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 published18.
1. Preparing culture plates
NOTE: Approximately 1 h prior to splitting hPSCs, coat 2 x 100 mm tissue culture plates with a stem cell qualified basement membrane matrix extract (BME). One may pre-coat the plates, seal them with a paraffin film and store at 4 &#176;C according to manufacturers&#39; instructions.
<ol>
	<li>Prepare 2 x 100 mm tissue culture-treated plates (1 for kidney organoid assay, 1 to maintain the cell line) and a 15 mL conical tube in the Class II biosafety hood.</li>
	<li>Aliquot 8 mL of cold, serum-free Dulbecco&#39;s Modified Eagle Medium (DMEM) into a 15 mL conical tube and ~4 mL into each of the 100 mm plates, enough to cover the bottom of each plate with medium.</li>
	<li>Take a 100 &#181;L aliquot of BME out of the freezer (-20 &#176;C). Using a 2 mL serological pipette, take ~1 mL of cold DMEM from the 15 mL conical tube. Slowly thaw the BME aliquot by gently pipetting up and down with the cold DMEM, avoiding making bubbles. 
	NOTE: Do not let BME aliquot sit at room temperature. Use immediately.</li>
	<li>Transfer the thawed DMEM/BME back into the 15 mL conical tube with the remaining DMEM. With a 10 mL serological pipette, gently mix the diluted BME by pipetting up and down at least 8 times to evenly disperse the BME, avoiding making bubbles.</li>
	<li>Transfer 4 mL of the diluted BME into each plate with DMEM and gently swirl the plate so that the BME is evenly distributed. Incubate the coated plate for 1 h at room temperature or 30 min at 37 &#176;C. 
	NOTE: Use 50 &#181;L of BME per 100 mm plate. Use of other hPSC culture media and cell lines may require different concentrations of BME.</li>
</ol>
2. Passaging hPSCs
NOTE: For routine hPSC culture, passage cell lines at 70-80% confluency.
<ol>
	<li>Aspirate the culture medium from the hPSC plate to be passaged. Add ~ 8 mL of Dulbecco&#39;s phosphate-buffered saline (DPBS) to the hPSC plate and gently swirl to wash the cells.</li>
	<li>Aspirate DBPS and add 2 mL of gentle cell dissociation reagent (GCDR) to the 100 mm plate, drop by drop on top to cover the cells. 
	NOTE: Other dissociation reagents may also be used. Adjust accordingly.</li>
	<li>Incubate at room temperature for ~6-8 min until the colonies are breaking up and cells are refractive under phase contrast (Figure 2A). 
	NOTE: The timing may vary between cell lines. Adjust accordingly.</li>
	<li>While incubating, prepare a 50 mL conical tube. Add 16 mL of hPSC medium (8 mL per 100 mm plate) and add Rho-associated kinase inhibitor (ROCKi) to a final concentration of 5 &#181;M.</li>
	<li>Aspirate DMEM from the BME-coated plates, and add 8 mL of hPSC medium plus ROCKi to each plate.</li>
	<li>When the cells are ready (as described in point 2.3, Figure 2A), aspirate the GCDR and tilt the plate ~45&#176; towards the experimenter and scrape the cells with a cell lifter. 
	NOTE: If cells are detatching, omit aspirating GCDR and proceed.</li>
	<li>Turn the plate ~90&#176; and scrape again to lift the remaining cells. Keep the plate ~45&#176; and wash the cells down with 3 mL of hPSC medium using a 10 mL serological pipette.</li>
	<li>Gently pipette up and down to break up large clumps (no more than 2-3 times) and seed the cells at the appropriate ratio for the cell line of interest onto the prepared plates. Place the plate with the cells in the incubator and move the plate gently in figure eight motions to distribute the cells evenly. 
	NOTE: In this experiment hPSC lines were split at a ratio of 1:5, this may vary for other cell lines and conditions. Leave the plate undisturbed over night.</li>
	<li>After ~ 24 h, examine the cells for attachment. Look for small individual colonies attached. Aspirate the spent medium and replenish with 8 mL of fresh hPSC medium (no ROCKi added).</li>
	<li>Continue observing and feeding daily until the cells reach ~60% confluency to start the kidney organoid assay (usually reached 48 to 72 h post passaging). The colonies will ideally be discrete and not merging (Figure 2B). 
	NOTE: It is very important to limit the cells to no more than 80% confluency in order to maintain their pluripotency state. Confluent cultures, rough handling or higher passages may lead to unwanted spontaneous differentiation or low efficiency of kidney organoid formation.</li>
</ol>
3. Day 0 - Setting up the kidney organoid assay
<ol>
	<li>Before starting, prepare both the E5-ILP and Stage II media as per formulations (Table 1 and Table 2). 
	NOTE: The media can be stored for up to 14 days at 4 &#176;C.</li>
	<li>For one kidney organoid assay (one 100 mm culture plate is needed for one 6-well plate), prepare complete E5-ILP medium in a 50 mL conical tube: 18 mL of E5-ILP supplemented with 8 &#181;M CHIR99021 (14.4 &#181;L), 3.3 &#181;M ROCKi (6 &#181;L), 0.1 mM beta-mercaptoethanol (32.7 &#181;L).</li>
	<li>Place 2 mL of complete E5-ILP medium into each well of a 6 well ultra-low attachment plate.</li>
	<li>Wash hPSCs at ~60 % confluency (Figure 2B) twice with ~ 8 mL of DPBS. Aspirate DPBS then add 2 mL of dispase per 100 mm plate, drop by drop to cover the cells and incubate for 6 min at 37 &#176;C. 
	NOTE: After 6 min, the edges of the colonies will start to curl up (Figure 2C, red arrows) while the rest of the colony remains attached. If this is not obtained after 6 min, place the cells back into the incubator for additional 30 s. Other hPSC media and matrix may not be compatible with this timing. Laminin based BME coating is not compatible with dispase. If laminin based BME are the standard hPSC matrix, coat one of the plates in section 1 with the BME described in this method to be used for the kidney organoid assay.</li>
	<li>Wash cells 3x with ~10 mL of DPBS. Aspirate DPBS then tilt the plate ~45&#176;&#160;and scrape down with a cell lifter. 
	NOTE: Dispase is not deactivated, hence it needs to be washed out thoroughly. Do not reduce the number of washes.</li>
	<li>Wash the colonies down from the top with 6 mL of complete E5-ILP medium using a 10 mL serological pipette. Pipette up and down gently to break up any large colonies (2 or 3 times is usually enough).</li>
	<li>Distribute the colony clusters evenly by adding 1 mL per well into the 6-well plate. Place the plate on an orbital shaker (settings: orbital = 30, reciprocal = 330&#176;, vibration = 5&#176; - 2 s) that is placed in the 37 &#176;C incubator (Figure 2D). 
	&#8203;NOTE: The vibration feature is important for adequate distribution of organoids and to prevent clumping.</li>
</ol>
4. Day 2 - Feeding by half-medium change
NOTE: Within the 48 h, colony clusters will form embryoid bodies.
<ol>
	<li>Prepare the complete medium: For one 6-well plate prepare 12 mL of E5-ILP medium + 8 &#181;M CHIR99021 in a 15 mL conical tube. 
	NOTE: Beta-mercaptoethanol and ROCKi are not required.</li>
	<li>Let the embryoid bodies settle at the bottom of the plate, tilt the plate ~45&#176; then aspirate the medium slowly from the top, leave ~1 mL per well. 
	NOTE: Embryoid bodies at this stage clump rapidly. Do not leave them to settle for &#62; 5 min.</li>
	<li>Add 2 mL of prepared complete medium (section 4.1) per well. Return the plate back onto the shaker.</li>
</ol>
5. Day 3 - Transfer of embryoid bodies to Stage II medium
<ol>
	<li>Prepare a 50 mL conical tube and DMEM (low glucose). Let the embryoid bodies settle at the bottom of the plate. Tilt the plate ~45&#176;&#160;and aspirate the medium from the top slowly, leave ~1 mL per well.</li>
	<li>Collect all the embryoid bodies carefully from each well using a 10 mL serological pipette and transfer them to the 50 mL conical tube.</li>
	<li>Wash each well to collect any remaining embryoid bodies with ~ 1 mL of DMEM (low glucose) and add them to the same 50 mL conical tube.</li>
	<li>Leave the embryoid bodies to settle to the bottom of the tube, ~5 min. While waiting, add 2 mL of Stage II medium to each well of the 6-well plate. Seive out large embryoid bodies (&#62;300 &#181;m) using a 200 &#181;m cell strainer (Figure 2E).
	<ol>
		<li>Use a new 50 mL conical tube and place the 200 &#181;m cell strainer on top. Pipette all of the embryoid bodies using a 10 mL serological pipette carefully over the cell strainer.</li>
		<li>Rinse the cell strainer with an additional ~5 mL of DMEM (low glucose) to collect any embryoid bodies stuck in the cell strainer. Allow the embryoid bodies to settle to bottom of the conical tube.</li>
	</ol>
	</li>
	<li>When the embryoid bodies are settled, aspirate the supernatant and wash with ~10 mL of DMEM (low glucose).</li>
	<li>Aspirate DMEM and re-suspend the embryoid bodies in 6 mL of Stage II medium.</li>
	<li>Transfer the embryoid bodies back into the 6 well ultra-low attachment plate, distributing them evenly among the 6 wells.</li>
	<li>Carry out half medium changes as described in steps 4.2 and 4.3 every other day. 
	&#8203;NOTE: From day 3 onwards, the embryoid bodies will have a &#39;golden&#39; and smooth, spherical appearance (Figure 2F). From ~ day 6, tubule formation in individual embryoid bodies will become apparent, with increasing numbers over the following days reaching optimum numbers and growth by day 14 (Figure 2G,H). To eliminate occasional clumping forming, upon visually observing the kidney organoids, or very small embryoid bodies without tubules, sieve out the &#60;200 and large &#62;500 &#181;m organoids with a 500 and 200 &#181;m cell strainers as described in steps 5.4.1 and 5.4.2.</li>
</ol>
6. Transfer to spinner flask and feeding
NOTE: A spinner flask may be used anytime from day 3 onwards for experiments that require large numbers of organoids. Routine transfer of organoids happens in our lab between days 6-8. Please see the Discussion section for alternatives if equipment is not available.
<ol>
	<li>Transfer embryoid bodies into a 125 mL spinner flask with 45 mL of Stage II medium. Set magnetic stirrer speed to 120 rpm and place into the incubator (Figure 2I).</li>
	<li>To feed embryoid bodies or kidney organoids, let the kidney organoids settle briefly to the bottom of the spinner flask. Lift the lid from one side arm of the flask and place the aspirating pipette inside, with the tip touching the opposite inside wall.</li>
	<li>Slowly angle the aspirating pipette down and aspirate approximately half of the medium. Replenish with 20 mL of fresh Stage II medium by pipetting it through the same opening.</li>
</ol>
7. Setting up 6-well magnetic stir plate (6MSP)
NOTE: The 6MSP format may be used in place of spinner flasks if multiple conditions need to be tested. Use the 6MSP for compound or nephrotoxin treatments. This saves the amount of medium used in the second stage while maintaining nutrient availability through diffusion.
<ol>
	<li>Clean the oval magnetic stir bars in a 50 mL conical tube by washing in a tissue culture suitable detergent briefly (if never used) or soak for &#62; 1 h if previously used.</li>
	<li>Briefly wash 3x in sterile DPBS.</li>
	<li>Wash 1x for 5 min in 70% ethanol, 1x in sterile DPBS.</li>
	<li>Rinse with anti-adherence solution and wash 1x in sterile DPBS and aspirate.</li>
	<li>Carefully, using long sterile forceps place one magnetic stir bar into each well of the 6-well plate with embryoid bodies or kidney organoids.</li>
	<li>Place the plate onto the 6MSP and set the speed to 120 rpm (Figure 2J). Maintain kidney organoids with half medium change as per section 4.2 and 4.3. 
	NOTE: In order for the magnetic stir bars to snap into position and start spinning, you may need to first put the power level to 100 briefly, then once they are all spinning, bring the power level down to 25.</li>
</ol>

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Authors have nothing to disclose.

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...

Watch this Scientific Journal Video about A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells at JoVE.com

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 ...

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Research

JoVE Journal

Developmental Biology

4.5K Views.  University of Pittsburgh, School of Medicine. 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.

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

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.