Name: guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions
Uploaded: 2020-07-02T13:00:00
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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...

<ol>
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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. 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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.

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

Research

JoVE Journal

Bioengineering

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 ...

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 ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

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 ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...