Name: kidney organoid generation at the air-liquid interface
Uploaded: 2020-07-16T14:36:46
Description: The prevalence of kidney diseases continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four ...

This work was supported in part by Merit Review Award #I01 BX002660 from the United States Department of Veterans Affairs, Biomedical Laboratory Research and Development Service to Jason A. Wertheim, National Institutes of Health grant number R24 DK106743 to Leif Oxburgh and National Institute of Diabetes and Digestive and Kidney Diseases award number F30DK123985 and National Institute of&#160;General Medical Sciences&#160;award number T32GM008152&#160;to Bilal A. Naved.&#160;The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the Department of Veterans Affairs, or the United States Government. The authors would like to acknowledge the Northwestern University Biological Imaging Facility, for fluorescent microscope imaging.

Cell line WA09 (H9) was approved by the National Institutes of Health (registration number 0062) and was tested negative for mycoplasma infection.
1. Medium and plate preparation for hESCs culture
<ol>
	<li>Dilute matrix (reduced growth factor basement membrane e.g., Geltrex) in DMEM/F12 (1:100) and add 1 mL/well in 2 wells of a 6 well plate (reagents utilized in the manuscript are summarized in the Table of Materials).</li>
	<li>Incubate coated plate undisturbed at 37 &#176...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolding+kidney+organoids+on+silk.">Scaffolding kidney organoids on silk.</a> Journal of Tissue Engineering and Regenerative Medicine. 13 (5), 812-822 (2019).</li><li>Takasato, M., Er, P. X., Chiu, H. S., Little, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+kidney+organoids+from+human+pluripotent+stem+cells.">Generation of kidney organoids from human pluripotent stem cells.</a> Nature Protocols. 11 (9), 1681-1692 (2016).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductive+epitheliomesenchymal+interaction+in+cultured+organ+rudiments+of+the+mouse.">Inductive epitheliomesenchymal interaction in cultured organ rudiments of the mouse.</a> Science. 118 (3054), 52-55 (1953).</li><li>Oxburgh, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+Nephron+Determination.">Kidney Nephron Determination.</a> Annual Review of Cell and Developmental Biology. 34, 427-450 (2018).</li><li>Gupta, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asynchronous+mixing+of+kidney+progenitor+cells+potentiates+nephrogenesis+in+organoids.">Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids.</a> Communications Biology. 3, 231 (2020).</li><li>Lindstr&#246;m, N. 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D., Oxburgh, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+niche+for+nephron+progenitor+cells.">A synthetic niche for nephron progenitor cells.</a> Developmental Cell. 34 (2), 229-241 (2015).</li><li>Subramanian, A., 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=Single+cell+census+of+human+kidney+organoids+shows+reproducibility+and+diminished+off-target+cells+after+transplantation.">Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation.</a> Nature Communications. 10 (1), 5462 (2019).</li><li>Taguchi, A., Nishinakamura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher-Order+Kidney+Organogenesis+from+Pluripotent+Stem+Cells.">Higher-Order Kidney Organogenesis from Pluripotent Stem Cells.</a> Cell Stem Cell. 21 (6), 730-746 (2017).</li></ol>

Asynchronous mixing of progenitors at the air-liquid interface (Figure 1) presents an efficient method to generate kidney organoids from hESCs. This work describes stepwise protocols for thaw and culture of hESCs, directed differentiation to kidney progenitors, making cell aggregates at the air-liquid interface, asynchronous mixing of progenitors to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal ...

Chronic kidney disease (CKD) is a worldwide healthcare problem with 13.4% global estimated prevalence1. Approximately 10% of the adult population of the United States suffers from CKD2. There is no curative treatment available for patients with CKD except renal transplantation. The lack of availability of transplantable organs warrants research into technologies to understand how new kidney tissues can be generated. In recent years, procedures have been reported to generate kidney organoids3,4 from human embryonic stem cells (hESCs) and human induced pluripotent cells but differentiation into off-target cells5,6 and lack of dense tubules within kidney organoids limit their use in modeling renal diseases, in-vitro chemical screening and transplantable renal tissue generation.
We followed a previously published protocol to generate kidney progenitors from hESCs3. The kidney functions as a 3D organ and an appropriate 3D environment allows kidney progenitors to self-organize to form a kidney organoid7. A 3D organotypic culture condition was selected to generate kidney organoids because it supports vigorous growth and differentiation of mammalian embryonic kidneys8. Using this approach, hESCs derived kidney progenitors were aggregated at the air-liquid interface, to provide a 3D organotypic culture environment for differentiation.
During kidney development, several stages of differentiating cells coexist within the embryonic kidney9, and their differentiation fate and spatial patterning along the nephron depends on the timing of their recruitment. To establish such a culture condition in-vitro, a method was developed for asynchronous mixing of kidney progenitors generated by directed differentiation3. Asynchronous mixing refers to the combination of two progenitor populations that are at different stages of differentiation. Directed differentiation cultures were staggered two days apart and newly differentiated cells were mixed with cells that have been cultured as aggregates in organotypic conditions for 2 days. Heterochronic mixing of two cell batches improve the fidelity of pluripotent stem cell-derived organoids10.
Here, we provide an efficient method of asynchronous mixing of the kidney progenitors at the air-liquid interface that potentiates nephrogenesis to produce tightly packed nephron epithelia with more tubular clusters in kidney organoids. These kidney organoids were filled with glomerular podocytes, proximal tubules, distal tubules, stromal cells, connecting tubule or collecting ducts. The protocol yields a complex and extensive network of endothelial cells. In addition, proximal tubules in kidney organoids were mature and functional, showing endocytic function confirmed by alexa flour 488 (AF488) labeled dextran uptake. In this protocol, a step by step methodology of asynchronous mixing of kidney progenitors to generate kidney organoids is presented which we recently published elsewhere10.

<table>
	<tbody>
		<tr>
			<td>15 mL falcon tube</td>
			<td>VWR</td>
			<td>62406-200</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>VWR</td>
			<td>29443-952</td>
			<td></td>
		</tr>
		<tr>
			<td>40 micron strainer</td>
			<td>VWR</td>
			<td>21008-949</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL falcon tube</td>
			<td>VWR</td>
			<td>21008-940</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>VWR</td>
			<td>29442-042</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well Clear Round Bottom Ultra-Low Attachment Microplate (U bottom)</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7920</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D systems</td>
			<td>338-AC-010</td>
			<td>Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Advanced RPMI 1640</td>
			<td>ThermoFisher Scientific</td>
			<td>12633-012</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>APEL2</td>
			<td>STEMCELL Technologies</td>
			<td>5270</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>BMP7</td>
			<td>R&#38;D systems</td>
			<td>354-BP-010</td>
			<td>Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>CHIR99021 IN SOLUTION</td>
			<td>Reprocell</td>
			<td>04-0004-10</td>
			<td>Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica Microsystems</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, AF 488, 10,000 MW</td>
			<td>Thermo fisher Scientific</td>
			<td>D22910</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo fisher Scientific</td>
			<td>11330-032</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>VWR</td>
			<td>45000-434</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>R&#38;D systems</td>
			<td>234-FSE-025</td>
			<td>Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>FGF9</td>
			<td>R&#38;D systems</td>
			<td>273-F9-025</td>
			<td>Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex</td>
			<td>Thermo fisher Scientific</td>
			<td>A1413301</td>
			<td>Aliquot and Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Thermo fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>H9 cells</td>
			<td>WiCell</td>
			<td>WA09</td>
			<td>Store in liquid Nitrogen</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3393-25KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopore membrane</td>
			<td>EMD Millipore</td>
			<td>VCTP01300</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>P6148-500G</td>
			<td>P6148-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>PFHM II</td>
			<td>Thermo fisher Scientific</td>
			<td>12040077</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Rock inhibitor Y-27632</td>
			<td>EMD Millipore</td>
			<td>688002-1mg</td>
			<td>Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>StemFit Medium</td>
			<td>amsbio</td>
			<td>SFB-500</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLe express</td>
			<td>Thermo fisher Scientific</td>
			<td>12563029</td>
			<td></td>
		</tr>
	</tbody>
</table>

kidney organoid generation at the air-liquid interface

The prevalence of kidney diseases continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters in the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells and their directed differentiation to kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in-vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cells types, interconnected nephron segments and physiologically functional renal tissues.

This protocol describes asynchronous mixing of kidney progenitor cells differentiated from hESCs (H9) at the air-liquid interface to generate kidney organoids with reproducible results and high success rates. We followed a previously published protocol to differentiate hESCs into kidney progenitors3 (Figure 1). The mix of cells that arises from the directed differentiation process is believed to represent the repertoire of developmental kidney progenitors that gives r...

This protocol describes asynchronous mixing of human embryonic stem cells derived kidney progenitors at the air-liquid interface to efficiently generate kidney organoids.

Kidney Organoid Generation at the Air-Liquid Interface

The prevalence of kidney diseases continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four ...