Name: isogenic kidney glomerulus chip engineered from human induced pluripotent stem cells
Uploaded: 2022-11-04T13:15:00.000+00:00
Duration: 10 min 23 s
Description: Watch this Scientific Journal Video about Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells at JoVE.com

This work was supported by the Pratt school of Engineering at Duke University, the Division of Nephrology at Duke Department of Medicine, a Whitehead Scholarship in Biomedical Research, and a Genentech Research Award for S. Musah. Y. Roye is a recipient of the Duke University-Alfred P. Sloan Foundation Scholarship and William M. &#34;Monty&#34; Reichert Graduate Fellowship from Duke University&#39;s Department of Biomedical Engineering. The DU11 (Duke University clone #11) iPS cell line was generated at the Duke iPSC Core Facility and provided to us by the Bursac Lab at Duke University. The authors thank N. Abutaleb, J. Holmes, R. Bhattacharya, and Y. Zhou for technical assistance and helpful discussions. The authors would also like to thank members of the Musah Lab for helpful comments on the manuscript. The authors thank the Segura Lab for the gift of an Acuri C6 flow cytometer.

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S.M. is an inventor on a patent related to podocyte differentiation from human iPS cells. The other author has nothing to disclose.

In this report, we outline a protocol to derive vascular endothelium and glomerular epithelium (podocytes) from an isogenic human iPS cell line and the use of these cells to engineer a 3D organ-on-a-chip system that mimics the structure, tissue-tissue interface, and molecular filtration function of the kidney glomerulus. This glomerulus chip is outfitted with endothelium and glomerular epithelium that, together, provide a barrier to selectively filter molecules. 
Researchers interested in adapting this protocol should make the following considerations: first, optimization may be necessary for cell seeding depending on the inherent growth characteristics of the stem cell lines being used. Cell seeding density may vary due to intrinsic differences in human iPS cell proliferation rates. It is recommended that researchers begin with the mesoderm seeding density suggested by the protocol, and then adjust if necessary. Similarly, it is recommended that the lateral mesoderm differentiation starts with the suggested cell seeding density before adjusting as needed to achieve a viEC yield and sorting efficiency of 50% or more. If sufficient cells are not differentiated after sorting, TGF-Beta inhibitor (SB431542) can be used to prevent quiescence and help exponentially expand viECs (past passage 3). However, several cellular processes or signaling pathways are dependent on TGF-Beta (e.g., pathogenesis of hyperglycemia/diabetes, immune homeostasis); as such, it is recommended that researchers consider the effects of TGF-Beta inhibition on downstream analysis or ensure adequate testing to avoid unintended experimental outcomes. 
Second, it is important to note possible variations in reagent quality and manufacturer specifications, particularly for components acquired from vendors other than those specified by the protocol. Thus, it is recommended that the researcher test reagents from different lot numbers, suppliers, and vendors to ensure reproducibility of experiments and results. Generally, the researcher should avoid excessive exposure of the human iPS cells to dissociation enzymes in the detachment buffers as this can lead to decreased cell viability and altered molecular profile of the cells. Additionally, the podocyte induction medium must be protected from light to prevent inactivation of all trans retinoic acid. Third, during organ-on-a-chip cell culture, it is critical to avoid air bubbles in the channels of the microfluidic device when perfusing the chips. The appearance of air bubbles can be minimized or prevented by regularly inspecting the chips during cell seeding, by maintaining liquid-liquid contact at every step involving chip perfusion, and by not pushing or pulling air into the fluidic channels when using pipette tips and/or aspiration. 
Previous efforts to engineer kidney glomerulus chips with genetically matched epithelium and endothelium have relied on the use of animal-derived cells1. While these animal-derived cell lines have traditionally been used for preclinical studies, they often fail to recapitulate human physiological responses, which contribute to the high failure rate (89.5%) of in-human clinical trials18. To help overcome some of these problems, functional in vitro models that more closely recapitulate human biology are desirable. Progress has been made to develop multicellular models of the human kidney; however, the glomerulus chips employed human cells from heterogeneous, non-isogenic sources. For example, we previously established a glomerulus chip reconstituted from human iPS cell-derived podocytes and primary tissue-derived endothelium10. Studies from other research groups employed a mixture of primary cells4,9, immortalized cells3, or amniotic fluid-derived cells3,6 that limit their use for studying patient-specific responses or applications in personalized medicine. 
The protocol described herein overcomes these limitations by enabling the derivation of both vascular endothelium (viECs) and glomerular epithelium (podocytes) from the same human iPS cell line and integrating these cells into compartmentalized microfluidic organ-on-a-chip devices to model the structure and function of the kidney glomerular capillary wall in vitro. Given the unlimited self-renewal of human iPS cells, combined with their ability to differentiate into almost any cell type, this protocol also provides an avenue for continuous sourcing of human podocytes and viECs for tissue engineering and other biomedical applications. This approach for the derivations of podocytes and viECs have been reproduced in multiple patient-specific human iPS cell lines, including PGP1- and DU112,10,12,17,19, thus enabling the establishment of personalized kidney glomerulus chips from the desired patient populations. 
The strategy for differentiating podocytes within the microfluidic chips enables mechanistic study of the developing human kidney glomerulus and disease modeling. However, the study of the developing human kidney glomerulus is limited by the viECs requiring sorting to enrich for the desired population. This work could benefit from the establishment of methods for differentiation of viECs without a need for subpopulation selection. This study is also limited by the thick PDMS membrane that separates the vascular endothelium and podocyte cell layers. Future work could integrate novel biomaterials to replace the thick PDMS to better mimic the molecular and biophysical properties of the glomerular basement membrane. For example, an alternative membrane could be engineered to possess biodegradable qualities with tunable porosity and be thinner (more GBM-like) than the 50 &#181;m thick PDMS membrane used in this protocol. 
Nevertheless, the glomerulus chip produced by this protocol can be applied to study the mechanisms of debilitating kidney diseases and serve as a platform for nephrotoxicity testing and drug discovery. Because human iPS cells maintain the genetic profile of the donor and the glomerulus chip is able to model kidney disease12, novel therapeutic targets could be discovered in the future to benefit those suffering from hereditary forms of kidney disease. Additionally, patient-specific biological responses to post-transplantation drugs can be more accurately evaluated using an isogenic kidney chip such as the one described in this study. Finally, this glomerulus chip is poised for studying the effects of fluid dynamics and differential mechanical strain-such as those observed in kidney disease patients with hypertension or cardio-renal syndrome-given the relative ease of modulating the rates of fluid flow, tissue stretching, or mechanical strain. It is conceivable that this protocol could advance the current understanding of human kidney development and disease mechanisms, as well as facilitate the development of personalized therapeutics in the future.

Organ-on-a-chip devices are dynamic 3D in vitro models that employ molecular and mechanical stimulation, as well as vascularization, to form tissue-tissue interfaces that model the structure and function of specific organs. Previously established organ-on-a-chip devices that aimed to recapitulate the kidney&#39;s glomerulus (glomerulus chips) consisted of animal cell lines1 or human primary and immortalized cell lines of heterogeneous sources2,3. The use of genetically heterogeneous cell sources present variations that significantly limit the studies of patient-specific responses and genetics or mechanisms of disease4,5. Addressing this challenge hinges on the availability of isogenic cell lines originating from specific individuals with preserved molecular and genetic profiles to provide a more accurate microenvironment for engineering in vitro models2,3,6.&#160;Isogenic cell lines of human origin can now be easily generated due to advancements in human iPS cell culture. Because human iPS cells are typically noninvasively sourced, can self-renew indefinitely, and differentiate into almost any cell type, they serve as an attractive source of cells for the establishment of in vitro models, such as the glomerulus chip7,8. The glomerular filtration barrier is the primary site for blood filtration. Blood is first filtered through vascular endothelium, the glomerular basement membrane, and finally through specialized epithelium named podocytes. All three components of the filtration barrier contribute to the selective filtration of molecules. Presented here is a protocol to establish an organ-on-a-chip device interfaced with vascular endothelium and glomerular epithelium from a single human iPS cell source. While this protocol is especially useful to engineer an isogenic and vascularized chip to recapitulate the glomerular filtration barrier, it also provides a blueprint for developing other types of personalized organs-on-chips and multi-organ platforms such as an isogenic &#39;body-on-a-chip&#39; system.
The protocol described herein begins with divergent differentiation of human iPS cells into two separate lineages - lateral mesoderm and mesoderm cells, which are subsequently differentiated into vascular endothelium and glomerular epithelium, respectively. To generate lateral mesoderm cells, human iPS cells were seeded on basement membrane matrix 1-coated plates and cultured for 3 days (without media exchange) in N2B27 medium supplemented with the Wnt activator, CHIR 99021, and the potent mesoderm inducer, bone-morphogenetic 4 (BMP4). The resulting lateral mesoderm cells were previously characterized by the expression of brachyury (T), mix paired-like homeobox (MIXL), and eomesodermin (EOMES)9. Subsequently, the lateral mesoderm cells were cultured for 4 days in a medium supplemented with VEGF165 and Forskolin to induce vascular endothelial cells that were sorted out based on VE-Cadherin and/or PECAM-1 expression using magnetic-activated cell sorting (MACS). The resulting vascular endothelial cells (viEC) were expanded by culturing them on basement membrane matrix 3-coated flasks until ready to seed in the microfluidic device.
To generate mesoderm cells, human iPS cells were seeded on basement membrane matrix 2-coated plates and cultured for 2 days in a medium containing Activin A and CHIR99021. The resulting mesoderm cells were characterized by the expression of HAND1, goosecoid, and brachury (T) as described previously2,10,11. To induce intermediate mesoderm (IM) cell differentiation, the mesoderm cells were cultured for 14 days in a medium supplemented with BMP-7 and CHIR99021. The resulting IM cells express Wilm&#39;s Tumor 1 (WT1), paired box gene 2 (PAX2), and odd-skipped related protein 1 (OSR-1)2,10,11.
A two-channel polydimethylsiloxane (PDMS)-based microfluidic chip was designed to recapitulate the structure of the glomerular filtration barrier in vitro. The urinary channel is 1,000 &#181;m x 1,000 &#181;m (w x h) and the capillary channel dimension is 1,000 &#181;m x 200 &#181;m (w x h). Cyclic stretching and relaxation cycles were facilitated by the hollow chambers present on each side of the fluidic channels. Cells were seeded onto a flexible, PDMS membrane (50 &#181;m thick) that separates the urinary and capillary channels. The membrane is outfitted with hexagonal pores (7 &#181;m diameter, 40 &#181;m apart) to help promote intercellular signaling (Figure 1A)2,12. Two days before IM induction was complete, the microfluidic chips were coated with basement membrane matrix 2. viECs were seeded into the capillary channel of the microfluidic chip using Endothelial Maintenance medium 1 day before IM induction was complete, and the chip was flipped upside down to enable cell adhesion on the basal side of the ECM-coated PDMS membrane. On the day IM induction was completed, the cells were seeded into the urinary channel of the microfluidic chip using a medium supplemented with BMP7, Activin A, CHIR99021, VEGF165, and all trans Retinoic Acid to induce podocyte differentiation within the chip. The following day, the media reservoirs were filled with Podocyte Induction medium and Endothelial Maintenance medium, and 10% mechanical strain at 0.4 Hz and fluid flow (60 &#181;L/h) were applied to the chips.
The cellularized microfluidic chips were cultured for 5 additional days using Podocyte Induction medium (in the urinary channel) and Endothelial Maintenance medium (in the vascular channel). The resulting kidney glomerulus chips were cultured for up to 7 additional days in maintenance media for both the podocyte and endothelial cells. The differentiated podocytes positively expressed lineage-specific proteins, including podocin and nephrin13,14, while viECs positively expressed the lineage identification proteins PECAM-1 and VE-Cadherin, all of which are essential molecules for maintaining the integrity of the glomerular filtration barrier15,16. The podocytes and viECs were both found to secrete the most abundant glomerular basement membrane protein, collagen IV, which is also important for tissue maturation and function.
The three-component system of the filtration barrier - endothelium, basement membrane, and epithelium - in the glomerulus chips were found to selectively filter molecules and respond to a chemotherapeutic, nephrotoxic drug treatment. Results from the drug treatment indicated that the glomerulus chip can be used for nephrotoxicity studies and for disease modeling. This protocol provides the general guideline for engineering a functional microfluidic kidney glomerulus chip from isogenic iPS cell derivatives. Downstream analyses of the engineered chip can be carried out as desired by the researcher. For more information on using the glomerulus chip to model drug-induced glomerular injury, refer to previous publications2,12.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies</td><td>Thermo/Life Technologies</td><td>A32744; A32754; A-11076; A32790; A21203; A11015</td><td></td></tr><tr><td>Collagen IV</td><td>Thermo/Life Technologies</td><td>14-9871-82</td><td></td></tr><tr><td>Nephrin</td><td>Progen</td><td>GP-N2</td><td></td></tr><tr><td>PECAM-1</td><td>R&amp;amp;D Systems</td><td>AF806</td><td></td></tr><tr><td>Podocin</td><td>Abcam</td><td>ab50339</td><td></td></tr><tr><td>VE-Cadherin</td><td>Santa Cruz</td><td>sc-9989</td><td></td></tr><tr><td>&lt;strong&gt;Basement membrane matrices&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Corning Fibronectin, Human</td><td>Corning</td><td>356008</td><td>Basement membrane (3)</td></tr><tr><td>iMatrix-511 Laminin-E8 (LM-E8) fragment</td><td>Iwai North America</td><td>N8922012</td><td>Basement membrane matrix (2)</td></tr><tr><td>Matrigel hESC-qualified matrix, 5-mL vial</td><td>BD Biosciences</td><td>354277</td><td>Basement membrane matrix (1); may show lot-to-lot variation</td></tr><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 iPSC Core Facility and provided to us by the Bursac Lab at Duke University. The line has been tested and found to be free of mycoplasma (last test in November 2021) and karyotype abnormalities (July 2019)</td></tr><tr><td>&lt;strong&gt;Culture medium growth factors and media supplements&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.5M EDTA, pH 8.0</td><td>Invitrogen</td><td>15575020</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Thermo/Life Technologies</td><td>21985023</td><td></td></tr><tr><td>Albumin from Bovine serum, Texas Red conjugate</td><td>Thermo/Life Technologies</td><td>A23017</td><td></td></tr><tr><td>All-trans retinoic acid (500 mg)</td><td>Stem Cell Technologies</td><td>72262</td><td></td></tr><tr><td>B27 serum-free supplement</td><td>Thermo/Life Technologies</td><td>17504044</td><td></td></tr><tr><td>B-27 supplement (50x) without Vitamin A</td><td>Thermo/Life Technologies</td><td>12587010</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>CHIR99021</td><td>Stemgent</td><td>04-0004</td><td>May show lot-to-lot variation</td></tr><tr><td>Complete medium kit with CultureBoost-R</td><td>Cell Systems</td><td>4Z0-500-R</td><td>Podocyte maintenance media</td></tr><tr><td>DMEM/F12</td><td>Thermo/Life Technologies</td><td>12634028</td><td></td></tr><tr><td>DMEM/F12 with GlutaMAX supplement</td><td>Thermo/Life Technologies</td><td>10565042</td><td>DMEM/F12 with glutamine</td></tr><tr><td>Forskolin (Adenylyl cyclase activator)</td><td>Abcam</td><td>ab120058</td><td></td></tr><tr><td>GlutaMAX supplement</td><td>Thermo/Life Technologies</td><td>35050061</td><td>glutamine supplement</td></tr><tr><td>Heat-inactivated FBS</td><td>Thermo/Life Technologies</td><td>10082147</td><td></td></tr><tr><td>Heparin solution</td><td>Stem Cell Technologies</td><td>7980</td><td></td></tr><tr><td>Human Activin A</td><td>Thermo/Life Technologies</td><td>PHC9544</td><td></td></tr><tr><td>Human BMP4</td><td>Preprotech</td><td>120-05ET</td><td></td></tr><tr><td>Human BMP7</td><td>Thermo/Life Technologies</td><td>PHC9544</td><td></td></tr><tr><td>Human VEGF</td><td>Thermo/Life Technologies</td><td>PHC9394</td><td></td></tr><tr><td>Inulin-FITC</td><td>Sigma-Aldrich</td><td>F3272</td><td></td></tr><tr><td>mTeSR1 medium</td><td>Stem Cell Technologies</td><td>05850</td><td>Human iPS cell culture media (CCM). Add 5x supplement according to the manufacturer. Human iPS CCM can be stored for up to 6 months at -20 &amp;deg;C.</td></tr><tr><td>N-2 Supplement (100x)</td><td>Thermo/Life Technologies</td><td>17502048</td><td></td></tr><tr><td>Neurobasal media</td><td>Thermo/Life Technologies</td><td>21103049</td><td>Lateral mesoderm basal media</td></tr><tr><td>PBS (Phosphate-buffered saline)</td><td>Thermo/Life Technologies</td><td>14190-250</td><td></td></tr><tr><td>Penicillin-streptomycin, liquid (100x)</td><td>Thermo/Life Technologies</td><td>15140-163</td><td></td></tr><tr><td>ROCK inhibitor (Y27632)</td><td>Tocris</td><td>1254</td><td></td></tr><tr><td>StemPro-34 SFM</td><td>Thermo/Life Technologies</td><td>10639011</td><td>Endothelial cell culture medium (CCM). Add supplement according to manufacturer. Endothelial CCM can be stored for up to two weeks at 4 &amp;deg;C or -20 &amp;deg;C for up to 6 months.</td></tr><tr><td>TGF-Beta inhibitor (SB431542)</td><td>Stem Cell Technologies</td><td>72234</td><td></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>Thermo/Life Technologies</td><td>A1110501</td><td>Cell detachment buffer</td></tr><tr><td>Dimethyl Suloxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2438</td><td></td></tr><tr><td>Ethanol solution, 70% (vol/vol), biotechnology grade</td><td>VWR</td><td>97065-058</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Thermo/Life Technologies</td><td>28906</td><td></td></tr><tr><td>Sterile distilled water</td><td>Thermo/Life Technologies</td><td>15230162</td><td></td></tr><tr><td>Triton X-100</td><td>VWR</td><td>97062-208</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trypsin EDTA, 0.05%</td><td>Thermo/Life Technologies</td><td>25300-120</td><td></td></tr><tr><td>(Orb) Hub module</td><td>Emulate</td><td>ORB-HM1</td><td></td></tr><tr><td>100mm x 15 mm round petri dish</td><td>Fisherbrand</td><td>FB087579B</td><td></td></tr><tr><td>120 x 120 mm square cell culture dish</td><td>VWR</td><td>688161</td><td></td></tr><tr><td>Accuri C6</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Aspirating pipettes, individually wrapped</td><td>Corning</td><td>29442-462</td><td></td></tr><tr><td>Aspirating Unit</td><td>SP Bel-Art</td><td>F19917-0150</td><td></td></tr><tr><td>Avanti J-15R Centrifuge</td><td>Beckman Coulter</td><td>B99516</td><td></td></tr><tr><td>Conical centrifuge tube, 15 mL</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>Conical centrifuge tube, 50 mL</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>EVOS M7000</td><td>Thermo/Life Technologies</td><td>AMF7000</td><td>Fluorescent microscope to take images of fixed and stained cells.</td></tr><tr><td>Hemocytometer</td><td>VWR</td><td>100503-092</td><td></td></tr><tr><td>Heracell VIOS 160i CO2 incubator</td><td>Thermo/Life Technologies</td><td>51030403</td><td></td></tr><tr><td>Inverted Zeiss Axio Observer equipeed with AxioCam 503 camera</td><td>Carl Zeiss Micrscopy</td><td>491916-0001-000(microscope) ; 426558-0000-000(camera)</td><td></td></tr><tr><td>Kimberly-Clark nitrile gloves</td><td>VWR</td><td>40101-346</td><td></td></tr><tr><td>Kimwipes, large</td><td>VWR</td><td>21905-049</td><td></td></tr><tr><td>Leoca SP8 Upright Confocal Microscope</td><td></td><td></td><td></td></tr><tr><td>Media reservoir (POD Portable Module)</td><td>Emulate</td><td>POD-1</td><td></td></tr><tr><td>Microplate shaker</td><td>VWR</td><td>12620-926</td><td></td></tr><tr><td>Organ-chip</td><td>Emulate</td><td>S-1 Chip</td><td></td></tr><tr><td>Organ-chip holder</td><td>Emulate</td><td>AK-CCR</td><td></td></tr><tr><td>P10 precision barrier pipette tips</td><td>Denville Scientific</td><td>P1096-FR</td><td></td></tr><tr><td>P100 barrier pipette tips</td><td>Denville Scientific</td><td>P1125</td><td></td></tr><tr><td>P1000 barrier pipette tips</td><td>Denville Scientific</td><td>P1121</td><td></td></tr><tr><td>P20 barrier pipette tips</td><td>Denville Scientific</td><td>P1122</td><td></td></tr><tr><td>P200 barrier pipette tips</td><td>Denville Scientific</td><td>P1122</td><td></td></tr><tr><td>Plasma Asher</td><td>Quorum tech</td><td>K1050X RF</td><td>This Plasma Etcher/Asher/Cleaner was used as a part of Duke University&#039;s Shared Materials Instrumentation Facility (SMiF).</td></tr><tr><td>Round bottom polystyrene test tube with cell strainer snap cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Serological pipette, 10 mL, indivdually wrapped</td><td>Corning</td><td>356551</td><td></td></tr><tr><td>Serological pipette, 25 mL, indivdually wrapped</td><td>Corning</td><td>356525</td><td></td></tr><tr><td>Serological pipette, 5 mL, indivdually wrapped</td><td>Corning</td><td>356543</td><td></td></tr><tr><td>Steriflip, 0.22 &amp;micro;m, PES</td><td>EMD Millipore</td><td>SCGP00525</td><td></td></tr><tr><td>Sterile Microcentrifuge tubes</td><td>Thomas Scientific</td><td>1138W14</td><td></td></tr><tr><td>T75cm&lt;sup&gt;2&lt;/sup&gt; cell culture flask with vent cap</td><td>Corning</td><td>430641U</td><td></td></tr><tr><td>Tissue culture-treated 12 well plates</td><td>Corning</td><td>353043</td><td></td></tr><tr><td>Tissue culture-treated 6 well plates</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Vacuum modulator and perstaltic pump (Zoe Culture Module)</td><td>Emulate</td><td>ZOE-CM1</td><td>Organ Chip Bioreactor</td></tr><tr><td>VE-Cadherin CD144 anti-human antibody - APC conjugated</td><td>Miltenyi Biotec</td><td>130-126-010</td><td></td></tr><tr><td>Wide-beveled cell lifter</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>&lt;strong&gt;MACS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD144 MicroBeads, human</td><td>Miltenyi Biotec</td><td>130-097-857</td><td></td></tr><tr><td>CD31 MicroBead Kit, human</td><td>Miltenyi Biotec</td><td>130-091-935</td><td></td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr></tbody></table>

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.

Here we show that a functional 3D in vitro model of the glomerulus can be vascularized and epithelialized from an isogenic source of human iPS cells. Specifically, this protocol provides instructions on how to apply human iPS cell technology, particularly their ability to differentiate into specialized cell types, to generate kidney glomerular epithelium (podocytes) and vascular endothelium (viECs) that can be integrated with microfluidic devices to model the structure and function of the human kidney at the patient-specific level. A schematic overview of this protocol and timeline (Figure 1A) describes how to culture mitotically active human iPS cells (Figure 1B) and then differentiate them (in parallel) into mesoderm and lateral mesoderm cell lineages (Figure 1C,D). The resulting mesoderm cells were found to express brachyury (T), while the lateral mesoderm cells expressed brachyury (T), MIXL, and EOMES2,9,10,11.
Subsequent differentiation of the mesoderm cells produced intermediate mesoderm (IM) cells, while differentiation of the lateral mesoderm cells produced viECs (Figure 1D)2,10,11,17. Flow cytometry analysis was used to examine the expression of CD144 in differentiated viECs (before and after MAC sorting) compared to negative controls (including stained and unstained undifferentiated human iPS cells and unstained endothelium). An optimized endothelial differentiation will result in 50% or greater CD31/CD144-positive cells before MAC sorting, which will significantly improve after cell sorting compared to controls. Representative results show 59% differentiation efficiency for CD144 before MAC sorting, which increased to 77% or more CD144-positive cells (not including CD31-positive cells) after MAC sorting (Figure 1E).
On day 14 of this protocol (before the completion of IM differentiation and viEC expansion), the organ-on-a-chip devices were prepared for cell seeding by plasma-treatment and functionalization with basement membrane matrix 2. The following day (day 15 of the protocol), viECs were seeded into the capillary (bottom) channel of the microfluidic device with viEC medium. The day after viEC seeding (day 16 of the protocol), IM cells were seeded into the urinary (top) channel of the microfluidic device with podocyte induction medium. The day following IM cell seeding (day 17 of this protocol), 60 &#181;L/h fluid flow rate and 10% strain at 0.4 Hz were applied to the glomerulus chips. These chips experience shear stress of 0.017dyn cm&#8722;2 and 0.0007dyn cm&#8722;2 in the capillary and urinary channels, respectively2,12. After up to 5 days of podocyte induction and 6 days of vascular endothelium propagation in the chip (day 21 of this protocol) (Figure 2A), the resulting cells within the glomerulus chips expressed lineage identification markers.
Specifically, the podocytes in the urinary channel expressed podocin and nephrin (Figure 2B, top panel), and the viECs in the capillary channel expressed PECAM-1 (CD31) and VE-Cadherin (CD144) (Figure 2B, bottom panel). Additionally, both the podocyte and viEC layers expressed collagen IV, the most abundant GBM protein (Figure 2B) in the kidney glomerulus. More collagen IV is expressed in the urinary channel because podocytes are the predominant producers of collagen IV, including the &#945;3&#945;4&#945;5 isoform, which is the main heterotrimer isoform of collagen in the mature glomerulus. In addition, the podocytes propagated in the glomerulus chips developed foot processes and secreted VEGF165, both of which are characteristic features of functional models of the kidney glomerulus2,12. This protocol also provides an assessment of the selective molecular filtration function of the kidney glomerulus using inulin and albumin, from which the glomerulus chips selectively filter small molecules (inulin) from the capillary into the urinary channel, while preventing large proteins (albumin) from leaving the capillary channel (Figure 2C)2,10,12.
As each human iPS cell line exhibits inherent differences in the doubling time, it is important to note that optimal cell seeding densities for different cell lines may vary and must therefore be optimized by the researcher. For endothelial cell differentiation, if the seeding density of human iPS cells is too low, the researcher may observe a lower yield of differentiated endothelial cells (&#60;30% efficiency). If the seeding density of human iPS cells is too high, the researcher may observe rapid cell overgrowth, detachment or poorer adhesion, increased cell death, and low yield (&#60;30% efficiency). During endothelial induction (days 4-7 of differentiation), an increase in the cell number resulting in a secondary layer of cells is normal but should be kept to a minimum (Figure 3A). For IM and podocyte differentiation, overseeding of human iPS cells (&#62;100,000 cells/well of a 12-well plate) may result in IM cells that grow in large clusters or form aggregates, which can impede differentiation and result in podocytes with a less mature morphological phenotype of aggregated cells and less secondary and/or tertiary foot processes10,11.
During microfluidic chip culture, unexpected fluid crossflow between the urinary and capillary channels (Figure 3B) may be observed if there is ruptured or inadequate bonding of the PDMS chip components, or if the path of fluid flow is blocked. This undesired fluid crossflow may also result from a compromised filtration barrier such as tissue models from inadequate (low) cell seeding or damaged cell layers. To prevent this problem, it is recommended that the researcher follows the recommended protocol and cell seeding densities, as well as visually inspects the chips for air bubbles in the channels at every stage of the process. If air bubbles are observed in the media reservoirs of the microfluidic chips that are under fluid flow, the pump can be stopped and the medium degassed under sterile conditions.
Together, this protocol and representative results describe the derivation of vascular endothelium (viECs) and glomerular epithelium (podocytes) from an isogenic human iPS cell line, and their reconstitution in a microfluidic organ-on-a-chip device to recapitulate the structure and function of the kidney glomerular filtration barrier in a patient-specific manner.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63821/63821fig1v2.jpg" /> 
Figure 1: Derivation of isogenic glomerular epithelium and vascular endothelium from human iPS cells. (A) Schematic timeline of intermediate mesoderm and viEC induction, organ-on-a-chip design and basement membrane matrix coating, cell seeding into the chip, and podocyte induction within the chip. (B)&#160;Representative brightfield images of PGP1 human iPS cells before dissociation at day 0 of the protocol. (C) Representative brightfield images of PGP1 mesoderm cells on day 2 of differentiation (left) and intermediate mesoderm cells on day 8 of differentiation (right). (D) Representative brightfield images of PGP1 lateral mesoderm cells on day 3 of differentiation (left) and PGP1 viECs on day 9 of differentiation (2 days of expansion) (right). Scale bars = 275 &#181;m (B-D). (E)&#160;Quantification of viEC differentiation via flow cytometry analysis for CD144-positive cells on day 7 of endothelial cell differentiation before MACS (blue) and on day 9 of endothelial cell expansion after MACS (pink) compared to CD144-stained human iPSCs (black) and unstained endothelial cells (red). This figure has been modified from12. Abbreviations: iPSCs = induced pluripotent stem cells; viEC = vascular endothelial cells; BMP4/7 = bone morphogenetic protein 4/7; RA = retinoic acid; VEGF = vascular endothelial growth factor; MACS = magnetic-activated cell sorting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63821/63821fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63821/63821fig02.jpg" /> 
Figure 2: Representative images of vascular endothelium and glomerular epithelium (podocyte layer) cultured within the microfluidic kidney glomerulus chip. Representative brightfield images of viECs (left) and glomerular epithelium (podocytes) (right) propagated in the glomerulus chip. Scale bars = 183 &#181;m. (B) Representative immunofluorescent images of glomerular epithelium (podocytes) and viEC showing the expression of lineage-specific markers. Scale bars = 100 &#181;m. (C)&#160;Representative data showing selective molecular filtration in the glomerulus chip. Error bars represent SD. p &#60; 0.0001. This figure has been reproduced from 12. Abbreviations: viECs = vascular endothelial cells; VE-cadherin = CD144; PECAM-1 (= CD31) = platelet endothelial cell adhesion molecule. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63821/63821fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63821/63821fig03.jpg" /> 
Figure 3: Images of suboptimal endothelium seeding density and uneven fluid flow in the microfluidic chips. (A) Representative brightfield images of optimal (left) and overseeded (right) cell cultures on day 6 of viEC differentiation. Scale bars = 275 &#181;m. (B) Representative images of outlet reservoirs from microfluidic chips with an even fluid flow and a functional barrier (left). Image from a chip with uneven fluid flow or dysfunctional barrier (right). Arrows denote fluid levels in outlet reservoirs for the capillary and urinary channels of the chips. Abbreviation: viECs = vascular endothelial cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63821/63821fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: Flow cytometry, ELISA for chip effluent, and mRNA isolation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63821/63821_Suppl File 1.zip" target="_blank">Please click here to download this File.</a>
Supplemental Figure S1: In-tissue culture hood material setups. (A) In-tissue culture hood material set up for MACS, including ice bucket with media, magnet on magnet stand, and conical tubes underneath the magnet. (B)&#160;In-tissue culture hood material setup of Petri dish for chips, with the top, facing downwards, under the Petri dish bottom. Abbreviation: MACS = magnetic-activated cell sorting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63821/20220520_Supplemental Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplemental Table S1: Media and buffers used in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63821/20220506_Media Buffers Solutions_YR_SM edit.zip" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells at JoVE.com

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

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 ...

isogenic-kidney-glomerulus-chip-engineered-from-human-induced

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.9K Views.  Duke University. De isogene glomeruluschip zal patiëntspecifieke ziektemodellering en nefrotoxiciteitstests en geavanceerde precisiegeneeskundetoepassingen mogelijk maken. Glomerulair epitheel en endotheel zijn afgeleid van een enkele populatie van door de mens geïnduceerde pluripotente stamcellen, of IPSC's. IPSC's bezitten onbeperkte zelfvernieuwing en kunnen zich onderscheiden in bijna elk celtype.Deze techniek kan worden gebruikt om pathologische cellulaire fenotypen en ziekten te beoordelen. De ...

Video: Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

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 ...

Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain degree. Unfortunately, their clinical applicability is hampered by the lack of a functional vasculature and consequently limited maturation in vitro. The transplantation of kidney organoids in the celomic cavity of chicken embryos induces vascularization by perfused blood vessels, including the formation of glomerular capillaries, and enhances their maturation. This technique is very efficient, allowing for the transplantation and analysis of large numbers of organoids. This paper describes a detailed protocol for the intracelomic transplantation of kidney organoids in chicken embryos, followed by the injection of fluorescently labeled lectin to stain the perfused vasculature, and the collection of transplanted organoids for imaging analysis. This method can be used to induce and study organoid vascularization and maturation to find clues for enhancing these processes in vitro and improve disease modeling.

Here, we present a detailed protocol for the transplantation of kidney organoids in the celomic cavity of chicken embryos. This method induces vascularization and enhanced maturation of the organoids within 8 days and can be used to study these processes in an efficient manner.

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain ...

Using Induced Pluripotent Stem Cell Suspension for Kidney Organoid Production

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

Author Spotlight: Optimizing iPSC Differentiation for Efficient Production to Generate Kidney Organoids 

This protocol presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells (iPSCs) using suspension culture conditions. The primary emphasis of this study lies in the determination of the initial cell density and the WNT agonist concentration, thereby benefiting investigators interested in kidney organoid research.

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

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

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

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

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

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Medicine

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.remotexs.ntu.edu.sg/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. ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

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