This work was supported by the Pratt school of Engineering at Duke University, the Division of Nephrology at Duke Medical School, A Chair&#8217;s Research Award from the Department of Medicine at Duke University, and a Burroughs Wellcome Fund PDEP Career Transition Ad Hoc Award to S.M.. M.B was supported by the National Science Foundation&#8217;s Graduate Research Fellowship Program. We thank the Bursac Lab for generously providing us with the DU11 stem cell line, and the Varghese Lab at Duke University for temporarily sharing their tissue culture facility with our group. This publication is dedicated to Prof. Laura L. Kiessling, Novartis Professor of Chemistry at the Massachusetts Institute of Technology, in celebration of her 60th birthday.

1. Preparation of reagents
<ol>
	<li>Dilute thawed 5x hiPS cell culture media (CCM) supplement in hiPS cell culture basal medium to obtain a 1x solution of hiPS CCM. 
	NOTE: Frozen 5x hiPS CCM supplement requires a slow thawing process, ideally in 4 &#730;C for overnight. Aliquots of the 1x hiPS CCM can be stored for up to 6 months at -20 &#730;C.</li>
	<li>Preparation of basement membrane (BM) matrix 1-coated plates for hiPS cell culture: Thaw BM matrix 1 overnight on ice at 4 &#730;C. Once thawed, prepare aliquots with appropriate dilution factors as suggested by the manufacturer. 
	NOTE: Typically, aliquots are prepared for subsequent dilution in 25 mL of cold DMEM/F12 in a 50 mL conical tube followed by thorough mixing to completely dissolve the BM matrix 1 and avoid the formation of residual crystals.</li>
	<li>Transfer 1 mL of the BM matrix 1 solution to each well of a 6 well plate and incubate at 37 &#730;C for 1-2 h or at 4 &#730;C for a minimum of 24 h, wrapped in paraffin film. The BM matrix 1 -coated plates can be stored at 4 &#730;C for up to 2 weeks.</li>
	<li>Preparation of Basement membrane (BM) matrix 2 - coated plates: Dilute appropriate amounts of BM matrix 2 in 9 mL sterile distilled water to prepare a final concentration of 5 &#181;g/mL. Add 700 &#181;L of the BM matrix 2 solution to each well of a 12 well plate and incubate the plate at room temperature for 2 h or overnight at 4 &#730;C.</li>
	<li>Prepare 100 &#181;g/mL stock solutions each of BMP7, Activin A, and VEGF as follows: reconstitute BMP7 in sterile distilled water containing 0.1% (wt/vol) BSA and reconstitute Activin A and VEGF separately in sterile PBS containing 0.1% (wt/vol) BSA. To avoid frequent freeze-thaw cycles, prepare 100 &#181;L aliquots from each stock solution and store at -20 &#730;C for up to 6 months.</li>
	<li>Prepare a 10 mM stock solution of Y27632 by dissolving 10 mg of Y27632 in 3.079 mL of sterile distilled water. Aliquot 100 &#181;L from the stock and store at -20 &#730;C for up to 6 months.</li>
	<li>Dissolve 2 mg of CHIR99021 in 143.4 &#181;L of sterile DMSO to prepare a 30 mM stock solution. Prepare 5 &#181;L aliquots and store at -20 &#730;C for up to 1 month (or according to the manufacturer&#8217;s recommendation).</li>
	<li>Dissolve 10 mg of all-trans retinoic acid in 3.33 mL sterile DMSO. Prepare 500 &#181;L aliquots and store at -20 &#730;C for up to 6 months.</li>
</ol>
2. Preparation of culture media
<ol>
	<li>Prepare the mesoderm differentiation medium by reconstituting corresponding stock solutions to a final concentration of 100 ng/mL Activin A, 3 &#181;M CHIR99021, 10 &#181;M Y27632, and 1x B27 serum-free supplement in an appropriate volume of DMEM/12 with glutamine supplement. 
	NOTE: The mesoderm differentiation medium should be freshly prepared before differentiation steps and at a volume appropriate for the scale of the experiment (typically, 50 mL of medium is adequate for two 12 well plates).</li>
	<li>Prepare intermediate mesoderm differentiation medium by reconstituting corresponding stock solutions to a final concentration of 100 ng/mL BMP7, 3 &#181;M CHIR99021, and 1x B27 serum-free supplement in DMEM/F12 with a glutamine supplement. 
	NOTE: If needed, the medium can be supplemented with 1% (vol/vol) Penicillin-Streptomycin. Adjust the volume of the medium by using DMEM/F12 with glutamine supplement. This medium can be prepared in large batches; however, it is recommended to store in smaller aliquots (e.g., 45 mL in 50 mL conical tubes) to avoid repeated freeze-thaw cycles. This media can be stored at -20 &#730;C for up to 3 months and can be thawed at 4 &#730;C overnight prior to use.</li>
	<li>Prepare the podocyte induction medium by reconstituting to a final concentration 100 ng/mL BMP7, 100 ng/mL activin A, 50 ng/mL VEGF, 3 &#181;M CHIR99021, 1x B27 serum-free supplement, and 0.1 &#181;M all-trans retinoic acid in DMEM/F12 with glutamine supplement. Protect medium from light (e.g., by wrapping container with foil paper). 
	NOTE: This medium can be prepared in large batches and stored in the dark at -20 &#730;C for up to 3 months. Frozen aliquots should be thawed overnight at 4 &#730;C prior to use.</li>
	<li>Prepare 25 mL of trypsin neutralizing solution by adding 10% (vol/vol) heat inactivated FBS in DMEM/F12 and filter under sterile conditions.</li>
	<li>For post-differentiation maintenance of the stem cell-derived podocytes, prepare Complete Medium with podocyte maintenance media by adding the supplement to the basal medium as per the manufacturer&#8217;s guidelines, and store at 4 &#730;C for up to two weeks.</li>
</ol>
3. Feeder-free hiPS cell culture using hiPS cell culture medium
<ol>
	<li>Aspirate the residual solution of BM matrix 1 from the pre-coated plates and wash the wells 3 times&#160;with 1 to 2 mL of warmed DMEM/F12.</li>
	<li>Aspirate spent hiPS CCM from the hiPS cells and rinse the cells 3 times&#160;with warmed DMEM/F12. Add 1 mL of warm cell detachment solution and incubate for 1 min at 37 &#730;C to help dissociate the cells. Perform visual inspection of cells under a tissue culture microscope and ensure that the edges of the cell colonies appear rounded, then quickly aspirate the cell detachment solution from the cells (ensuring that the cell colonies are still attached to the plate, albeit loosely). Gently rinse cells once with DMEM/F12 to remove the cell detachment solution.</li>
	<li>Add 3 mL of hiPS CCM to the hiPS cells (in each well of a 6-well plate) that were treated with the cell detachment solution. Scrape colonies by using a cell lifter, and gently pipette cell suspension up and down to dislodge the loosely adhered cells. Wash the plate thoroughly to ensure all the cells are harvested. 
	NOTE: Use of a 5 mL pipette is recommended to avoid excess shear on the cells.</li>
	<li>Transfer 0.5 mL of the cell suspension into each well of a new BM matrix 1-coated 6-well plate containing 2 mL of hiPS CCM per well. Move the plate in figure-eight fashion to distribute cell colonies within wells and incubate at 37 &#730;C in a 5 % CO2 incubator. Refresh medium daily until the cells are ready to be passaged or used for the differentiation experiment (approximately 70 % confluency). 
	NOTE: The ideal colony size for routine passaging of iPSCs is between 200-500 &#181;m under feeder free conditions using hiPS CCM (without ROCK inhibitor). If cells are individualized during treatment with dissociation enzymes or buffers, the hiPS CCM can be supplemented with ROCK inhibitor (e.g., 10 &#181;M Y27632) to improve cell viability.</li>
</ol>
4. Differentiation of hiPS cells into mesoderm cells (days 0-2)
<ol>
	<li>While the hiPS cell cultures are in the exponential growth phase (approximately within 4 days of culture after passaging, and around 70% confluency), visually inspect for the presence of spontaneously differentiated cells within and around the edges of the colonies. If necessary, aseptically scrape-off areas of differentiation.</li>
	<li>Aspirate hiPS CCM from hiPS cells and rinse the cells 3 times&#160;with warm DMEM/F12. Incubate the cells with 1 mL of enzyme-free cell dissociation buffer for 10 min at 37 &#730;C and check for the dissociation under a microscope. Due to inherent differences between different hiPS cell lines, the actual incubation time for the cell dissociation buffer must be determined for a given cell line.</li>
	<li>Gently scrape the well with a cell lifter to dislodge loosely adhered cells and transfer the cell suspension to a 15 mL conical tube, followed by pipetting up and down several times to individualize the hiPS cells.
	<ol>
		<li>Bring cell suspension to the 15 mL volume with warm DMEM/F12 and centrifuge for 5 min at 290 x g at room temperature.</li>
		<li>Gently aspirate the supernatant and resuspend the cells with warm DMEM/F12 for another round of centrifugation to remove residual BM matrix 1 and dissociation buffer components.</li>
	</ol>
	</li>
	<li>Aspirate the supernatant and resuspend cells in 1 mL of mesoderm induction medium as described in above. Count the total number of cells using a hemocytometer or coulter counter to determine the appropriate volume of mesoderm differentiation medium necessary to achieve a concentration of 1 x 105 cells/mL.</li>
	<li>Aspirate ECM solution from the BM matrix 2-coated plates and rinse the plates twice with warm DMEM/F12. Mix the hiPS cell suspension gently by pipetting a few times. Transfer 1 mL of the cell suspension to each well of the BM matrix 2-coated 12-well plates and then gently shake the plates to distribute the cells more evenly.</li>
	<li>Incubate the plate at 37 &#730;C in a 5% CO2 incubator. Refresh the mesoderm induction medium the next day. 
	NOTE: After 2 days, hiPS cell-derived mesoderm cells would be ready for intermediate mesoderm induction.</li>
</ol>
5. Differentiation of hiPS cell-derived mesoderm cells into intermediate mesoderm (days 2-16)
<ol>
	<li>On the day 2 of the differentiation protocol, aspirate mesoderm induction medium and replenish with 1 mL per well intermediate mesoderm induction medium.</li>
	<li>Refresh medium every day to maintain an accurate threshold of growth factors and small molecules for the metabolically active cells. If there is substantial cell growth and rapid depletion of media nutrients (indicated by the yellowing of the media), the volume of the intermediate mesoderm differentiation medium can be increased to 1.3 mL per well of the 12 well plates.</li>
	<li>Culture cells for additional 14 days to obtain intermediate mesoderm cells. By day 16, these cells can be cryopreserved for later use.</li>
</ol>
6. Differentiation of hiPS cell-derived intermediate mesoderm cells into podocytes (days 16 to 21)
<ol>
	<li>Rinse the intermediate mesoderm cells with warm DMEM/F12 followed by incubation of the cells with 0.5 mL per well of 0.05 % trypsin-EDTA for 3 min at 37 &#730;C. Perform visual inspection to ensure that the cells are beginning to dissociate.</li>
	<li>Scrape cells using a cell lifter and pipette the cell suspension several times using a 1,000 &#181;L pipette tip to obtain individualized (or small clumps of) cells. 
	NOTE: At this stage, ensure that the cells are fully dissociated as aggregated cells may fail to acquire a terminally differentiated phenotype within the timeline of the protocol.</li>
	<li>Add about 2 mL per well of trypsin neutralizing solution to stop the activity of trypsin.</li>
	<li>Transfer cells to a 50 mL conical tube and bring the volume up to 50 mL using DMEM/F12, and then centrifuge the cell suspension for 5 min at 201 x g at room temperature.</li>
	<li>Aspirate the supernatant and resuspend cells in the podocyte induction medium. For optimal results, ensure a final seeding density of approximately 100,000 cells/well of a 12 well plate. Add the cell suspension to the BM matrix 2-coated plates and gently shake the plate to help distribute the cells more evenly.</li>
	<li>Incubate cells at 37 &#730;C and 5% CO2 and refresh medium daily for up to 5 days to obtain podocytes by day 21. 
	NOTE: The resulting hiPS cell-derived podocytes can be maintained in culture for 2-4 additional weeks by using complete medium with podocyte maintenance media. Once in podocyte maintenance media, the cells can be fed every other day and may be used for subsequent studies or downstream analyses.</li>
</ol>

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S.M. is an author on a patent pending for methods for the generation of kidney podocytes from pluripotent stem cells (US patent application 14/950859). The remaining authors declare that they have no competing interests.

In this report, we describe a protocol for the generation of kidney glomerular podocytes from hiPS cells. The hiPS cell-derived podocytes exhibit morphological and molecular features associated with the mature kidney podocyte phenotype13. In previous publications, we showed that the hiPS cell-derived podocytes can mimic the structure and selective filtration function of the kidney glomerulus when co-cultured with glomerular microvascular endothelial cells in a perfusable microfluidic organ-on-a-ch...

Advances in human pluripotent stem cell culture are poised to revolutionize regenerative medicine, disease modeling, and drug screening by providing researchers with a renewable, scalable source of biological material that can be engineered to obtain almost any cell type within the human body1. This strategy is especially useful for deriving specialized and functional cell types that would otherwise be difficult to obtain. Human induced pluripotent stem (hiPS) cells2,3,4,5 are particularly attractive due to their somatic cell origin and the potential they represent for personalized medicine. However, developing methods to derive other cell lineages from hiPS cells remain challenging due to the frequent use of poorly defined culture conditions which leads to low efficiency and non-specific generation of heterogenous cell populations6,7.
Presented here is a method for the derivation of mature kidney podocytes from hiPS cells with specificity and high efficiency under chemically defined conditions. By considering the roles of multiple factors within the cellular microenvironment, a stem cell differentiation strategy was developed that involved the optimization of soluble factors presented in the cell culture medium as well as insoluble factors, such as extracellular matrix components or adhesive substrates. Given the importance of integrin signaling in podocyte development and function, the expression of integrin receptors on the cell surface was initially examined. &#946;1 integrins were highly expressed not only in hiPS cells, but also in their derivatives including mesoderm and intermediate mesoderm cells8,9,10. Subsequent experiments confirmed that ligands that bind to &#946;1 integrins (including laminin 511 or laminin 511-E8 fragment) support the adhesion and differentiation of hiPS cells into podocytes when used in conjunction with the soluble inductive media described below.
Induction of cell lineage commitment was initiated by first confirming that hiPS cells cultured on the laminin-coated surfaces for two days in the presence of a medium containing Activin A, CHIR99021, and Y27632 Rock inhibitor can differentiate into cells that express the early mesoderm markers HAND1, goosecoid, and brachyury8,11. Treatment of the mesoderm cells for 14 days with a medium supplemented with bone morphogenetic protein 7 (BMP-7) and CHIR99021 enabled the derivation of intermediate mesoderm cells that expressed the nephron-progenitor cell markers Wilm&#8217;s Tumor 1 (WT1), odd-skipped related protein 1 (OSR1)8,11, and paired box gene 2 protein (PAX2)12. To derive the mature kidney glomerular podocytes, the intermediate mesoderm cells were treated for 4&#8211;5 days with a novel medium consisting of BMP-7, Activin A, vascular endothelial growth factor (VEGF), all-trans retinoic acid, and CHIR99021. Flow cytometry and immunostaining were used to confirm that &#62;90% of the resulting cells exhibited the molecular, morphological, and functional characteristics of the mature kidney podocyte8,11,13. These characteristics include the development of primary and secondary foot processes; the expression of podocyte lineage-specific genes including SYNPO, PODXL, MAF, EFNB28 and the expression of proteins including podocin, nephrin, and WT114,15,16. Additionally, it was found that the hiPS cell-derived podocytes can be maintained in culture for up to four weeks in vitro by using a commercially available medium8,11 which provides an additional flexibility in the timing of downstream experiments. For more information regarding the flow cytometry panels used for determining the purity of the hiPS-podocytes, please refer to our previous publication11.

<table><tbody><tr><td>&lt;strong&gt;Cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DU11 human iPS cells</td><td></td><td></td><td>The DU11(Duke University clone #11) iPS cell line was generated at the Duke University iPSC Core Facility and provided to us by the Bursac Lab at Duke University. This line has been tested for mycoplasma and was last karyotyped in July 2019 by our lab, and found to be karyotypically normal.</td></tr><tr><td>&lt;strong&gt;Growth Factors and Media Supplements&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>All-trans retinoic acid (500 mg)</td><td>72262</td><td>Stem Cell Technologies</td><td></td></tr><tr><td>B27 serum-free supplement</td><td>17504044</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>CHIR99021</td><td>04-0004</td><td>Stemgent</td><td>May show lot-to-lot variation</td></tr><tr><td>Complete Medium Kit with CultureBoost-R</td><td>4Z0-500-R</td><td>Cell Systems</td><td>Podocyte maintenance media</td></tr><tr><td>DMEM/F12</td><td>12634028</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>DMEM/F12 with GlutaMAX supplement</td><td>10565042</td><td>Thermo/Life Technologies</td><td>DMEM/F12 with glutamine supplement</td></tr><tr><td>Heat-inactivated FBS</td><td>10082147</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>Human activin A</td><td>PHC9564</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>Human BMP7</td><td>PHC9544</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>Human VEGF</td><td>PHC9394</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>mTeSR1 medium</td><td>05850</td><td>Stem Cell Technologies</td><td>hiPS cell culture media (CCM)</td></tr><tr><td>Penicillin&amp;ndash;streptomycin, liquid (100&amp;times;)</td><td>15140-163</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>Y27632 ROCK inhibitor</td><td>1254</td><td>Tocris</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488&amp;ndash; and Alexa Fluor 594&amp;ndash;conjugated secondary antibodies</td><td>A32744; A32754; A-11076; A32790</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>Brachyury(T)</td><td>ab20680</td><td>Abcam</td><td></td></tr><tr><td>Nephrin</td><td>GP-N2</td><td>Progen</td><td></td></tr><tr><td>OCT4</td><td>AF1759</td><td>R&amp;amp;D Systems</td><td></td></tr><tr><td>PAX2</td><td>71-6000</td><td>Invitrogen</td><td></td></tr><tr><td>WT1</td><td>MAB4234</td><td>Millipore</td><td></td></tr><tr><td>&lt;strong&gt;ECM Molecules&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>iMatrix-511 Laminin-E8 (LM-E8) fragment</td><td>N-892012</td><td>Iwai North America</td><td>Basement membrane (BM) matrix 2</td></tr><tr><td>Matrigel hESC-qualified matrix, 5-mL vial</td><td>354277</td><td>BD Biosciences</td><td>Basement membrane (BM) matrix 1. May show lot-to-lot variation</td></tr><tr><td>&lt;strong&gt;Enzymes and Other Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Accutase</td><td>A1110501</td><td>Thermo/Life Technologies</td><td>Cell detachment solution</td></tr><tr><td>BSA</td><td>A9418</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>D2438</td><td>Sigma-Aldrich</td><td>DMSO is toxic. Should be handled in chemical safety hood</td></tr><tr><td>Enzyme-free cell dissociation buffer, Hank&amp;rsquo;s balanced salt</td><td>13150016</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>Ethanol solution, 70% (vol/vol), biotechnology grade</td><td>97065-058</td><td>VWR</td><td>Ethanol is flammable and toxic</td></tr><tr><td>FBS</td><td>431097</td><td>Corning</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>28906</td><td>Thermo/Life Technologies</td><td>PFA should be handled in a chemical fume hood with proper personal protection equipment, including gloves, lab coat, and safety eye glasses. Avoid inhalation and contact with skin.</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>14190-250</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>Sterile Distilled Water</td><td>15230162</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>Triton X-100</td><td>97062-208</td><td>VWR</td><td></td></tr><tr><td>Trypsin-EDTA, 0.05%</td><td>25300-120</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aspirating pipettes, individually wrapped</td><td>29442-462</td><td>Corning</td><td></td></tr><tr><td>Avanti J-15R Centrifuge</td><td>B99516</td><td>Beckman Coulter</td><td></td></tr><tr><td>Conical centrifuge tube, 15 mL</td><td>352097</td><td>Corning</td><td></td></tr><tr><td>Conical centrifuge tube, 50 mL</td><td>352098</td><td>Corning</td><td></td></tr><tr><td>Cryoboxes</td><td>3395465</td><td>Thermo/Life Technologies</td><td>For storing frozen aliquots</td></tr><tr><td>EVOS M7000</td><td>AMF7000</td><td>Thermo/Life Technologies</td><td>Flourescent microscope used to acquire images of fixed and stained iPS cells and their derivatives</td></tr><tr><td>Hemocytometer</td><td>100503-092</td><td>VWR</td><td></td></tr><tr><td>Heracell VIOS 160i CO2 incubator</td><td>51030403</td><td>Thermo/Life Technologies</td><td>For the routine culture and maintenace of iPS cells and their derivatives</td></tr><tr><td>Inverted Zeiss Axio Observer equipped with AxioCam 503 camera</td><td>491916-0001-000(microscope) ; 426558-0000-000(camera)</td><td>Carl Zeiss Microscopy</td><td>Used to acquire phase contrast images of live iPS cells and their derivatives at each stage of podocyte differentiation</td></tr><tr><td>Kimberly-Clark nitrile gloves</td><td>40101-346</td><td>VWR</td><td></td></tr><tr><td>Kimwipes, large</td><td>21905-049</td><td>VWR</td><td></td></tr><tr><td>Kimwipes, small</td><td>21905-026</td><td>VWR</td><td></td></tr><tr><td>P10 precision barrier pipette tips</td><td>P1096-FR</td><td>Denville Scientific</td><td></td></tr><tr><td>P100 barrier pipette tips</td><td>P1125</td><td>Denville Scientific</td><td></td></tr><tr><td>P1000 barrier pipette tips</td><td>P1126</td><td>Denville Scientific</td><td></td></tr><tr><td>P20 barrier pipette tips</td><td>P1121</td><td>Denville Scientific</td><td></td></tr><tr><td>P200 barrier pipette tips</td><td>P1122</td><td>Denville Scientific</td><td></td></tr><tr><td>Serological pipette, 10 mL, individually wrapped</td><td>356551</td><td>Corning</td><td></td></tr><tr><td>Serological pipette, 25 mL, individually wrapped</td><td>356525</td><td>Corning</td><td></td></tr><tr><td>Serological pipette, 5 mL, individually wrapped</td><td>356543</td><td>Corning</td><td></td></tr><tr><td>Steriflip, 0.22 &amp;mu;m, PES</td><td>SCGP00525</td><td>EMD Millipore</td><td></td></tr><tr><td>Sterile Microcentrifuge Tubes</td><td>1138W14</td><td>Thomas Scientific</td><td>For aliquoting growth factors</td></tr><tr><td>Tissue culture&amp;ndash;treated 12-well plates</td><td>353043</td><td>Corning</td><td></td></tr><tr><td>Tissue culture&amp;ndash;treated six-well plates</td><td>353046</td><td>Corning</td><td></td></tr><tr><td>VWR white techuni lab coat</td><td>10141-342</td><td>VWR</td><td></td></tr><tr><td>Wide-beveled cell lifter</td><td>3008</td><td>Corning</td><td></td></tr></tbody></table>

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.

The goal of this protocol was to demonstrate that mature human podocytes can be derived from hiPS cells under chemically defined conditions. The data presented in this manuscript were generated by using the DU11 hiPS cell line17, which were first tested for, and found to be free of mycoplasma. Chromosomal analysis was also performed, and the cells were found to be karyotypically normal. Starting with the undifferentiated DU11 hiPS cells, the differentiation strategy (Figure 1<...

Watch this Scientific Journal Video about Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions at JoVE.com

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

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 ...

guided-differentiation-mature-kidney-podocytes-from-human-induced

Research

JoVE Journal

Bioengineering

4.5K Views.  Duke University. Hier wordt een chemisch gedefinieerd protocol gepresenteerd voor de afleiding van menselijke nierpodocyten uit geïnduceerde pluripotente stamcellen met een hoge efficiëntie (>90%) en onafhankelijk van genetische manipulaties of subpopulatieselectie. Dit protocol produceert het gewenste celtype binnen 26 dagen en kan nuttig zijn voor nefrotoxiciteitstests en ziektemodellering.

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

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

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

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

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

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

Developmental Biology

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

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 ...

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 ...

Using Skin Biopsies to Generate Patient-Derived Podocytes

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

Author Spotlight: Generation of Patient-Derived Podocytes from Skin Biopsies  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...

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/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. ...

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

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