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 aliquo...

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Expression+of+Nephrin+in+Human+and+Pig.">Tissue Expression of Nephrin in Human and Pig.</a> Pediatric Research. 55, 774-781 (2004).</li><li>Guo, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WT1+is+a+key+regulator+of+podocyte+function:+reduced+expression+levels+cause+crescentic+glomerulonephritis+and+mesangial+sclerosis.">WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis.</a> Human Molecular Genetics. 11, 651-659 (2002).</li><li>Roselli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podocin+localizes+in+the+kidney+to+the+slit+diaphragm+area.">Podocin localizes in the kidney to the slit diaphragm area.</a> American Journal of Pathology. 160, 131-139 (2002).</li><li>Shadrin, I. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiopatch+platform+enables+maturation+and+scale-up+of+human+pluripotent+stem+cell-derived+engineered+heart+tissues.">Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues.</a> Nature Communications. 8, 1825 (2017).</li><li>Satoh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=aPKC&#955;+maintains+the+integrity+of+the+glomerular+slit+diaphragm+through+trafficking+of+nephrin+to+the+cell+surface.">aPKC&#955; maintains the integrity of the glomerular slit diaphragm through trafficking of nephrin to the cell surface.</a> The Journal of Biochemistry. 156, 115-128 (2014).</li><li>Mae, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+and+robust+induction+of+nephrogenic+intermediate+mesoderm+from+human+pluripotent+stem+cells.">Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells.</a> Nature Communications. 4, 1367 (2013).</li><li>Reya, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+in+stem+cells+and+cancer.">Wnt signalling in stem cells and cancer.</a> Nature. 434, 843-850 (2005).</li><li>Clevers, H., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt&#946;-Catenin+Signaling+and+Disease.">Wnt&#946;-Catenin Signaling and Disease.</a> Cell. 149, 1192-1205 (2012).</li><li>Ciampi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+podocytes+from+human+induced+pluripotent+stem+cells.">Generation of functional podocytes from human induced pluripotent stem cells.</a> Stem Cell Research. 17, 130-139 (2016).</li><li>Fuente Mora, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+Podocyte+and+Proximal+Tubule-Like+Cells+from+a+Mouse+Kidney-Derived+Stem+Cell+Line.">Differentiation of Podocyte and Proximal Tubule-Like Cells from a Mouse Kidney-Derived Stem Cell Line.</a> Stem Cells and Development. 21, 296-307 (2011).</li><li>Chuah, J. K. C., Zink, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+kidney+cells+and+organoids:+Recent+breakthroughs+and+emerging+applications.">Stem cell-derived kidney cells and organoids: Recent breakthroughs and emerging applications.</a> Biotechnology Advances. 35, 150-167 (2017).</li><li>Becker, G. J., Hewitson, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+chronic+kidney+disease:+useful+but+not+perfect.">Animal models of chronic kidney disease: useful but not perfect.</a> Nephrology Dialysis Transplantation. 28, 2432-2438 (2013).</li></ol>

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>Cells</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>Growth Factors and Media Supplements</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&#8211;streptomycin, liquid (100&#215;)</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>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488&#8211; and Alexa Fluor 594&#8211;conjugated secondary antibodies</td>
			<td>A32744; A32754; A-11076; A32790</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Brachyury(T&#65289;</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&#38;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>ECM Molecules</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>Enzymes and Other Reagents</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&#8217;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>Equipment</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 &#956;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&#8211;treated 12-well plates</td>
			<td>353043</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;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 ...

Protocols for the differentiation of stem cells into functional kidney cells remain elusive. This method produces mature podocytes with specificity and efficiency, providing new tools for studying kidney disease mechanisms. This method employs cell culture medium and extracellular matrix proteins with a chemically-defined composition to produce human iPS cell derived podocytes with a high purity without using subpopulation selection or genetic manipulation.There are limited therapeutic options for patients with kidney disease. This method has implications for modeling and understanding disease mechanisms, which could facilitate the development of novel biomarkers and therapeutics. To set up a feeder-free human iPSC culture, aspirate the residual solution of basement membrane matrix 1 from pre-coated plates and wash the wells three times with one to two milliliters per well of warmed basal medium.Aspirate the spent cell culture medium from a human iPSC culture and rinse the cells three times with warmed medium. Add one milliliter of warm cell detachment solution to each well for a one-minute incubation at 37 degrees Celsius. After confirming rounded cell colony edges under a tissue culture microscope, quickly aspirate the cell detachment solution and gently rinse the cells with cell culture medium.Add three milliliters of human iPSC culture medium to the human-induced pluripotent stem cells and use a cell lifter to scrape the colonies. Gently pipette the cell suspension to dislodge any loosely adhered cells and wash the plate thoroughly to ensure harvesting of all of the cells. Transfer 500 microliters of cells into each well of a new basement membrane matrix 1 coated six-well plate containing two milliliters of human-induced pluripotent stem cells per well and move the plate in a figure eight fashion to evenly distribute the cell colonies within the wells.Place the plate in the cell culture incubator, refreshing the medium daily until the cells reach approximately 70%confluency. If necessary, aseptically scrape off areas of differentiation before aspirating the supernatant from the wells. After washing the wells three times, dissociate the cells as demonstrated and pull the resulting cell suspensions in a 15 milliliter conical tube.After mixing by pipetting, bring the final volume in the tube to 15 milliliters with warm medium and collect the cells by centrifugation. Resuspend the pellet in fresh medium for a second centrifugation and resuspend the cells in one milliliter of mesoderm induction medium for counting. After counting, resuspend the cells to a one times 10 to the fifth cells per milliliter of mesoderm induction medium concentration and aspirate the extracellular matrix solution from the basement membrane matrix 2 coated plates.Rinse the plates two times with warm medium and mix the human-induced pluripotent stem cell suspension with gentle pipetting. Add one milliliter of cells to each well of the basement membrane matrix 2 coated 12-well plates and gently shake the plates to distribute the cells more evenly. Then place the plate into the cell culture incubator.On days 2-15 of the differentiation, replace the mesoderm induction medium with one milliliter of intermediate mesoderm induction medium per well. If substantial cell growth and a rapid depletion of nutrients is observed as indicated by yellowing of the medium, the volume of the intermediate mesoderm differentiation medium can be increased to 1.3 milliliters per well. On day 16 of culture, rinse the intermediate mesoderm cells with warm medium and incubate the cells with 500 microliters of 0.05%Trypsin EDTA per well for three minutes at 37 degrees Celsius.When the cells begin to dissociate, scrape the cells with a cell lifter and gently mix the cells by pipetting. Stop the reaction with about two milliliters of Trypsin neutralizing solution per well and transfer the cells to a 50 milliliter conical tube. Bring the volume up to 50 milliliters with medium and collect the cells by centrifugation.Resuspend the pellet in podocyte induction medium at a one times 10 to the fifth cells per milliliter of medium concentration and add the cells to basement membrane matrix 2 coated plates. Then gently shake the plate to help distribute the cells more evenly and place the cells into the incubator for up to five days. Using the culture strategy as demonstrated, the stem cells can be first differentiated into mesoderm cells that express Brachyury, followed by differentiation into PAX2 positive intermediate mesoderm cells, and eventually mature kidney glomerular podocytes.Stem cell derived podocytes stain positive for WT1, as well as for the lineage identification marker nephrin. Intriguingly, the subcellular localization of nephrin is predominantly within the podocyte foot processes and cell cytoplasm, consistent with a mature podocyte phenotype. Cells seeded at densities that significantly exceed the recommended density of one times 10 to the fifth cells per well of a 12-well plate result in large clusters of cells that lack the expected morphological phenotype of mature podocytes within the standard timeline of the protocol.Seeding at the densities recommended in this protocol, however, results in cultures of cells with the desired morphology. We recommend using the appropriate basement membrane protein for cell detachment and maintaining the correct cell seeding density for mesoderm and podocyte induction. This podocyte differentiation method can be integrated with organs-on-chips microfluidic systems or 3D bioprinting technologies to develop functional models of the human kidney for drug screening and nephrotoxicity testing.The lack of appropriate cell culture models has impeded progress in understanding kidney disease mechanisms. This protocol provides researchers with an inexhaustible source of patient-derived podocytes for disease modeling.

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Bioengineering

4.4K Görüntüleme.  Duke University. Protocols for the differentiation of stem cells into functional kidney cells remain elusive. This method produces mature podocytes with specificity and efficiency, providing new tools for studying kidney disease mechanisms. This method employs cell culture medium and extracellular matrix proteins with a chemically-defined composition to produce human iPS cell derived podocytes with a high purity without using subpopulation selection or genetic manipulation.There are limited therapeutic...