3) On day 0, wash the cells 2 times with 5.0 mL of DPBS each time. After aspirating the DPBS, add 3.0 mL of Dispase digestive enzyme and incubate in a 37 °C incubator for 6 min. When the edges of the cell clones are observed to curl up under the microscope, terminate the digestion, aspirate the Dispase, and wash the cells 3 times with 5.0 mL of DPBS each time.

[0083] (4) Add the remaining 6.0 mL of BPEL complete medium to a 10 cm dish, and scrape the cell clones from the bottom of the dish along the same direction with a cell scraper. Pipette the cell suspension 3 - 5 times with a 10 mL pipette, and the cell clumps should be uniform and about 100 μm in size. Spread the cell suspension evenly onto an ultra-low attachment 6-well plate, 1.0 mL per well. Place the plate in a 37 °C incubator, shake it well up, down, left, and right, and then let the plate stand completely still for 48 h.

[0084] (5) On day 2, change the medium. First, prepare 12 mL of medium by adding 9.6 μL of 10 mM CHIR99021 (final concentration 8.0 μM). Then tilt the plate at a 45-degree angle. After the formed embryoid bodies settle to the bottom of the wells, aspirate 2.0 mL from each well and retain 1.0 mL of the medium supernatant. Supplement each well with 2.0 mL of freshly prepared BPEL medium. Then put it back into the 37 °C incubator, shake it well again, and continue the culture.

[0085] (6) On day 3, for the second stage of culture, change the medium to StageⅡ medium. Transfer the embryoid bodies together with the medium to a 50 mL centrifuge tube using a 10 mL pipette. Wash the culture well plate with 1.0 mL of DMEM medium per well and transfer all of it to the 50 mL centrifuge tube.

[0086] (7) After all the embryoid bodies settle to the bottom of the centrifuge tube, aspirate the medium supernatant until about 5.0 mL remains, then add 10 mL of DMEM medium for washing. After the embryoid bodies settle to the bottom of the centrifuge tube again, aspirate the supernatant.

[0087] (8) Add 2.0 mL / well of Stage II medium to the original ultra-low attachment 6-well plate. Add 6.0 mL of Stage II medium to the 50 mL centrifuge tube containing embryoid bodies, and then transfer the mixture of embryoid bodies and medium with a pipette, and evenly distribute it into the original ultra-low attachment 6-well plate at 1 mL / well.

[0088] (9) Put the well plate back into the 37 °C incubator, shake it well up, down, left and right, and continue the culture. Change the medium every 1 day. When changing the medium, tilt the well plate at 45 degrees. After the formed organoids have fully settled, aspirate 2.0 mL of the supernatant from each well, and then supplement 2.0 mL of Stage II medium to each well. Until the end point of the 14-day culture.

[0089] 3. Experimental results

[0090] Detect the expression of renal unit organoid-specific markers through immunofluorescence experiments. The experimental operations are shown in Example 4.

[0091] The results are as Figure 1 shown. Immunofluorescence identification shows that renal unit organoids have the expression of marker proteins of cell types such as glomerular podocyte NPHS1, proximal tubule LTL, distal tubule CDH1, vascular endothelium CD31, and interstitial cells MEIS1 / 2. The renal unit organoids are successfully prepared.

[0092] Example 2 Differentiation, construction and culture method of ureteric bud organoids

[0093] In this example, H1 cells are used to differentiate ureteric bud organoids. After monolayer differentiation, ureteric bud precursor cells are sorted by flow cytometry, and then suspended into 3D cell spheres for further differentiation to construct human ureteric bud organoids in vitro. The differentiation, construction and culture method are operated according to the following steps.

[0094] 1. Preparation of culture medium

[0095] The ME, UB-I, UB-II and hUBCM media required for the differentiation and culture process of ureteric bud organoids are prepared according to Tables 8, 9, 10, and 11 below respectively. After filtration and sterilization through a 0.22 μm filter membrane, they are stored in a 4 °C refrigerator.

[0096] Table 8 Basic differentiation medium

[0097]

[0098] Table 9 ME medium (supplement)

[0099]

[0100] Table 10 UB-I medium (supplement)

[0101]

[0102] Table 11 UB-II Medium (Supplements)

[0103]

[0104] Table 12 hUBCM Medium (Supplements)

[0105]

[0106]

[0107] 2. Culture Method

[0108] Take the differentiation of a batch of cells in one well of a 6-well cell culture plate as an example:

[0109] (1) Cell preparation. Passage H1 cells into one well of a 6-well cell culture plate for culture. The next day, wait for the cells to adhere. After reaching ~30% confluence, start the subsequent operations.

[0110] (2) Preparation of ME medium. Add CHIR99021 with a final concentration of 4.5 μM and LDN-193189 with a final concentration of 10 nM to 3 mL of the basic differentiation medium. After mixing, replace mTeSR 1plus in the 6-well plate with the ME medium to start the differentiation on the first day.

[0111] (3) On the 3rd day, prepare UB-I medium. Add FGF2 with a final concentration of 200 ng / mL, TTNPB with a final concentration of 0.1 μM, LDN-193189 with a final concentration of 30 nM, and A83-01 with a final concentration of 0.2 μM to 3 mL of the basic differentiation medium. After mixing, replace ME in the 6-well plate with UB-I medium to start the differentiation on the third day.

[0112] (4) On the 5th day, prepare UB-II medium. Add FGF2 with a final concentration of 200 ng / mL, TTNPB with a final concentration of 0.1 μM, and LDN-193189 with a final concentration of 30 nM to 3 mL of the basic differentiation medium. After mixing, replace UB-I in the 6-well plate with UB-II medium to start the differentiation on the fifth day.

[0113] (5) On the 7th day, the cells have reached 100% confluence. Wash the cells 2 times with 1.0 mL of DPBS each time. After aspirating the DPBS, add 1.0 mL of Accutase digestive enzyme and incubate in a 37°C incubator for 6 min. When most cells are observed to be digested into single cells under the microscope, terminate the digestion, transfer to a centrifuge tube, and centrifuge at 300×g for 3 min to collect the cells and count.

[0114] (6) The ureteric bud progenitor cells sorted by flow cytometry and collected above were added with PE-CD117 (c-kit) antibody at a concentration of 1:100, incubated in the dark on ice for 30 min, terminated the incubation with FACS (DPBS + 10% FBS), filtered the cells through a 40-μm cell strainer, sorted the PE-positive cells with a FACS Aria IIU flow cytometer, centrifuged to collect the cells and then counted. Every 20,000 cells were seeded into one well of an ultra-low attachment 96-well plate, and 100 μL / well of hUBCM medium was added and incubated statically in a cell culture incubator overnight.

[0115] (7) The cells aggregated into small spheres with clear boundaries at the bottom of the wells. After aspirating the medium, each cell sphere was suspended and wrapped with 10 μL of liquid Matrigel and transferred to one well of a new ultra-low attachment 96-well plate. After placing it at 37 °C for 10 min, the Matrigel solidified, and 100 μL of hUBCM medium was added to each well. Incubated statically in a 37 °C incubator, and the medium was changed every other day.

[0116] (8) On the 14th - 18th day, after examining the diameter of the ureteric bud organoids under a microscope and finding it to be about 1 mm, the organoids were aspirated with a Pasteur pipette under a stereomicroscope, cut into 8 - 10 small fragments with sterile surgical scissors and blades, and each small fragment was wrapped with 10 μL of liquid Matrigel for passage and amplification. Or digested with Accumax digestive solution at 37 °C for 30 min, observed that most of them had been digested into single cells, then centrifuged to collect the cells and counted. Every 20,000 cells were seeded into one well of an ultra-low attachment 96-well plate, and 100 μl / well of hUBCM medium was added and incubated statically in a cell culture incubator overnight. After forming spheres, each cell sphere was suspended and wrapped with 10 μL of liquid Matrigel and transferred to one well of a new ultra-low attachment 96-well plate for passage and amplification.

[0117] (9) Cryopreservation of ureteric bud organoids: After cutting the organoids into small fragments, every 8 - 10 small fragments were cryopreserved in one cryotube, 500 μL of cryopreservation solution (hUBCM + 10% DMSO) was added, placed in a -80 °C freezer overnight with a programmable freezing container, and then transferred to a liquid nitrogen tank for long-term cryopreservation.

[0118] (10) Resuscitation of ureteric bud organoids: Take out the cryopreservation tube from the liquid nitrogen tank, quickly thaw it in a 37°C resuscitator, then take out the cryopreservation tube, wipe the outer surface of the tube with 75% alcohol, transfer the cell suspension to a 6-cm cell culture dish, add 0.5 mL of F12 medium to dilute the cryopreservation solution. Small fragments of ureteric bud organoids can be seen with the naked eye. Aspirate the small fragments with a pipette tip and wrap each small fragment with 10 μL / particle of Matrigel, then seed it into a well of an ultra-low attachment 96-well plate, and add 100 μl / well of hUBCM medium, and let it stand and culture overnight in the cell culture incubator. Change the medium the next day and passage as needed according to the growth of the organoids.

[0119] 3. Experimental results

[0120] Detect the expression of ureteric bud organoid-specific markers by fluorescence PCR experiment. The experimental steps are shown in Example 4.

[0121] The results are as Figure 2 shown. qPCR identified that the ureteric bud organoid iUB highly expressed WNT11, RET, GATA3, SOX9, and FOXL1 specifically. These organoids can also be continuously passaged and amplified in the UB medium hUBCM, and can still self-assemble into a stable ring structure after being digested into single cells or cut into fragments. The preparation of ureteric bud organoids was successful.

[0122] Example 3 In vitro fusion method of renal unit and ureteric bud organoids

[0123] 1. Micromanipulation

[0124] (1) Prepare the renal unit organoids differentiated and cultured from hiPSC, and the ureteric bud organoids obtained from stable passage and amplification of H1 differentiation.

[0125] (2) Under a stereomicroscope, use sterile surgical forceps to pick up a 3D-cultured renal unit organoid, and manually dissect a small piece of ureteric bud organoid cultured for 7-10 days and place it beside.

[0126] (3) Use a pair of fine dissecting forceps to clamp / stabilize the renal unit organoid from one side, then use a sterile needle to pierce a small hole in the renal unit organoid from the other side, and carefully push the small piece of ureteric bud organoid beside into the gap with the needle.

[0127] (4) All these operations are carried out in a 10-μL droplet of fusion renal organoid medium in a 10-cm cell culture dish, which contains 10 μM Y-27632. Carefully place the culture dish after this operation into the cell culture incubator. After overnight culture, the renal unit organoid will slowly and autonomously wrap the embedded ureteric bud organoid, and the ureteric bud and the renal unit will fuse into a whole.

[0128] 2. Fusion time

[0129] In this fusion system, single circular structures obtained by chopping ureteric buds in stable subculture and precursor parts of renal units were used for in vitro fusion. Renal organoids at day 5, day 8, day 11, day 14, and day 17 were fused with ureteric bud organoids in stable subculture respectively. The fusion efficiency and the differentiation and maturation of podocytes and distal tubules in the renal unit part after fusion were compared.

[0130] The results of the optimal culture time are as Figure 3 、 4 shown. After fusing renal organoids that were still in the NPC stage on the 8th day of differentiation with UB, co-differentiation culture could obtain a larger proportion of differentiated and mature podocytes and distal tubules. The qPCR results showed that the expression levels of NPHS1 in podocytes, SLC12A1 in the thin descending limb of the loop of Henle, and CDH1 in the distal tubule were the highest under the D8 fusion condition, and the area ratio of immunofluorescence quantitative podocytes was also the highest. Therefore, this fusion system selects renal organoids differentiated to the 8th day and fuses them with UB in stable culture.

[0131] 3. Selection of differentiation medium for co-culture after fusion

[0132] According to the above recipes of StageII and hUBCM media, two media were prepared respectively, and then the media for co-inducing the differentiation of fused renal organoids were set with different mixing ratios, from StageII:hUBCM = 1:3 to StageII:hUBCM = 4:1, and whether to add FBS (see Table 13).

[0133] Table 13 Formulation of fusion differentiation medium

[0134]

[0135] According to the above ratios, the fused renal organoids were cultured to continue differentiation, and the fusion efficiency and the differentiation and maturation of cell types in the renal unit part were compared.

[0136] The optimal medium ratio is as Figure 4 、 5 、6 shown. From the qPCR results, the expression levels of specific cell types from two lineage sources obtained by differentiation in the StageII:hUBCM = 3:1 differentiation medium were relatively high. The immunofluorescence results showed that there were more abundant podocytes in the renal unit part obtained by differentiation in the StageII:hUBCM = 3:1 differentiation medium, and there were more fusion points between UB and the distal tubule of the renal unit. Therefore, in this example, StageII:hUBCM = 3:1 was selected as the differentiation medium for fused renal organoids.

[0137] 4. Continue to induce differentiation and maturation after fusion

[0138] Based on the experimental results of the optimal fusion time and the optimal continued differentiation medium obtained from the above experiments, the StageII: hUBCM = 3:1 medium was used to continue inducing the differentiation of the renal organoids after the fusion of day8 renal unit organoids and ureteric bud organoids. After micromanipulation overnight, the two fused organoids were wrapped with 10 μL of Matrigel and transferred to a well of a new ultra-low adhesion U-bottom 96-well plate. After the Matrigel solidified, 100 μL of the StageII: hUBCM = 3:1 medium was added to each well to continue inducing the differentiation of the fused organoids.

[0139] After fusion, the samples were collected at 5 days, 8 days, 10 days, and 13 days of continued differentiation to identify the fusion status of the renal unit and ureteric bud and the maturation status of their respective lineage development after fusion.

[0140] The results are as Figure 8 shown. From the immunofluorescence staining comparison results of the continued differentiation time of the fused renal organoids, the renal unit organoids fused at day8. After fusion, they were continued to be differentiated for 5 days, 8 days, 10 days, and 13 days. After 8 days of continued differentiation, the differentiation of both the renal unit and ureter lineages was in a relatively mature state. If the differentiation time was too short, the podocytes in the renal unit part were not rich enough, and if the differentiation time was longer, it would lead to apoptosis of a large number of cell types such as glomerular podocytes. Therefore, in this example, the fused mature renal organoids were obtained by continuing the differentiation culture for 8 days after fusion.

[0141] Example 4 Characterization and Detection of Human Renal Organoid Samples

[0142] 1. Optimized Fusion Method

[0143] 1) Micromanipulation: Prepare kidney unit organoids differentiated from hiPSCs on the 8th day; for convenient observation, use ureteric bud organoids obtained from the stable passaged and amplified H1 differentiated after editing in our laboratory and stably expressing GFP protein; Fusion kidney organoid medium StageII: hUBCM = 3:1 + 10 μM Y-27632. Under a stereomicroscope, pick up a 3D-cultured kidney unit organoid, manually dissect a small piece of ureteric bud organoid cultured for 7 - 10 days with a sterile needle and place it beside. Hold / stabilize the kidney unit organoid with a pair of fine dissecting forceps from one side, then pierce a small hole in the kidney unit organoid with a sterile needle from the other side, and carefully push the small piece of ureteric bud organoid into the gap with the needle. All these operations are carried out in a 10 μL droplet of fusion kidney organoid medium in a 10 cm cell culture dish containing 10 μM Y-27632. Carefully place the cultured dish after this operation into the cell incubator. After overnight culture, the kidney unit organoid will slowly and autonomously wrap the embedded ureteric bud organoid, and the ureteric bud and the kidney unit will fuse into a whole.

[0144] 2) Continue to induce differentiation and maturation after fusion: Wrap the two fused organoids with 10 μL Matrigel, transfer to a low-adhesion U-bottom 96-well plate. After the Matrigel solidifies, add 100 μL of StageII: hUBCM = 3:1 medium to each well to continue to induce differentiation of the fused organoid, and collect samples after 8 days.

[0145] 2. For gene transcriptional level expression analysis

[0146] Aspirate an appropriate amount of fused human kidney organoids (about 50 per sample) into a 1.5 mL centrifuge tube; aspirate and discard the culture medium supernatant, wash with DPBS, and aspirate and discard the DPBS; add 0.75 mL of TRIzol reagent, transfer to a -80 °C refrigerator for storage until RNA extraction.

[0147] 2.1 Cell lysis and RNA extraction

[0148] (1) After thawing the samples, add 2 2-mm steel beads to each collected fused human kidney organoid sample tube, place in a pre-cooled tissue grinder, grind at 40 Hz for 1 min, and let stand at room temperature for 5 min.

[0149] (2) Add 0.2 times the volume of 1,3-bromochloropropane, shake vigorously for 15 s, and place on ice for 15 min.

[0150] (3) Centrifuge at 4 °C, 12000×g for 12 min.

[0151] (4) Aspirate the upper aqueous phase into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, invert and mix well for 15 s, and place at -20 °C for 10 min.

[0152] (5) Centrifuge at 4℃, 12000×g for 10 min.

[0153] (6) Discard the supernatant, add 1.0 mL of 75% ethanol prepared with RNase-free H2O, invert the tube up and down, centrifuge at 4℃, 7500×g for 5 min, aspirate and discard the ethanol, and repeat this step 2 times.

[0154] (7) After aspirating and discarding the ethanol, air-dry at room temperature for 5 min until the ethanol has completely evaporated and the precipitate becomes transparent.

[0155] (8) Add 30 - 50 μL of RNase-free H2O to dissolve the precipitate and place it on ice.

[0156] (9) After measuring the concentration of the obtained RNA with SPECTRA MAX 190, perform reverse transcription or transfer it to a -80℃ refrigerator for storage.

[0157] 2.2 Reverse Transcription

[0158] Use GoScript TM Reverse Transcription Mix, Oligo(dT) Kit:

[0159] (1) Take 1.0 μg of RNA for each sample in a PCR tube and supplement with Nuclease-Free Water to a total volume of 10.0 μL.

[0160] (2) After preheating at 70℃ for 5 min on a PCR instrument, quickly place it on ice for 5 min. Centrifuge briefly in a microcentrifuge for 5 s and place it on ice.

[0161] (3) Perform the operation on ice and prepare the Reverse Transcription Mix (for 1 reaction system) according to the following composition:

[0162] Nuclease-Free Water 4.0 μL;

[0163] GoScript TM Reaction Buffer;

[0164] Oligo(dT) 4.0 μL;

[0165] GoScript TM Enzyme Mix 2.0 μL;

[0166] Total volume 10.0 μL.

[0167] (4) After mixing 10.0 μL of the RNA sample and 10.0 μL of the Reverse Transcription Mix evenly, place them in a PCR instrument and perform reverse transcription according to the set program. Reverse transcription program: 25°C for 5 min, 42°C for 60 min, 70°C for 15 min, 4°C forever. (5) After the reaction is completed, store the obtained cDNA sample at -20°C.

[0168] 2.3 Real-time fluorescence quantitative PCR

[0169] Use the SsoAdvanced Universal SYBR Green Supermix kit to perform real-time fluorescence quantitative PCR (qRT-PCR) detection:

[0170] (1) Dilute the cDNA sample obtained by reverse transcription at a ratio of 1:20 and use it as the template for qRT-PCR. The template amount of single-well cDNA is 10.0 ng.

[0171] (2) For each sample, make 3 replicate wells for different genes. The reaction system for each well is as follows:

[0172] Primer (F / R each 2.5 μM) 0.8 μL;

[0173] cDNA (2.5 ng / μL) 4.0 μL;

[0174] Nuclease-Free Water 0.2 μL;

[0175] 2×SYBR Green 5.0 μL;

[0176] Total volume 10.0 μL.

[0177] (3) Adopt the Standard reaction program of the Applied Biosystems StepOnePlus real-time fluorescence quantitative PCR instrument: 95°C for 10 min, 95°C for 15 s, 60°C for 1 min, 40 cycles

[0178] During detection, generally use GAPDH as the internal reference. Use the ΔΔCt method to process the experimental results.

[0179] 3. Immunofluorescence staining analysis

[0180] Absorb an appropriate amount of fused human kidney organoids (about 15 per sample) into a 24-well plate; aspirate the culture medium supernatant, wash with DPBS, and aspirate the DPBS; add 1.0 mL / well of 4% paraformaldehyde and fix the fused human kidney organoids at room temperature for about 20 min; aspirate the 4% paraformaldehyde, wash with DPBS multiple times, and aspirate the DPBS; add 1.0 mL / well of 30% sucrose solution and dehydrate at 4°C in the refrigerator for 24 h; transfer the fused human kidney organoids into an embedding mold, aspirate the excess solution, embed with OCT embedding medium, and then transfer to a -20°C refrigerator for storage until frozen sectioning.

[0181] 3.1 Frozen Sectioning

[0182] (1) Install the microtome knife and set the temperature of the cryostat to -20°C. Place the embedded sample in the cryostat to equilibrate the temperature.

[0183] (2) Take out the sample block from the embedding mold and fix the frozen sample block on the specimen holder with OCT.

[0184] (3) Fix the cooled sample to the machine specimen head, use the sample fast-forward button to move the sample closer to the knife edge, and start trimming the block with the slow-forward button to adjust the plane to be cut.

[0185] (4) Adjust the anti-rolling plate, set the section thickness to 16 μm, rotate the cryostat wheel, and start collecting sections. The sections should be complete and smooth.

[0186] (5) Bring the glass slide close to the cut section and adhere the tissue to the glass slide. Generally, 3 - 5 samples are attached to one glass slide.

[0187] (6) Store the glass slides in a slide box and place them in a 4°C refrigerator for short-term storage for subsequent experiments, or place them in an -80°C refrigerator for long-term storage.

[0188] 3.2 Immunofluorescence Staining

[0189] (1) Take the glass slides out of the refrigerator and warm them to room temperature.

[0190] (2) Carefully add PBS to the sample sections from the edge of the section sample with a Pasteur pipette and wash multiple times until the OCT is completely dissolved and washed away.

[0191] (3) Block and permeabilize. Prepare the blocking and permeabilizing solution (PBS + 5% serum from the corresponding secondary antibody species source + 0.3% TritonX-100). Use an immunohistochemical PAP pen to draw a circle around the section sample on the glass slide and add an appropriate amount of the blocking and permeabilizing solution to the circle. Place the glass slide in a wet box and incubate at room temperature for 1 - 2 h.

[0192] (4) Dilute the primary antibody proportionally with a primary antibody diluent (PBS + 1% BSA + 0.3% Triton X-100). Use absorbent paper to suck out the blocking and permeabilizing solution from the edge of the sample circle, then add an appropriate amount of the primary antibody diluent, place the glass slide in a humidified chamber, and incubate overnight at 4°C. The specific antibody dilution ratio is shown in Appendix Table S2.

[0193] (5) Use absorbent paper to suck out the primary antibody diluent from the edge of the sample circle, add PBS and wash 3 times, 5 min each time.

[0194] (6) Dilute the secondary antibody corresponding to the primary antibody species (1:1000) and DAPI (1:5000) with PBS proportionally, add an appropriate amount of the secondary antibody mixture, and incubate in a humidified chamber at room temperature in the dark for 1 - 1.5 h.

[0195] (7) Use absorbent paper to suck out the secondary antibody mixture from the edge of the sample circle, add PBS and wash 3 times, 5 min each time.

[0196] (8) Use absorbent paper to suck out the liquid in the sample circle, add an appropriate amount of anti-fluorescence quenching agent when the sample is not dry. Then cover with a coverslip and detect and photograph with a two-photon laser confocal microscope Zeiss LSM 710.

[0197] 4 Single-cell RNA sequencing and analysis

[0198] 4.1 Preparation of fused human kidney organoid samples

[0199] (1) Observe under a microscope and aspirate well-fused 16-day-old fused human kidney organoids. Place about 10 kidney organoids in a 1.5 mL centrifuge tube. Aspirate the culture medium and wash with PBS.

[0200] (2) Add 1.0 mL of the digestive enzyme Accumax, place in an incubator at 37°C, take it out every 15 min and invert the centrifuge tube, and gently pipette with a 1 mL pipette tip to help digest and lyse the kidney organoids. After about 1 h, it is digested into a single-cell suspension.

[0201] (3) After digestion, centrifuge at 300×g for 3 min to remove the enzyme. Then add 1.0 mL of PBS to wash the cells, centrifuge at 300×g for another 3 min, and aspirate the PBS.

[0202] (4) Resuspend the cells with 200 μL of PBS, filter the cells through a 40 μm cell strainer, and detect the cell viability. A cell viability greater than 80% meets the requirements for subsequent library construction and sequencing.

[0203] 4.2 Single-cell RNA sequencing

[0204] The samples were sent to Novogene Tianjin Co., Ltd. for subsequent library construction and sequencing. An appropriate amount of cell suspension was loaded onto the Chromium microfluidic chip, and 10,000 target cells were captured; Barcodes were added using a 10x Chromium Controller (10xGenomics); the RNA of the cells with added Barcodes was reverse-transcribed, and library construction was performed using the Chromium SingleCell 30v2 kit (10x Genomics); sequencing was carried out using an Illumina NovaSeq 6000 instrument.

[0205] 4.3 Bioinformatics analysis

[0206] (1) Sequencing data quality control. The FastQC software was used to perform quality detection on the original Fastq data; the 10xGenomics built-in software Cell Ranger (v.5.0.1) was used to align the sequencing reads to the GRCh38 reference genome, and the single-cell gene expression matrix information was obtained using the Barcode of each GEM droplet and the UMI (unique molecular identity) of each gene.

[0207] (2) Secondary data filtering. The Seurat package based on the R language (Stuart T, Butler A, Hoffman P, et al. Comprehensive Integration of Single-Cell Data. Cell. 2019, 177(7):1888-1902.e21.) was used to filter out low-quality cells and genes, cells with fewer than 200 gene expressions in a single cell, and genes expressed in fewer than 3 cells, and some foreign cells were filtered out.

[0208] (3) Cell clustering analysis and annotation of cell populations. The Seurat package was used to perform data normalization, dimensionality reduction, clustering, differential expression gene analysis, and Marker gene screening on the data. The FindVariableFeatures function was used to screen out the top 2000 highly variable genes for downstream analysis; the NormalizedData function was used to normalize the data; the FindCluster function was used to perform clustering to obtain different cell subsets (clusters); the FindAllMarkers function was used to calculate the differential genes of different cell subsets; the cell type of each cell subset was annotated through the expression patterns of specific reference genes obtained by referring to the literature and the SingleR software; the RunUMAP function was used to perform dimensionality reduction visualization analysis and display of the expression patterns and distribution of specific genes under investigation.

[0209] (4) Integrative analysis of the integration of human kidney organoid samples and single-cell transcriptome data of human embryonic kidney. Use the R package Harmony to integrate the data of multiple samples; use the FindTransferAnchors and IntegrateData functions of the Seurat package to perform comparative analysis on the integrated data. Combine the validated marker genes reported in relevant literature (Kuppe C, Ibrahim M M, Kranz J, et al. Decoding Myofibroblast Origins in Human Kidney Fibrosis. Nature. 2021, 589(7841): 281–286., Howden SE, Wilson S B, Groenewegen E, et al. Plasticity of Distal Nephron Epithelia from Human Kidney Organoids Enables the Induction of Ureteric Tip and Stalk. Cell Stem Cell. 2020, 1–14., Stewart BJ, Ferdinand J R, Young M D, et al. Spatiotemporal Immune Zonation of the Human Kidney. Science. 2019, 365(6460): 1461–1466.) to annotate the key cell types that make up the kidney, such as podocytes (Pod), proximal tubule cells (PT), loop of Henle (LOH), distal tubule cells (DT), stromal cells, endothelial cells (Endo), and ureteric bud (UB) in the fused human kidney organoids.

[0210] 5. Experimental results

[0211] The fused organoids were identified by immunofluorescence and qPCR to have all cell types developed from two lineages, the renal unit and the ureteric bud, with the expression of the marker markers of cell types such as glomerular podocyte PODXL, distal tubule ECAD, vascular endothelium CD31, and ureteric bud GATA3. Moreover, the ureteric bud expressing GFP and the red-fluorescently labeled distal tubules of the renal unit extended alternately during the continuous differentiation after the fusion of the two types of organoids, indicating that the two types of organoids fused and grew with each other during the continuous differentiation process. Figure 9)。Single-cell sequencing analysis also demonstrated that the fused kidney organoids contained all cell types differentiated from two lineages, the nephron and the ureteric bud ( Figure 10 )。

[0212] The above results indicated that this fusion technique successfully achieved the fusion of the nephron and a single ureteric bud in vitro and their continued differentiation and maturation, which was similar to the in vivo developmental process: starting from a single ureteric bud, being surrounded by nephron precursor cells, and finally developing into a multi-lineage kidney organoid with glomeruli, proximal tubules, thin segments of the loop of Henle, distal tubules, connecting tubules, and a single ureteric bud with a single outlet. The present invention realized the in vitro culture of multi-lineage human kidney organoids, laying a foundation for the in vitro reproduction of a complete and orderly kidney organ.

Claims

1. A method for constructing multi-lineage human kidney organoids, characterized in that: 1) Construct kidney unit organoids and ureteric bud organoids respectively; 2) Create a notch on the surface of the kidney unit organoids and place fragments of the ureteric bud organoids into the notch; 3) Fuse and differentiate.

2. The method according to claim 1, characterized in that: The method for constructing the kidney unit organoids includes the following steps: 1) Cultivate induced pluripotent stem cells derived from human urine cells in BPEL medium until embryoid bodies are formed; 2) Cultivate the embryoid bodies in Stage II medium until 3D-shaped kidney unit organoids are formed.

3. The method according to claim 2, characterized in that: The BPEL medium includes IMDM, F12, PFHMII, BSA, ITS-X, αMTG, AA2P, Glutamax, CD Lipid concentrate, and PVA; The Stage II medium includes DMEM, KOSR, NEAA, Glutamax, HEPES, and PVA.

4. The method according to claim 1, characterized in that: The method for constructing the ureteric bud organoids includes the following steps: 1) Induce the differentiation of human embryonic stem cell lines using basal differentiation medium, ME medium, UB-I medium, and UB-II medium respectively, and flow-sort to obtain the + ureteric bud precursor cells of KIT; 2) Cultivate ureteric bud progenitor cells in hUBCM medium to obtain ureteric bud organoids with a diameter of 0.5 - 2 mm, and divide them into 8 - 10 fragments.

5. The method according to claim 4, characterized in that: The basic differentiation medium includes F12, Glutamax, NEAA, β-Mercaptoethanol, B-27 supplement minus vitamin A, ITS; The ME medium adds CHIR99021 and LDN-193189 on the basis of the basic differentiation medium; The UB-I medium adds FGF2, TTNPB, LDN-193189, and A83-01 on the basis of the basic differentiation medium; The UB-II medium adds FGF2, TTNPB, and LDN-193189 on the basis of the basic differentiation medium; The hUBCM medium adds LDN-193189, TTNPB, CHIR99021, JAK inhibitor, GDNF, A83-01, R-Spondin 1, FGF7, SB202190, and EGF on the basis of the basic differentiation medium.

6. The method according to claim 4, characterized in that: The differentiation time of the ME medium, UB-I medium, and UB-II medium is 1 - 2 days respectively.

7. The method according to claim 1, characterized in that: In step 3): The fusion time is 7 - 9 days; The differentiation time is 5 - 10 days.

8. The method according to claim 1, characterized in that: The medium used for fusion is hUBCM medium containing Y-27632; The medium used for differentiation is a mixed medium with a volume ratio of Stage II and hUBCM of (2.5 - 3.5):

1.

9. A multi-lineage human kidney organoid prepared by the method according to any one of claims 1 to 8.

10. Use of the method according to claims 1 to 8 and the organoid according to claim 9 in the construction of disease models, screening of therapeutic drugs, and screening of drug nephrotoxicity.

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