Name: generating kidney organoids in suspension from induced pluripotent stem cells
Uploaded: 2023-09-01T13:20:00.000+00:00
Duration: 7 min 22 s
Description: Watch this Scientific Journal Video about Optimizing iPSC Differentiation for Efficient Production to Generate Kidney Organoids at JoVE.com

We are extremely grateful to all Mao and Hu Lab members, past and present, for the interesting discussions and great contributions to the project. We thank the National Clinical Research Center for Child Health for the great support. This study was financially supported by the National Natural Science Foundation of China (U20A20351 to Jianhua Mao, 82200784 to Lidan Hu), the Natural Science Foundation of Zhejiang Province of China (No. LQ22C070004 to Lidan Hu), and the Natural Science Foundation of Jiangsu Province (Grants No. BK20210150 to Gang Wang).

The iPSCs used for the present study were obtained from a commercial source. The cells were maintained with mTeSR medium on commercially available basement membrane matrix-coated plates (see Table of Materials). Table 1 contains all the medium compositions utilized in the study.
1. Plating iPSCs for differentiation and inducing posterior primitive streak (PS)
<ol>
	<li>Wash iPSCs on the membrane matrix-coated 6-well plate with 2 mL DPBS. Aspirate the DPBS using a pipette.</li>
	<li>Add 1 mL of the commercially available cell detachment solution (see Table of Materials) to detach iPSCs and incubate at 37 &#176;C for 5 min.</li>
	<li>Add 1 mL of mTeSR to iPSCs, and ensure cells have lifted off the plastic surface.</li>
	<li>Centrifuge the cells at 400 x g for 3 min at room temperature (RT). Resuspend the cell pellet and count the cell number using a hemocytometer.</li>
	<li>Seed a range of cell densities onto a 24-well- or 6-well plate coated with membrane matrix and culture with mTeSR supplemented with 10 &#181;M Rho kinase inhibitor (Y-27632) (see Table of Materials). Culture them overnight in a 37 &#176;C CO2 incubator. 
	NOTE: To induce PS, the optimal concentration of CHIR99021 and the suitable cell densities vary from different iPSC lines. Seed a range of densities (e.g., 0.6, 0.9, 1.2, 1.5, 1.8, 2.4 * 103 single cells per square centimeter) and a range of concentration of CHIR99021 from 7&#160;&#181;M-12 &#181;M. Initiate the differentiation of each density and each CHIR on the same day. Find the optimal concentration of CHIR99021 and the suitable cell densities in a 24-well plate and then expand the culture in 6-well plates. Here we provide a protocol based on a 6-well plate.</li>
	<li>Next day, aspirate the mTeSR and wash the cells with 2 mL of DPBS.</li>
	<li>Add 2 mL of stage I medium (stem-cell differentiation medium containing 8-12 &#181;M CHIR99021) (Table 1), refer to as Day 0.</li>
	<li>Culture them in a 37 &#176;C incubator for 4 days, refresh stage I medium every two days.</li>
</ol>
2. Inducing nephrogenic intermediate mesoderm (IM)
<ol>
	<li>On Day 4, remove the stage I medium and wash with 2 mL of DPBS.</li>
	<li>Add 2 mL of stage II medium to the cells (stem-cell differentiation medium containing 200 ng/mL FGF9, 1 &#181;g/mL Heparin, and 1 &#181;M CHIR99021) (Table 1).</li>
	<li>Culture them in a 37 &#176;C incubator, and refresh the medium every 2 days until Day 7.</li>
</ol>
3. Generating kidney organoids in suspension culture
NOTE: During the observation period from Day 0 to Day 7, the state of the cells is critical. If the cells exhibit successful expansion and heap up without significant cell debris, it demonstrates the successful derivation of the intermediate mesoderm (IM), indicating the readiness to proceed to the next stage. However, if the cells show signs of initial apoptosis, followed by necrosis or breakage, it may be indicative of inappropriate concentrations of CHIR99021 or cell densities.
<ol>
	<li>On Day 7, remove the stage II medium and wash the cells with 2 mL of DPBS.</li>
	<li>Add 1 mL of the cell detachment solution to each well and incubate at 37 &#176;C for 5 min until cells are refractive under phase contrast.</li>
	<li>Pipette 1 mL of stage II medium to cells, mix, and ensure the cells have lifted off from the surface.</li>
	<li>Collect the cell suspension in a 15 mL tube and centrifuge at 400 x g for 3 min at room temperature.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 2 mL of stage III medium (stem-cell differentiation medium containing 200 ng/mL FGF9, 1 &#181;g/mL Heparin, and 1 &#181;M CHIR99021 in 0.1% Methylcellulose (MC), 0.1% Polyvinylalcohol (PVA)) supplemented with 10 &#181;M Rho kinase inhibitor (Y-27632) (see Table of Materials). Mix it gently.</li>
	<li>Mix the cell suspension and seed into a 6-well low-adhesion plate in a ratio of 1:3. Put the plate on an orbital shaker (CO2 resistant, rotating at 60 rpm) in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>24 h later, remove Rho kinase inhibitor (Y-27632) and add stage III media (stem-cell differentiation medium supplement with 200 ng/mL FGF9, 1 &#181;g/mL Heparin, 1 &#181;M CHIR99021, 0.1% MC, 0.1% PVA). Culture for 2 days. Change the medium in the suspension culture by following the steps below:
	<ol>
		<li>Cut off the tip of the 1 mL pipette with aseptic scissors.</li>
		<li>Gently shake the 6-well plate to arrange the organoids in the middle of the plate.</li>
		<li>Aspirate the organoids with the prepared pipettes into a 15 mL tube and allow them to stand for 5 min.</li>
		<li>Aspirate the supernatant and leave the organoids at the bottom of the tube.</li>
		<li>Add the fresh medium to resuspend the organoids and replate back into a 6-well low-adhesion plate.</li>
	</ol>
	</li>
	<li>Continue shaking the low-adhesion plate at 60 rpm in the incubator. Change the stage III medium every two days until Day 12.</li>
	<li>On Day 12, change the medium to stage IV medium (stem-cell differentiation medium containing 0.1% MC and 0.1% PVA).</li>
	<li>Change the medium every two days until Day 25. The organoids can gradually develop into mature nephron structures from Day 12-25.</li>
</ol>
4. Immunofluorescence staining of kidney organoids
<ol>
	<li>Collect kidney organoids into a 15 mL tube and wash with 2 mL PBS.</li>
	<li>After allowing it to set for 5-10 min, fix the kidney organoids in freshly prepared 2% PFA for 20 min at 4 &#176;C.</li>
	<li>Remove the PFA and wash the organoids with 1 mL PBS containing 0.3% Triton X-100 (0.3% PBST), shake it evenly on a shaker (with a rotation speed not exceeding 60 rpm) for 8 min, allow it to stand for 5 min, and then remove the supernatant. Repeat three times. 
	NOTE: The organoids can be stored in 0.3% PBST at 4&#176; C for 2-4 weeks.</li>
	<li>Incubate the fixed organoids with 10% donkey serum in PBST (blocking buffer) (see Table of Materials) overnight at 4 &#176;C.</li>
	<li>Dilute the primary antibodies (listed in the Table of Materials) in a blocking buffer with a ratio of 1:300.</li>
	<li>Incubate the organoids with primary antibodies overnight at 4 &#176;C with agitation. 
	NOTE: Ensure that glomeruli and tubules can be stained simultaneously on one organoid.</li>
	<li>Wash the organoids with 1 mL 0.3% PBST, shake it evenly on a shaker (with a rotation speed not exceeding 60 rpm) for 8 min, allow it to stand for 5 min, and then remove the supernatant. Repeat three times.</li>
	<li>Incubate the organoids with secondary antibodies (listed in the Table of Materials) and DAPI overnight at 4 &#176;C with agitation. Then repeat step 4.7. 
	NOTE: Both secondary antibody and DAPI are diluted in blocking solution, secondary antibody (1:400) and DAPI (1:1000).</li>
	<li>After the staining, dehydrate the organoids with methanol (25%, 50%, 75%, and 100% for 5 min each), then permeate with benzyl alcohol and benzyl benzoate (BABB, 1:2 ratio) (see Table of Materials) for 5 min.</li>
	<li>Moute the cleared organoids on a glass-bottom dish (see Table of Materials) and examine them by confocal microscopy. 
	&#8203;NOTE: When imaging, choose the Petri dish at the bottom of the glass dish, and when permeating, put the BABB directly in the Petri dish to keep the bottom of the dish moist.</li>
</ol>
5. In vitro dextran uptake assay
<ol>
	<li>On Day 25, incubate the organoids with 100 &#181;g/mL of fluorescence-labeled dextran (see Table of Materials) for 4 h in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>4 hours later, switch to a fresh medium and capture live cell culture images using a wide-field fluorescence microscope.</li>
</ol>

Using Induced Pluripotent Stem Cell Suspension for Kidney Organoid Production

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	<li>Tekguc, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+organoids:+a+pioneering+model+for+kidney+diseases.">Kidney organoids: a pioneering model for kidney diseases.</a> Translational Research. 250, 1-17 (2022).</li><li>Hill, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+prevalence+of+chronic+kidney+disease+-+a+systematic+review+and+meta-analysis.">Global prevalence of chronic kidney disease - a systematic review and meta-analysis.</a> PLOS ONE. 11 (7), 0158765 (2016).</li><li>Rossi, G., Manfrin, A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+potential+in+organoid+research.">Progress and potential in organoid research.</a> Nature Reviews Genetics. 19 (11), 671-687 (2018).</li><li>Takasato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+human+embryonic+stem+cell+differentiation+towards+a+renal+lineage+generates+a+self-organizing+kidney.">Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.</a> Nature Cell Biology. 16 (1), 118-126 (2014).</li><li>Morizane, R., Lam, A. Q., Freedman, B. S., Kishi, S., Valerius, M. T., Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+organoids+derived+from+human+pluripotent+stem+cells+model+kidney+development+and+injury.">Nephron organoids derived from human pluripotent stem cells model kidney development and injury.</a> Nature Biotechnology. 33 (11), 1193-1200 (2015).</li><li>Freedman, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+kidney+disease+with+CRISPR-mutant+kidney+organoids+derived+from+human+pluripotent+epiblast+spheroids.">Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.</a> Nature Communications. 6 (1), 8715 (2015).</li><li>Takasato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+organoids+from+human+iPS+cells+contain+multiple+lineages+and+model+human+nephrogenesis.">Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.</a> Nature. 526 (7574), 564-568 (2015).</li><li>Mugford, J. W., Sipil&#228;, P., McMahon, J. A., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osr1+expression+demarcates+a+multi-potent+population+of+intermediate+mesoderm+that+undergoes+progressive+restriction+to+an+Osr1-dependent+nephron+progenitor+compartment+within+the+mammalian+kidney.">Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney.</a> Developmental biology. 324 (1), 88-98 (2008).</li><li>SaxOn, L., Sariola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+organogenesis+of+the+kidney.">Early organogenesis of the kidney.</a> Pediatric Nephrology. 1, 385-392 (1987).</li><li>Kobayashi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six2+defines+and+regulates+a+multipotent+self-renewing+nephron+progenitor+population+throughout+mammalian+kidney+development.">Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development.</a> Cell Stem Cell. 3 (2), 169-181 (2008).</li><li>Boyle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+using+Cited1-CreERT2+mice+demonstrates+that+the+cap+mesenchyme+contains+self-renewing+progenitor+cells+and+gives+rise+exclusively+to+nephronic+epithelia.">Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia.</a> Developmental Biology. 313 (1), 234-245 (2008).</li><li>Nishinakamura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+kidney+organoids:+progress+and+remaining+challenges.">Human kidney organoids: progress and remaining challenges.</a> Nature Reviews Nephrology. 15 (10), 613-624 (2019).</li><li>Kumar, S. 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The authors reported no potential conflict of interest.

A detailed protocol has been described for generating kidney organoids from iPSCs, involving minor modifications to the basal medium, initial cell density, and concentration of CHIR99021. In various experiments, the critical factors for successful kidney organoid generation were found to be the initial differentiation of the intermediate mesoderm (IM) and the cell state on Day 7. Moreover, different iPSC lines exhibited variations in cell proliferation and differentiation potential, resulting in varying optimal cell dens...

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.

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Incubator</td><td>Thermo SCIENTIFIC</td><td>Cat #370</td><td></td></tr><tr><td>Geltre LDEV-Free</td><td>Gibco</td><td>Cat #A1413202</td><td></td></tr><tr><td>Glass Bottom Culture Dishes</td><td>NEST</td><td>Cat #801002</td><td></td></tr><tr><td>Goat anti-human CUBN (1:300)</td><td>Santa Cruz Biotechnology</td><td>Cat #sc-20607</td><td></td></tr><tr><td>Heparin Solution (Cell culture supplement)</td><td>STEMCELL Technologies</td><td>Cat #07980</td><td></td></tr><tr><td>Human Recombinant FGF-9</td><td>STEMCELL Technologies</td><td>Cat #78161</td><td></td></tr><tr><td>Inverted Microscope</td><td>OLYMPUS</td><td>Cat #CKX53</td><td></td></tr><tr><td>Laser Scanning Confocal Microscope</td><td>OLYMPUS</td><td>Cat #FV3000</td><td></td></tr><tr><td>Methyl cellulose</td><td>Sigma-Aldrich</td><td>Cat #M7027</td><td></td></tr><tr><td>Micro Centrifuge</td><td>HENGNUO</td><td>Cat #2-4B</td><td></td></tr><tr><td>Mouse anti-human CD31 (1:300)</td><td>BD Biosciences</td><td>Cat #555444</td><td></td></tr><tr><td>Mouse anti-human ECAD (1:300)</td><td>BD Biosciences</td><td>Cat #610182</td><td></td></tr><tr><td>Mouse anti-human Integrin beta 1 (1:300)</td><td>Abcam</td><td>Cat #ab30394</td><td></td></tr><tr><td>Mouse anti-human MEIS 1/2/3 (1:300)</td><td>Thermo SCIENTIFIC</td><td>Cat #39795</td><td></td></tr><tr><td>Mowiol 4-88 (Polyvinylalcohol 4-88)</td><td>Sigma-Aldrich</td><td>Cat #81381</td><td></td></tr><tr><td>mTeSR1 5X Supplement</td><td>STEMCELL Technologies</td><td>Cat #85852</td><td></td></tr><tr><td>mTeSR1 Basal Medium</td><td>STEMCELL Technologies</td><td>Cat #85851</td><td></td></tr><tr><td>Nunc CryoTube Vials</td><td>Thermo SCIENTIFIC</td><td>Cat #377267</td><td></td></tr><tr><td>Others</td><td></td><td></td><td></td></tr><tr><td>Rabbit anti-human GATA3 (1:300)</td><td>Cell Signaling Technology</td><td>Cat #5852S</td><td></td></tr><tr><td>Rabbit anti-human LRP2 (1:300)</td><td>Sapphire Bioscience</td><td>Cat #NBP2-39033</td><td></td></tr><tr><td>Rabbit anti-human Synaptopodin (1:300)</td><td>Abcam</td><td>Cat #ab224491</td><td></td></tr><tr><td>Rabbit anti-human WT1 (1:300)</td><td>Abcam</td><td>Cat #ab89901</td><td></td></tr><tr><td>Rabbit anti-mouse PDGFR (1:300)</td><td>Abcam</td><td>Cat #ab32570</td><td></td></tr><tr><td>Recombinant Human Serum Albumin (rHSA)</td><td>YEASEN</td><td>Cat #20901ES03</td><td></td></tr><tr><td>Sheep anti-human NPHS1 (1:300)</td><td>R&amp;amp;D Systems</td><td>Cat #AF4269</td><td></td></tr><tr><td>STEMdiff APEL 2 Medium</td><td>STEMCELL Technologies</td><td>Cat #05275</td><td></td></tr><tr><td>Streptavidin Cy3 (1:400)</td><td>Gene Tex</td><td>Cat #GTX85902</td><td></td></tr><tr><td>Versene (1X)</td><td>Gibco</td><td>Cat #15040066</td><td></td></tr><tr><td>Y-27632 (Dihydrochloride)</td><td>STEMCELL Technologies</td><td>Cat #72304</td><td></td></tr></tbody></table>

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.

The production of IM is achieved by activating canonical WNT signaling using the GSK3 inhibitor CHIR99021, followed by FGF9/heparin. From Day 0 to Day 4, iPSCs rapidly expand and take on rhomboid or triangular shapes. The confluence reaches 90%-100% and accumulates evenly until Day 7. Upon suspension culture, the aggregates spontaneously form nephron structures after dissociating on Day 7. The kidney organoids created through suspension culture display tubular-like structures and are easily observed in bright field image...

Watch this Scientific Journal Video about Optimizing iPSC Differentiation for Efficient Production to Generate Kidney Organoids at JoVE.com

Author Spotlight: Optimizing iPSC Differentiation for Efficient Production to Generate Kidney Organoids 

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.

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 ...

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JoVE Journal

Developmental Biology

2.3K Views.  Zhejiang University School of Medicine. Ons onderzoek presenteert een uitgebreide en efficiënte methode voor het produceren van nierorganoïden uit geïnduceerde pluripotente stamcellen, met behulp van suspensiecultuuromstandigheden. We benadrukken het belang van initiële celdichtheid en WNT-agonistconcentratie tijdens de inducerende fase, die nieuwe organoïde-onderzoekers zal opleiden. Momenteel is het moeilijk om een enkele cellijn met hoge dichtheid voor iPSC's te verkrijgen.De impact van de batch was merkbaar, met nam...

Video: Auteur in de schijnwerpers: iPSC-differentiatie optimaliseren voor efficiënte productie om nierorganoïden te genereren 

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain degree. Unfortunately, their clinical applicability is hampered by the lack of a functional vasculature and consequently limited maturation in vitro. The transplantation of kidney organoids in the celomic cavity of chicken embryos induces vascularization by perfused blood vessels, including the formation of glomerular capillaries, and enhances their maturation. This technique is very efficient, allowing for the transplantation and analysis of large numbers of organoids. This paper describes a detailed protocol for the intracelomic transplantation of kidney organoids in chicken embryos, followed by the injection of fluorescently labeled lectin to stain the perfused vasculature, and the collection of transplanted organoids for imaging analysis. This method can be used to induce and study organoid vascularization and maturation to find clues for enhancing these processes in vitro and improve disease modeling.

Here, we present a detailed protocol for the transplantation of kidney organoids in the celomic cavity of chicken embryos. This method induces vascularization and enhanced maturation of the organoids within 8 days and can be used to study these processes in an efficient manner.

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Bioengineering

Generation of Human Kidney Tubuloids from Tissue and Urine

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high efficiency. Normal kidney tubuloids recapitulate many aspects of their tissue of origin. They represent distinct nephron segments - most notably of the proximal tubule, loop of Henle, distal tubules, and collecting duct - and can be used to study normal kidney physiology. Furthermore, tubuloid technology facilitates disease modeling, e.g., for infectious diseases as well as for cancer. Obtaining kidney epithelial cells for tubuloid generation is, however, dependent on leftover surgical material (such as partial) nephrectomies) or needle biopsies. The ability to grow tubuloids from urine would provide an alternative, less invasive source of healthy kidney epithelial cells. It has been previously shown that tubuloid cultures can be successfully generated from only a few milliliters of freshly collected urine. This article describes the protocols to generate and propagate ASC-derived human kidney tubuloid cultures from tissue and urine samples.

Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high ...

Biology

Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids

Source: Koch, L. S., et al. <a href="https://app.jove.com/v/64586">Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research.</a> J. Vis. Exp. (2023).
This video demonstrates an assay to generate cerebral organoids from induced pluripotent stem cells (iPSCs). Human iPSCs are induced to form embryoid bodies (EBs). The EBs are cultured over a polymer scaffold using an induction medium, promoting differentiation into neural stem cells, progenitor cells, and immature neurons. Finally, the EBs are embedded in a matrix and transferred to a maintenance medium for cerebral organoid formation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Figure 1 illustrates a schematic ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Deriving Human Kidney Podocytes from Induced Pluripotent Stem Cells: A Procedure for Directed Differentiation of Mature Kidney Podocytes from Stem Cells

After counting, resuspend the cells to a 1 times 10 to the fifth cells per milliliter of mesoderm induction medium concentration and aspirate the extracellular matrix solution from the basement membrane matrix 2-coated plates. Rinse the plates two times with warm medium and mix the human-induced pluripotent stem cell suspension with gentle pipetting.
Add 1 milliliter of cells to each well of the basement membrane matrix 2-coated 12-well plates and gently shake the plates to distribute the cells more evenly. Then, place the plate into the cell culture incubator. On days 2 to 15 of the differentiation, replace the mesoderm induction medium with 1 milliliter of intermediate mesoderm induction medium per well.
If substantial cell growth and a rapid depletion of nutrients is observed as indicated by yellowing of the medium, the volume of the intermediate mesoderm differentiation medium can be increased to 1.3 milliliters per well. On day 16 of culture, rinse the intermediate mesoderm cells with warm medium and incubate the cells with 500 microliters of 0.05% trypsin-EDTA per well for 3 minutes at 37 degrees Celsius.
When the cells begin to dissociate, scrape the cells with a cell lifter and gently mix the cells by pipetting. Stop the reaction with about 2 milliliters of trypsin neutralizing solution per well and transfer the cells to a 50-milliliter conical tube. Bring the volume up to 50 milliliters with medium and collect the cells by centrifugation.
Resuspend the pellet and podocyte induction medium at a 1 times 10 to the fifth cells per milliliter of medium concentration and add the cells to basement membrane matrix 2-coated plates. Then, gently shake the plate to help distribute the cells more evenly and place the cells into the incubator for up to 5 days.

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

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