Name: isogenic kidney glomerulus chip engineered from human induced pluripotent stem cells
Uploaded: 2022-11-04T13:15:00
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.

1. Prepare basement membrane matrix solutions and coated substrates
<ol>
	<li>Thaw basement membrane matrix 1 overnight on ice at 4 &#176;C. Aliquot according to the manufacturer&#39;s suggestion for dilution ratio. Using a 50 mL conical tube and pipette, thoroughly mix an appropriate amount of basement membrane matrix 1 into 25 mL of cold DMEM/F12 until completely thawed and dissolved.
	<ol>
		<li>To dissolve a frozen aliquot, take ~200 &#181;L from the 25 mL of cold DMEM/F12 and transfer it to the fr...

<ol>
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J., Wu, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+heterogeneity:+when+do+differences+make+a+difference.">Cellular heterogeneity: when do differences make a difference.</a> Cell. 141 (4), 559-563 (2010).</li><li>Da Sacco, 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=A+novel+source+of+cultured+podocytes.">A novel source of cultured podocytes.</a> PloS One. 8 (12), 81812 (2013).</li><li>Singh, V. K., Kalsan, M., Kumar, N., Saini, A., Chandra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+applications+in+regenerative+medicine,+disease+modeling,+and+drug+discovery.">Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery.</a> Frontiers in Cell and Developmental Biology. 3, 2 (2015).</li><li>Ebert, A. D., Liang, P., Wu, J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+induced+pluripotent+stem+cells+into+mature+kidney+podocytes+and+establishment+of+a+Glomerulus+Chip.">Directed differentiation of human induced pluripotent stem cells into mature kidney podocytes and establishment of a Glomerulus Chip.</a> Nature Protocols. 13 (7), 1662-1685 (2018).</li><li>Burt, M., Bhattachaya, R., Okafor, A. E., Musah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+differentiation+of+mature+kidney+podocytes+from+human+induced+pluripotent+stem+cells+under+chemically+defined+conditions.">Guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions.</a> Journal of Visualized Experiments. (161), e61299 (2020).</li><li>Roye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+personalized+glomerulus+chip+engineered+from+stem+cell-derived+epithelium+and+vascular+endothelium.">A personalized glomerulus chip engineered from stem cell-derived epithelium and vascular endothelium.</a> Micromachines. 12 (8), 967 (2021).</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> The American Journal of Pathology. 160 (1), 131-139 (2002).</li><li>Ruotsalainen, V., 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=Nephrin+is+specifically+located+at+the+slit+diaphragm+of+glomerular+podocytes.">Nephrin is specifically located at the slit diaphragm of glomerular podocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (14), 7962-7967 (1999).</li><li>Privratsky, J. R., Newman, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PECAM-1:+regulator+of+endothelial+junctional+integrity.">PECAM-1: regulator of endothelial junctional integrity.</a> Cell and Tissue Research. 355 (3), 607-619 (2014).</li><li>Menon, M. C., Chuang, P. Y., He, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glomerular+filtration+barrier:+components+and+crosstalk.">The glomerular filtration barrier: components and crosstalk.</a> International Journal of Nephrology. 2012, 749010 (2012).</li><li>Abutaleb, N. O., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+characterization+of+human+iPSC-derived+vascular+endothelial+cells+under+physiological+shear+stress.">Differentiation and characterization of human iPSC-derived vascular endothelial cells under physiological shear stress.</a> STAR Protocols. 2 (2), 100394 (2021).</li><li>Pammolli, F., Magazzini, L., Riccaboni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+productivity+crisis+in+pharmaceutical+R&amp;D.">The productivity crisis in pharmaceutical R&amp;D.</a> Nature Reviews. Drug Discovery. 10 (6), 428-438 (2011).</li><li>Atchison, L., 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=iPSC-derived+endothelial+cells+affect+vascular+function+in+a+tissue-engineered+blood+vessel+model+of+Hutchinson-Gilford+progeria+syndrome.">iPSC-derived endothelial cells affect vascular function in a tissue-engineered blood vessel model of Hutchinson-Gilford progeria syndrome.</a> Stem Cell Reports. 14 (2), 325-337 (2020).</li></ol>

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 adap...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</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&#38;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>Basement membrane matrices</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>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 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>Culture medium growth factors and media supplements</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 &#176;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 &#176;C or -20 &#176;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>Enzymes and other reagents</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>Equipment</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&#39;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 &#181;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>T75cm2 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>MACS</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 pat...

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 ...

The isogenic glomerulus chip will enable patient specific disease modeling and nephrotoxicity testing and advanced precision medicine applications. Glomerular epithelium and endothelium are derived from a single population of human-induced pluripotent stem cells, or IPSCs. IPSCs possess unlimited self-renewal and can differentiate into almost any cell type.This technique can be used to assess pathological cellular phenotypes and disease. The patient-specific organ chip can also serve as a platform for drug screening and mechanistic studies. This model can be applied to the development of a body on a chip system personalized to a specific patient.This system has the potential to predict patient-specific responses prior to clinical testing. Begin by gently adding 25 microliters of basement membrane matrix 2 solution to the urinary channel and 20 microliters into the capillary channel of the chip. Take two sterile 15 milliliter conical tube caps and fill them with 500 microliters of sterile distilled water.Place the cap in the Petri dish to prevent drying and place the lid on the dish. Incubate it at 37 degrees Celsius overnight. Proceeding, aspirate the medium from T75 flasks containing a 15 day culture of vascular endothelial cells at 90%confluency.Add five milliliters of cell detachment buffer and incubate at 37 degrees Celsius for five to seven minutes. Then transfer the cells to a 15 milliliter conical tube and add five milliliters of DMEM/F-12. Centrifuge at 200 times G for five minutes.Remove the supernatant and resuspend the cells in 300 microliters of endothelial maintenance medium. Count the cells with a hemocytometer to obtain approximately 2 million cells. Using a P200 barrier tip, flush both the top and bottom channels of the microfluidic chip with 200 microliters of DMEM/F-12 while simultaneously aspirating from the periphery of the outlet.Hold the chip firmly with the P200 barrier tip attached to the aspirator away from the outlet of the bottom channel. Firmly inject 20 microliters of the vascular endothelial cell suspension into the capillary channel of the chip and carefully aspirate the medium from the periphery of the outlet. Check under the microscope for bubbles or an uneven seeding density.Gently invert the chip so that the cells can adhere to the basal side of the flexible PDMS membrane. Place the chip into the holder cartridge and add three milliliters of PBS into the cartridge to prevent the membrane from drying. Incubate the chip at 37 degrees Celsius for three hours.Check the bottom channel under the microscope for a confluent layer of cells attached to the flexible PDMS membrane. Wash the unattached cells by adding 200 microliters of endothelial maintenance medium on the inlet of the bottom channel while aspirating from the periphery of the capillary channel outlet. Place the chip back into the holder cartridge and incubate at 37 degrees Celsius overnight.On the next day, gently flush the capillary channel with 200 microliters of endothelial maintenance medium. Flush the urinary channel with 200 microliters of DMEM/F-12 and drop 50 microliters of DMEM/F-12 on the inlet and outlet ports. Proceeding intermediate mesoderm cells, replace the intermediate mesoderm induction medium from each well of the 12 well plate with one milliliter of Trypsin EDTA and incubate at 37 degrees Celsius for five minutes.Gently scrape the cells using a cell lifter and dissociate the cells using pipetting. Add two milliliters of Trypsin neutralization solution to each well and transfer the cells to a 50 milliliter conical tube. Make up the volume of the cell suspension to 50 milliliters with DMEM/F-12 and centrifuge at 200 times G for five minutes.Aspirate the supernatant and resuspend the cells in 500 microliters of intermediate mesoderm induction medium to obtain approximately 3 million cells. Count the cells with a hemocytometer. Hold the chip and firmly inject 25 microliters of cell suspension into the urinary channel of the chip while carefully aspirating the medium from the periphery of the outlet.Check for bubbles or uneven cell seeding density. Add three milliliters of PBS into the chip holder cartridge and incubate at 37 degrees Celsius for three hours. After incubation, flush both channels with 200 microliters of their respective cell culture medium.Attach empty P200 barrier tips into both outlets of the urinary and capillary channels. Pipette 200 microliters of endothelial maintenance medium and inject half of it into the capillary channel inlet. Release the pipette tip inside the inlet such that both the inlet and outlet of the channel are attached to pipette tips filled with medium.Repeat this procedure for the urinary channel inlet with 200 microliters of intermediate mesoderm maintenance medium. Incubate the chips with tips embedded at 37 degrees Celsius overnight. To connect the chips to organ chip bioreactor, first, remove P200 tips from the channels.Add droplets of respective media to the inlet and outlet of the urinary and capillary channels to prevent drying. Add three milliliters of warm podocyte induction medium to the urinary inlet reservoir and three milliliters of warm endothelial maintenance medium to the capillary inlet reservoir. Add 300 microliters of the respective media to the outlet reservoirs directly over the outlet port.Slide the pods onto the tray and into the organ chip bioreactor. Use the rotary dial to select and start the prime cycle for two minutes. Visually inspect the underside of the pod for small droplets at all four fluidic ports.To achieve fluid to fluid contact between the pod underside and microfluidic chip ports, gently slide the chip carrier into the pod. Gently press the chip carrier tab in and up. Aspirate excess medium from the chip's surface.Set the organ chip bioreactor flow rate to 60 microliters per hour. Set the cyclic strain to 10%at 0.4 hertz. Use the rotary dial on the organ chip bioreactor to select the regulate cycle and run for two hours.Visually inspect the outlet reservoirs for an increase in the level of medium. Use the rotary dial on the organ chip bioreactor to select the regulate cycle. After day 17 of culture, daily aspirate the medium from the urinary channel outlet reservoirs diagonally away from the port but keep some medium in the reservoir.Replenish the urinary channel inlet reservoir with up to three milliliters of podocyte induction medium every two days for five days. After five days, aspirate the medium from the urinary channel but keep some medium in the reservoir. Replenish the urinary channel inlet reservoir daily with three milliliters of podocyte maintenance medium.Similarly, aspirate the medium from the capillary channel outlet and replenish the inlet reservoirs daily with endothelial maintenance medium. Using this protocol human induced pluripotent stem cells were used to develop a functional in vitro model of the glomerulus by propagating vascular endothelial cells and podocytes. On day 21 after podocyte induction and vascular endothelium propagation, the cells within the chips expressed lineage identification markers such as Podocin and Nephrine for podocytes, VE Cadherin and PECAM-1 for vascular endothelial cells.Both the podocyte and vascular endothelial cell layers expressed Collagen IV which is the most abundant GBM protein in the kidney glomerulus. The glomerulus chips selectively filtered small molecules such as inulin from the capillary into the urinary channel, while preventing large proteins like albumin from leaving the capillary channel, showing a selective molecular filtration function. During endothelial induction on days four to seven, an increase in the cell number may result in a secondary layer of cells but over seeding may cause aggregation which can impede differentiation.Unexpected fluid cross flow between the urinary and capillary channels may be observed in case of inadequate bonding of the PDMS chip components, blockage in the fluid flow path, or compromised filtration barrier. When seeding endothelium and epithelium and maintaining the chips from days 15 onward, it is important to visually inspect for air bubbles in the channels at every step. In the isogenic glomerulus chip, clinically relevant drug candidates can be administered to test their therapeutic potential or toxicity levels.This technique now opens up opportunities to understand the genetic basis of human kidney disease and develop personalized therapeutics.

isogenic-kidney-glomerulus-chip-engineered-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 ...

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 ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

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 ...

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 ...

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 ...