Name: a simplified method for generating kidney organoids from human pluripotent stem cells
Uploaded: 2021-04-13T14:00:00.000+00:00
Duration: 7 min 39 s
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 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><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&amp;nbsp;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&amp;nbsp;Sterile Aspirating Pipet 2mL</td><td>Fisher Scientific</td><td>14-955-135</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp; Cell Lifters - Cell lifter</td><td>Fisher Scientific</td><td>08-100-240</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;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>&lt;strong&gt;BME&lt;/strong&gt;. 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>&lt;strong&gt;GCDR&lt;/strong&gt;</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>&lt;strong&gt;ITSE&lt;/strong&gt;</td></tr><tr><td>KnockOut&amp;nbsp; Serum Replacement - Multi-Species</td><td>Thermo Fisher</td><td>A3181502</td><td>&lt;strong&gt;KOSR&lt;/strong&gt;. 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&amp;nbsp; 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>&lt;strong&gt;6MSP&lt;/strong&gt;</td></tr><tr><td>MP Biomedicals&amp;nbsp; 7X Cleaning Solution</td><td>Fisher Scientific</td><td>MP0976670A4</td><td>&lt;strong&gt;Tissue culture suitable detergent.&lt;/strong&gt; Make a 5% solution in water</td></tr><tr><td>mTeSR1</td><td>STEMCELL Technologies</td><td>85850</td><td>&lt;strong&gt;hPSC medium.&lt;/strong&gt;TeSR-E8, NutriStem XF, and mTeSR Plus medium have also been tested and are suitable alternatives.&amp;nbsp;</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>&lt;strong&gt;Anti-mycoplasma reagent.&lt;/strong&gt; Aliquot and freeze</td></tr><tr><td>pluriStrainer&amp;reg; 200 &amp;micro;m</td><td>Fisher Scientific</td><td>NC0776417</td><td>Cell strainer</td></tr><tr><td>pluriStrainer&amp;reg; 500 &amp;micro;m</td><td>Fisher Scientific</td><td>NC0822591</td><td>Cell strainer</td></tr><tr><td>Poly(vinyl alcohol) 87-90% hydrolyzed&amp;nbsp; (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&amp;nbsp; - 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 &lt;strong&gt;E5-ILP&lt;/strong&gt;</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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Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

4.8K 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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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 ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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 ...

Using Induced Pluripotent Stem Cell Suspension for Kidney Organoid Production

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.

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.

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

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Bioengineering

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 ...

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

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

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

Source: Burt, M. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/61299" target="_blank">Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions.</a> J. Vis. Exp. (2020)
In this video, we describe the protocol to derive human kidney podocytes from induced pluripotent stem cells through cell differentiation under defined chemical conditions. The generated podocytes can be used for nephrotoxicity testing and disease modeling.

Source: Burt, M. et al. Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions. ...