Name: a simplified method for generating kidney organoids from human pluripotent stem cells
Uploaded: 2021-04-13T14:00:00
Description: Watch this Scientific Journal Video about A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells at JoVE.com

This research was funded by the National Institutes of Health R01 DK069403, UC2 DK126122 and P30-DK079307 and ASN Foundation for Kidney Research Ben J. Lipps Research Fellowship Program to AP.

All experiments using hPSCs were performed in compliance with institutional guidelines, and were carried out in a Class II biosafety hood with appropriate personal protective equipment. All reagents are cell culture-grade unless stated otherwise. All cultures are incubated at 37 &#176;C, 5% CO2 air atmosphere. At all stages of the assay, embryoid bodies or kidney organoids can be collected, and fixed or prepared for analysis. The hPSC lines used to generate this data have been fully characterized and published...

<ol>
	<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>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, 8715 (2015).</li><li>Morizane, 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=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>Taguchi, 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=Redefining+the+in+vivo+origin+of+metanephric+nephron+progenitors+enables+generation+of+complex+kidney+structures+from+pluripotent+stem+cells.">Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.</a> Cell Stem Cell. 14 (1), 53-67 (2013).</li><li>Przepiorski, 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=A+simple+bioreactor-based+method+to+generate+kidney+organoids+from+pluripotent+stem+cells.">A simple bioreactor-based method to generate kidney organoids from pluripotent stem cells.</a> Stem Cell Reports. 11 (2), 470-484 (2018).</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 Communication. 6, 8715 (2015).</li><li>Morizane, 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=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>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>Taguchi, A., 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=Higher-order+kidney+organogenesis+from+pluripotent+stem+cells.">Higher-order kidney organogenesis from pluripotent stem cells.</a> Cell Stem Cell. 21 (6), 730-746 (2017).</li><li>Uchimura, K., Wu, H., Yoshimura, Y., Humphreys, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pluripotent+stem+cell-derived+kidney+organoids+with+improved+collecting+duct+maturation+and+injury+modeling.">Human pluripotent stem cell-derived kidney organoids with improved collecting duct maturation and injury modeling.</a> Cell Reports. 33 (11), 108514 (2020).</li><li>Howden, S. E., Little, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+kidney+organoids+from+human+pluripotent+stem+cells+using+defined+conditions.">Generating kidney organoids from human pluripotent stem cells using defined conditions.</a> Methods in Molecular Biology. 2155, 183-192 (2020).</li><li>Tanigawa, 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=Activin+is+superior+to+BMP7+for+efficient+maintenance+of+human+iPSC-derived+nephron+progenitors.">Activin is superior to BMP7 for efficient maintenance of human iPSC-derived nephron progenitors.</a> Stem Cell Reports. 13 (2), 322-337 (2019).</li><li>Sander, V., 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=Protocol+for+large-scale+production+of+kidney+organoids+from+human+pluripotent+stem+cells.">Protocol for large-scale production of kidney organoids from human pluripotent stem cells.</a> STAR Protocols. 1 (3), 100150 (2020).</li><li>Ekblom, P., Thesleff, I., Miettinen, A., Saxen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis+in+a+defined+medium+supplemented+with+transferrin.">Organogenesis in a defined medium supplemented with transferrin.</a> Cell Differentiation. 10 (5), 281-288 (1981).</li><li>Thesleff, I., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transferrin+in+branching+morphogenesis,+growth+and+differentiation+of+the+embryonic+kidney.">Role of transferrin in branching morphogenesis, growth and differentiation of the embryonic kidney.</a> Journal of Embryology and Experimental Morphology. 82, 147-161 (1984).</li><li>Freund, C., 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=Insulin+redirects+differentiation+from+cardiogenic+mesoderm+and+endoderm+to+neuroectoderm+in+differentiating+human+embryonic+stem+cells.">Insulin redirects differentiation from cardiogenic mesoderm and endoderm to neuroectoderm in differentiating human embryonic stem cells.</a> Stem Cells. 26 (3), 724-733 (2008).</li><li>Nishikawa, 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=An+optimal+serum-free+defined+condition+for+in+vitro+culture+of+kidney+organoids.">An optimal serum-free defined condition for in vitro culture of kidney organoids.</a> Biochemistry and Biophysics Research Communication. 501 (4), 996-1002 (2018).</li><li>Oh, J. K., 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=Derivation+of+induced+pluripotent+stem+cell+lines+from+New+Zealand+donors.">Derivation of induced pluripotent stem cell lines from New Zealand donors.</a> Journal of the Royal Society of New Zealand. , 1-14 (2020).</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 (2013).</li><li>Lam, A. 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Previous studies have shown that the initial protocol steps are critical for intermediate mesoderm differentiation5,19,20 and, therefore, it is essential to implement a stringent medium composition at this stage. Removing undefined components such as serum, albumin, protein free hybridoma medium II from the first stage of the protocol may help to improve consistent differentiation efficiency between assays21</s...

In recent years, a number of protocols to differentiate human pluripotent stem cells into kidney organoids have been developed1,2,3,4,5. Kidney organoids have provided an important tool to aid research into new regenerative medicine approaches, model kidney-related diseases, perform toxicity studies and therapeutic drug development. Despite their wide applicability, kidney organoids have limitations such as lack of maturation, limited long-term culture capacity in vitro, and a paucity of several cell types found in the human kidney6,7,8. Recent work has focused on improving the level of organoid maturation, extending the culture periods and expanding the complexity of kidney cell populations by modifying the existing protocols9,10,11,12. In this present iteration of our established protocol5,13, we have modified the medium components in the first stage of the protocol to a serum-free base medium supplemented with insulin-transferrin-selenium-ethanolamine (ITSE), lipids, polyvinyl alcohol (E5-ILP) and CHIR99021 (Figure 1). These changes provide a fully-defined, serum-free, low-protein medium, with less components than our previous medium composition5,13 and without additional growth factors. As a result, the first stage medium is less labor-intensive to prepare than our previously published version, and may reduce batch-to-batch variability5. Previous studies have shown that both insulin and transferrin are important in serum-free culture14,15, however, high levels of insulin can be inhibitory to mesoderm differentiation16. We have maintained the low insulin levels as provided in the original protocol, and further reduced levels of KOSR (containing insulin) in second stage of the assay. In line with other protocols for kidney organoid formation, lower levels of KOSR are beneficial to maintaining a balance between proliferation and differentiation of the kidney tissue17. In addition, we have lowered the glucose concentration in our Stage II medium13.
Our method describes a setup for suspension assay of kidney organoids, yielding up to ~1,000 organoids from an initial ~60% confluent hPSC 100 mm culture plate as described in the original publication5,13. This protocol can be easily scaled up to starting with multiple 100 mm or 150 mm plates to further increase the organoid numbers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo Fisher</td>
			<td>21-985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-adherence rinsing solution</td>
			<td>STEMCELL Technologies</td>
			<td>7010</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>STEMCELL Technologies</td>
			<td>72054</td>
			<td>10 mM stock in DMSO</td>
		</tr>
		<tr>
			<td>Corning&#160;disposable spinner flasks</td>
			<td>Fisher Scientific</td>
			<td>07-201-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Ultra-Low Attachment 6-well plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-601</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Slow-Speed Stirrers</td>
			<td>Fisher Scientific</td>
			<td>11-495-03</td>
			<td>Multi plate magnetic stirrer for spinner flask culture</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>STEMCELL Technologies</td>
			<td>7923</td>
			<td>Aliquot and freeze</td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td>
			<td>Thermo Fisher</td>
			<td>11054020</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1x, no calcium, no magnesium</td>
			<td>Thermo Fisher</td>
			<td>14-190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg / Oval Stirring Bars</td>
			<td>2mag</td>
			<td>PI20106</td>
			<td></td>
		</tr>
		<tr>
			<td>Excelta General-Purpose Tweezers</td>
			<td>Fisher Scientific</td>
			<td>17-456-103</td>
			<td>Keep sterile in the cell culture hood</td>
		</tr>
		<tr>
			<td>EZBio Single Use Media Bottle, 250mL</td>
			<td>Foxx Life Sciences</td>
			<td>138-3211-FLS</td>
			<td>Used to make PVA 10%</td>
		</tr>
		<tr>
			<td>Falcon Standard Tissue Culture Dishes (100 mm)</td>
			<td>Thermo Fisher</td>
			<td>08-772E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Sterile Aspirating Pipet 2mL</td>
			<td>Fisher Scientific</td>
			<td>14-955-135</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160; Cell Lifters - Cell lifter</td>
			<td>Fisher Scientific</td>
			<td>08-100-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Multi Function 3D Rotators</td>
			<td>Fisher Scientific</td>
			<td>88-861-047</td>
			<td>Orbital shaker</td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Thermo Fisher</td>
			<td>A1413302</td>
			<td>BME. Aliquot on ice and freeze. Another suitable matrix alternative is Matrigel or Cultrex.</td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>STEMCELL Technologies</td>
			<td>7174</td>
			<td>GCDR</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher</td>
			<td>35-050-061</td>
			<td>L-glutamine supplement.</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Thermo Fisher</td>
			<td>15-630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-Ethanolamine</td>
			<td>Thermo Fisher</td>
			<td>51-500-056</td>
			<td>ITSE</td>
		</tr>
		<tr>
			<td>KnockOut&#160; Serum Replacement - Multi-Species</td>
			<td>Thermo Fisher</td>
			<td>A3181502</td>
			<td>KOSR. Aliquot and freeze</td>
		</tr>
		<tr>
			<td>Lipid Mixture 1, Chemically Defined</td>
			<td>Millipore-Sigma</td>
			<td>L0288-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliporeSigma Stericup Quick Release-GP Sterile Vacuum Filtration System 500mL</td>
			<td>Fisher Scientific</td>
			<td>S2GPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliporeSigma&#160; Stericup Quick Release-GP Sterile Vacuum Filtration System 250mL</td>
			<td>Fisher Scientific</td>
			<td>S2GPU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>MIXcontrol MTP / Variomag TELEcontrol MTP Control Unit</td>
			<td>2mag</td>
			<td>VMF 90250 U</td>
			<td></td>
		</tr>
		<tr>
			<td>MIXdrive 6 MTP / Variomag TELEdrive 6 MTP Microplate Stirring Drive</td>
			<td>2mag</td>
			<td>VMF 40600</td>
			<td>6MSP</td>
		</tr>
		<tr>
			<td>MP Biomedicals&#160; 7X Cleaning Solution</td>
			<td>Fisher Scientific</td>
			<td>MP0976670A4</td>
			<td>Tissue culture suitable detergent. Make a 5% solution in water</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td>hPSC medium.TeSR-E8, NutriStem XF, and mTeSR Plus medium have also been tested and are suitable alternatives.&#160;</td>
		</tr>
		<tr>
			<td>Nunc 50 mL Conical, Sterile Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>12-565-270</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 15mL Conical Sterile Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>12-565-268</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15-140-122</td>
			<td>Aliquot and freeze</td>
		</tr>
		<tr>
			<td>Plasmocin</td>
			<td>Invivogen</td>
			<td>ant-mpt</td>
			<td>Anti-mycoplasma reagent. Aliquot and freeze</td>
		</tr>
		<tr>
			<td>pluriStrainer&#174; 200 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>NC0776417</td>
			<td>Cell strainer</td>
		</tr>
		<tr>
			<td>pluriStrainer&#174; 500 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>NC0822591</td>
			<td>Cell strainer</td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol) 87-90% hydrolyzed&#160; (PVA)</td>
			<td>Millipore-Sigma</td>
			<td>P8136-250G</td>
			<td>10% in DPBS stirring at 98 degrees C until disolves, make in 138-3211-FLS</td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632 (ROCKi)</td>
			<td>STEMCELL Technologies</td>
			<td>72304</td>
			<td>10 mM stock in DPBS</td>
		</tr>
		<tr>
			<td>Sterile Disposable Serological Pipets&#160; - 10mL</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Serological Pipets - 25mL</td>
			<td>Fisher Scientific</td>
			<td>13-678-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Serological pipette - 5 mL</td>
			<td>Fisher Scientific</td>
			<td>13-678-12D</td>
			<td></td>
		</tr>
		<tr>
			<td>TeSR-E5</td>
			<td>STEMCELL Technologies</td>
			<td>5916</td>
			<td>Serum-free, low protein base medium for E5-ILP</td>
		</tr>
		<tr>
			<td>Variomag distriBOX 2 Distributor</td>
			<td>2mag</td>
			<td>VMF 90512</td>
			<td>If you use more than one MIXdrive</td>
		</tr>
	</tbody>
</table>

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.

In this most recent version of our protocol, kidney organoid differentiation is initiated in a defined, low protein medium. The assays are performed entirely in suspension and rely on the innate ability of hPSCs differentiation and organization for initiation of tubulogenesis. A single assay originating from a 100 mm ~60% confluent hPSC culture plate routinely yields 500-1,000 kidney organoids, as shown in our previous publication5. Due to such high numbers of organoids generated, this protocol is...

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

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 ...

This method can be performed in any laboratory equipped for cell culture experiments. Using this method, we can further our understanding of renal development and disease pathways. This is a relatively simple and inexpensive method compared to others for generating kidney organoids.The biggest challenge of this technique is maintaining high-quality human pluripotent stem cell culture. It is critical that the cells are maintained at less than 80%confluency and split regularly. Researchers new to human pluripotent stem cell culture should familiarize themselves with the stem cell lines before proceeding to differentiation.Begin the kidney organoid assay by adding two milliliters of complete E5-ILP medium into each well of a six-well ultra-low attachment plate. Culture human pluripotent stem cells, or hPSCs, in 100-millimeter tissue culture-treated plates until they reach 60%confluency. Wash the hPSCs twice with approximately eight milliliters of DPBS.Aspirate the DPBS, then add two milliliters of Dispase dropwise to cover the cells, and incubate them for six minutes at 37 degrees Celsius. Wash the cells three times with approximately 10 milliliters of DPBS. Aspirate the DPBS, then tilt the plate by 45 degrees, and scrape the cells down with a cell lifter.Wash the colonies down from the top with six milliliters of complete E5-ILP medium using a 10-milliliter serological pipette. Then, holding the pipette vertically, break up large colonies by gently pipetting up and down, taking care not to touch the sides of the culture plate. Distribute the colony clusters evenly by adding one milliliter per well into the six-well plate, then place the plate on an orbital shaker inside a 37-degree Celsius incubator.Alternatively, if an orbital shaker is not available, the cells can be statically cultured. On day two, after letting the embryoid bodies settle at the bottom of the plate, tilt the plate by 45 degrees, and aspirate the medium slowly from the top, leaving one milliliter per well. Add two milliliters of prepared complete medium per well, and return the plate back to the shaker.On day three, after aspirating the medium as previously demonstrated, collect all the embryoid bodies carefully from each well using a 10-milliliter serological pipette, and transfer them to a 50-milliliter conical tube. To collect any remaining embryoid bodies, rinse each well with one milliliter of low-glucose DMEM, and add them to the same 50-milliliter conical tube. While waiting for the embryoid bodies to settle at the bottom of the tube, add two milliliters of Stage II medium to each well of the six-well plate.Next, sieve out large embryoid bodies by placing a 200-micrometer cell strainer on top of a new 50-milliliter conical tube and pipetting all the embryoid bodies carefully over the cell strainer. Rinse the cell strainer with an additional five milliliters of DMEM to collect any embryoid bodies stuck in the strainer. At later stages, first use a 500-micrometer strainer to sieve out very large embryoid bodies, then pass the filtrate through a 200-micrometer strainer to sieve out very small embryoid bodies without tubules.Collect the correct size embryoid bodies, between 200 to 500 micrometers, by inverting the 200-micrometer strainer into a new 50-milliliter tube and washing the strainer with DPBS. After straining, allow the embryoid bodies to settle to the bottom of the conical tube, then aspirate the supernatant, and wash with approximately 10 milliliters of DMEM. Then, resuspend the embryoid bodies in six milliliters of Stage II medium.Transfer the embryoid bodies back into the six-well ultra-low attachment plate, distributing them evenly among the wells. Since most of the organoids collect at the top of the pipette, leave approximately half a milliliter at the end, then gently touch the tip to the media in each well, allowing organoids to flow into the well. Transfer the embryoid bodies into a 125-milliliter spinner flask with 45 milliliters of Stage II medium.Set the magnetic stir speed to 120 rpm, and place the flask into the incubator. To feed the embryoid bodies or kidney organoids, let them settle briefly to the bottom of the spinner flask, then lift the lid from one side arm of the flask, and place the aspirating pipette inside with the tip touching the opposite inside wall. Slowly angle the pipette down, and aspirate approximately half of the medium.Replenish with 20 milliliters of fresh Stage II medium by pipetting it through the same opening. Use long sterile forceps to carefully place one magnetic stir bar into each well of a six-well plate with embryoid bodies or kidney organoids. Place the plate onto the six-wheel magnetic stir plate, and set the speed to 120 rpm.For the magnetic stir bars to snap into position and start spinning, first set the power level to 100. Once all bars start spinning, bring the power level down to 25. Maintain the kidney organoids by changing half the medium as previously demonstrated.Immunofluorescence of paraffin sections of day 14 kidney organoids show presence of nephron segments such as renal tubules expressing hepatocyte nuclear factor-1 beta and Lotus tetragonolobus lectin, as well as podocyte clusters expressing V-maf musculoaponeurotic fibrosarcoma oncogene homolog B and nephrin. The modifications in this protocol can support expansion of endothelial cells, as confirmed by staining with platelet and endothelial cell adhesion molecule-1 at day 26 of culture. Human pluripotent stem cells need to be treated with Dispase for the appropriate amount of time, then thoroughly washed to remove all traces.If Dispase is not completely removed, it can lead to cell death and clustering of embryoid bodies. This method has helped us examine the development of congenital kidney diseases, as well as various kidney injury pathways and potential for therapeutic interventions in vitro.

Research

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