ipsc differentiation into posterior primitive streak and nephrogenic intermediate mesoderm

使用诱导多能干细胞悬液生产肾脏类器官

The kidney plays a critical role in maintaining physiological homeostasis, depending on its functional unit. Nephrons, which excrete waste products, can regulate the composition of body fluids. Chronic kidney disease (CKD), caused by hereditary mutations or other high-risk factors, will eventually progress to end-stage kidney disease (ESKD)1,2. ESKD is apparently due to the limited regeneration capacity of nephrons. Thus, renal replacement therapy is required. Directed differentiation of human iPSCs enables the in vitro generation of patient-specific 3D kidney organoids, which can be used to study kidney development, model patient-specific diseases, and perform nephrotoxic drug screening3,4.
During embryonic development, kidneys originate from the intermediate mesoderm (IM), which differentiates from the primitive streak (PS). The classical WNT signaling pathway may induce additional differentiation of IM with the coordinated participation of FGF (FGF9, FGF20) and BMP (Bmp7 signaling through JNK)5,6,7. They produce two important cell populations of nephric progenitor cells (NPC): the ureteral bud (UB), and the metanephric mesenchyme (MM), forming the collecting ducts and the nephron, respectively8,9. Each nephron consists of glomerular and tubular segments, such as the proximal and distal tubules, and the loop of Henle10,11. According to the theory mentioned above, currently published protocols mimic the signal cascades and growth factor stimulation to induce kidney organoids5,12.
Over the past several years, many protocols have been developed to differentiate human iPSCs into kidney organoids5,6,7,12. Takasato et al.7 optimized the duration of CHIR (WNT agonist) treatment before replacement by FGF9. According to their protocol, CHIR exposure for 4 days, followed by FGF9 for 3 days, is the most effective way to induce IM from iPSCs. Transwell filters were utilized as the culture format in their procedure; however, this method is difficult for beginners. Therefore, Kumar et al.13 tried to change the culture format and chose to suspend the culture. They dissociated the adherent cells on Day 7 for seeding in low adherent plates to help them assemble into embryoid bodies (EBs) that contain nephron-like structures. However, the batch effect of these methods was apparent, especially in different iPSCs. Additionally, different literature reported that the concentration of CHIR varied from 7 &#181;M to 12 &#181;M5,13,14.
We speculated that the concentration of cell density and the CHIR might affect the generation of organoids in different iPSCs, and this has been verified numerous times in our experiments. The present protocol has slightly modified the study method of Kumar et al.13 and provided users with a step-by-step procedure. The schedule and schematic of the approach are shown in Figure 1.

作者聚焦：优化 iPSC 分化以实现高效生产以生成肾脏类器官 

从诱导多能干细胞中产生悬浮液中的肾脏类器官

Our research presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells, using suspension cultural conditions. We emphasize the importance of initial cell density and WNT agonist concentration during the inducing phase, which will educate new organoid researchers. Currently, it is difficult to obtain a single high-density cell line for iPSCs.The batch impact was noticeable, particularly in various iPSC lines. Each batch's kidney organoids, may have a varied form and size. Several attempts are necessary to get the best initial cell density and concentration of CHIR for one iPSC line.We have established that the initial differentiation of the intermediary method is important for successful kidney organoid formation. Our protocol describes in detail, how to choose the optimal cell densities and CHIR concentrations during the process because different iPSC lines showed variances in cell proliferation and differentiation ability. Researchers can quickly generate the specific kidney organoids from their iPSCs.According to our protocol, by adjusting the cell density and the concentration of CHIR, it's undoubtedly beneficial for all our researchers to focus on their kidney disease. Our lab future research has to optimize the method of the protocol in disease models, regenerate new medicine and the drug screening. In recent five years, our lab will focus on the epigenetics and the cell topic drug development of various hereditary kidney disease, based on patient development, iPSC and the kidney organized disease models.

Watch this Scientific Journal Video about IPSC Differentiation into Posterior Primitive Streak and Nephrogenic Intermediate Mesoderm at JoVE.com

IPSC Differentiation into Posterior Primitive Streak and Nephrogenic Intermediate Mesoderm  

iPSC 分化为后原始条纹和肾源性中间中胚层

首先用两毫升DPBS洗涤膜基质包被的6孔板上的诱导多能干细胞或iPSC。使用移液器吸出DPBS。接下来，加入 1 毫升市售细胞分离溶液以分离 iPSC。然后将细胞放入 37 度的培养箱中 5 分钟。现在将一毫升mTeSR移液到iPSC上，确保细胞已与塑料表面分离。在室温下以 400g 离心这些细胞 3 分钟。重悬细胞沉淀，然后使用血细胞计数器计数细胞数。接下来，在涂有膜基质的 6 孔板上接种一系列细胞密度。用补充有 10 微摩尔 Rho 激酶抑制剂的 mTeSR 培养这些。将培养板在37摄氏度的二氧化碳培养箱中培养过夜。第二天，吸出mTeSR并用两毫升DPBS洗涤细胞。接下来，向细胞中加入两毫升第 1 阶段培养基。然后将细胞置于 37 摄氏度的培养箱中四天。第四天，取出第 1 阶段培养基并用 2 毫升 DPBS 洗涤细胞。然后向细胞中加入两毫升第 2 阶段培养基，然后像以前一样孵育细胞。从第0天到第4天，iPSC迅速扩增并呈现菱形或三角形。汇合度达到 90% 至 100%，并均匀累积到第七天。悬浮培养后，聚集体在第 7 天解离后自发形成肾单位结构。

ipsc-differentiation-into-posterior-primitive-streak-nephrogenic

Research

JoVE Journal

Developmental Biology

iPSC Differentiation into Posterior Primitive Streak and Nephrogenic Intermediate Mesoderm

180 次观看.  Zhejiang University School of Medicine. 首先用两毫升DPBS洗涤膜基质包被的6孔板上的诱导多能干细胞或iPSC。使用移液器吸出DPBS。接下来，加入 1 毫升市售细胞分离溶液以分离 iPSC。然后将细胞放入 37 度的培养箱中 5 分钟。现在将一毫升mTeSR移液到iPSC上，确保细胞已与塑料表面分离。在室温下以 400g 离心这些细胞 3 分钟。重悬细胞沉淀，然后使用血细胞计数器计数细胞数。接下来，在涂有膜基质的 6 孔?...

视频: IPSC Differentiation into Posterior Primitive Streak and Nephrogenic Intermediate Mesoderm  

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

This protocol presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells (iPSCs) using suspension culture conditions. The primary emphasis of this study lies in the determination of the initial cell density and the WNT agonist concentration, thereby benefiting investigators interested in kidney organoid research.

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease ...

科研

发育生物学

Begin by washing the induced pluripotent stem cells or iPSCs on the membrane matrix coated 6-well plate with two milliliters DPBS. Aspirate the DPBS using a pipette. Next, add one milliliter of the commercially available cell detachment solution to detach the iPSCs.Then place the cell into an incubator at 37 degrees for five minutes. Now pipette one milliliter of mTeSR onto the iPSCs, ensuring that the cells have separated from the plastic surface. Centrifuge these cells at 400g for three minutes at room temperature.Resuspend the cell pellet, then count the cell numbers using a hemocytometer. Next seed a range of cell densities on a 6-well plate that has been coated with a membrane matrix. Culture these with mTeSR supplemented with 10 micromolar Rho-kinase inhibitor.Culture the plates overnight in a carbon dioxide incubator set at 37 degrees Celsius. The next day aspirate the mTeSR and wash the cells with two milliliters of DPBS. Next, add two milliliters of stage 1 medium to the cells.Then place the cells in a 37 degrees Celsius incubator for four days. On day four, remove the stage 1 medium and wash the cells with two milliliters of DPBS. Then add two milliliters of stage 2 medium to the cells before incubating the cells as before.From day zero to day four, iPSCs rapidly expanded and took on rhomboid or triangular shapes. The confluence reached 90 to 100%and accumulated evenly until day seven. Upon suspension culture, the aggregates spontaneously form nephron structures after dissociating on day seven.

Generating Kidney Organoids from Induced Pluripotent Stem Cell Suspension Culture

Begin by removing stage II medium from the iPSC cell suspension on the seventh culture day. Now wash the cells with two milliliters of DPBS. Pipette one milliliter of cell detachment solution to each well.Then place the plate in an incubator at 37 degrees for five minutes. After incubation, add one milliliter of stage II medium to the cells and mix thoroughly until the cells have detached from the surface. Collect the cell suspension in a 15 milliliter tube, then centrifuge it at 400g for three minutes at room temperature.After centrifugation, discard the supernatant and resuspend the cell pellet in two milliliters of stage III medium. Mix the solution gently. Now seed the suspension into a six-well, low-adhesion plate at a one-to-three ratio.Place the plate on an orbital shaker in an incubator set at 37 degrees Celsius with 5%carbon dioxide. To remove the medium in the suspension culture, first cut off the tip of a one milliliter pipette using aseptic scissors. Then gently agitate the six-well plate to centralize the organoids.Next, aspirate the organoids with the prepped pipettes and place them in a 15 milliliter tube, letting them stand for five minutes. Aspirate the supernatant, ensuring that the organoids remain at the tube's base. Then pipette stage III medium into the plate.Pipette the mixture to resuspend the organoids. Replate the organoids back into a six-well, low-adhesion plate. Continue shaking the low-adhesion plate at 60 rotations per minute in the incubator.On the 12th day, replace the medium with stage IV medium. The kidney organoids display tubular like structures and are easily observed in bright field images after 18 days of aggregation. CD31 endothelial cells were induced.Dextran was taken into the proximal tubules of the kidney organoids.