Name: establishing a high throughput epidermal spheroid culture system to model keratinocyte stem cell plasticity
Uploaded: 2021-01-30T14:00:00
Description: Watch this Scientific Journal Video about Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity at JoVE.com

The UofSC School of Medicine Instrumentation Resource Facility (IRF) provided access to imaging and cell sorting equipment and technical assistance. This work was supported in part by grant 1R21CA201853. The MCF and the IRF receive partial support from NIH grant P20GM103499, SC INBRE. The MCF also receives support from NIH grant P20GM109091. Yvon Woappi was supported in part by NIH grants 2R25GM066526-06A1 (PREP) and R25GM076277 (IMSD), and by a Fellowship by the Grace Jordan McFadden Professors Program at UofSC. Geraldine Ezeka and Justin Vercellino were supported by NIH grants 2R25GM066526-10A1 (PREP) at UofSC. Sean M. Bloos was supported by the 2016 Magellan Scholar Award at UofSC.

The protocol for the collection and handling of skin specimens and isolation of human keratinocytes has been reviewed by the University of South Carolina (UofSC) IRB and classified as &#34;research not involving human subjects&#34;, as the foreskin specimens were surgical discards produced during routine surgical procedures (circumcision of neonate boys) and were completely devoid of identifying information. The protocol was also reviewed and approved by the UofSC Biosafety Committee on a regular basis, and all laborator...

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphere-forming+capacity+as+an+enrichment+strategy+for+epithelial-like+stem+cells+from+equine+skin.">Sphere-forming capacity as an enrichment strategy for epithelial-like stem cells from equine skin.</a> Cellular Physiology and Biochemistry. 34 (4), 1291-1303 (2014).</li><li>Vollmers, 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=Two-+and+three-dimensional+culture+of+keratinocyte+stem+and+precursor+cells+derived+from+primary+murine+epidermal+cultures.">Two- and three-dimensional culture of keratinocyte stem and precursor cells derived from primary murine epidermal cultures.</a> Stem Cell Reviews and Reports. 8 (2), 402-413 (2012).</li><li>Kang, B. M., Kwack, M. H., Kim, M. K., Kim, J. C., Sung, Y. 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E., Pirisi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Properties+of+Normal+Human+Keratinocytes+Determine+Transformation+Responses+to+Human+Papillomavirus+16+DNA.">Stem Cell Properties of Normal Human Keratinocytes Determine Transformation Responses to Human Papillomavirus 16 DNA.</a> Journal of Virology. 92 (11), (2018).</li><li>Woappi, Y., Altomare, D., Creek, K., Pirisi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+3D+spheroid+cultures+of+human+neonatal+keratinocytes+have+enhanced+regenerative+properties.">Self-assembling 3D spheroid cultures of human neonatal keratinocytes have enhanced regenerative properties.</a> Stem Cell Research. , (2020).</li><li>Toma, J. G., McKenzie, I. A., Bagli, D., Miller, F. 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The use of 3D spheroid culture systems has had broad utility in assessing cell stemness. These systems have been demonstrated to enhance enrichment of tissue stem cells13, yet their utility for the study of human epidermal stem cells has been limitedly explored. Here, we describe a strategy for enriching human keratinocyte stem cells using 3D culture techniques. In this system, NHKc are cultivated as self-assembling multicellular spheroid suspensions, comprised of several keratinocyte subtypes sus...

The mammalian stratified epithelium is the most complex epithelial architecture in all living systems and is most often subject to damage and injury. As a protective tissue, stratified epithelium has evolved to generate a complex and effective tissue damage response. Upon injury, these cells must activate lineage plasticity programs, which enable them to migrate to the injured site and carry out repair1,2,3. This multifaceted response occurs in several sequential steps which remain poorly understood.
A major obstacle in studying the intricate process of epithelial regeneration lies in the dearth of high throughput model systems that can capture dynamic cellular activities at defined stages of cell regeneration. While in vivo mouse models offer relevant insight into wound healing and most closely recapitulate the human regenerative process, their development require laborious efforts and significant cost, limiting their throughput capacity. There exists, therefore, a critical need for establishing systems that enable functional investigation of human epithelial tissue regeneration at high throughput scale.
In recent years, several attempts have been made to meet the scalability challenge. This is seen through great expansion of innovative in vitro&#160;and ex vivo cell-based models that closely mimic the in vivo regenerative context. This include advances in organ-on-chip4, spheroid5, organoid6, and organotypic cultures7. These 3D cell-based systems each offer unique advantages and present distinct experimental limitations. To date, spheroid culture remains the most cost-effective and widely used 3D cell culture model. And while several reports have indicated that spheroid cultures can be used to study skin stem cell characteristics, these studies have largely been conducted with animal tissue8,9, or with dermal fibroblasts10, with virtually no reports thoroughly characterizing the regenerative properties of human epidermal spheroid cultures. In this protocol we detail the functional development, culture, and maintenance of epidermal spheroid cultures from normal human keratinocytes (NHKc). We equally describe the utility of this system to model the sequential phases of epidermal regeneration and keratinocyte stem cell plasticity in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Affymetrix platform</td>
			<td>Affymetrix</td>
			<td></td>
			<td>For microarray experiments</td>
		</tr>
		<tr>
			<td>Affymetrix&#8217;s HuGene-2_0-st library file</td>
			<td>Affymetrix</td>
			<td></td>
			<td>Process</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td></td>
			<td>For microarray experiments</td>
		</tr>
		<tr>
			<td>All Prep DNA/RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td>Used for RNA isolation</td>
		</tr>
		<tr>
			<td>Analysis Console Software version 3.0.0.466</td>
			<td></td>
			<td></td>
			<td>analyze cell type specific transcriptional responses using one-way between-subject analysis of variance</td>
		</tr>
		<tr>
			<td>BD FACSAria II flow cytometer</td>
			<td>Beckman</td>
			<td></td>
			<td>For flow cytometry</td>
		</tr>
		<tr>
			<td>Console Software version 3.0.0.466/Expression console Software</td>
			<td>Affymetrix/Thermo Fisher Scientific</td>
			<td></td>
			<td>For confirming data quality</td>
		</tr>
		<tr>
			<td>Cytokeratin 14</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53253</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Sigma-Aldrich</td>
			<td>D4818</td>
			<td>For cell media</td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-integrin&#945;6</td>
			<td>Abcam</td>
			<td>ab30496</td>
			<td>For FACS analysis</td>
		</tr>
		<tr>
			<td>GeneChip Command Console 4.0 software</td>
			<td>Affymetrix/Thermo Fisher Scientific</td>
			<td></td>
			<td>For confirming data quality</td>
		</tr>
		<tr>
			<td>GeneChip Fluidics Stations 450 (Affymetrix/Thermo Fisher Scientific)</td>
			<td>Affymetrix/Thermo Fisher Scientific</td>
			<td></td>
			<td>For washing and staining of hybridized arrays</td>
		</tr>
		<tr>
			<td>GeneChip HuGene 2.0 ST Arrays</td>
			<td>Affymetrix/Thermo Fisher Scientific</td>
			<td></td>
			<td>For hybridization and amplifycation of total RNA</td>
		</tr>
		<tr>
			<td>GeneChip Hybridization Oven 640</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>For hybridization and amplifycation of total RNA | Amplify labeled samples</td>
		</tr>
		<tr>
			<td>GeneChip Hybridization Wash, and Stain Kit (Affymetrix/Thermo Fisher Scientific).</td>
			<td>Affymetrix/Thermo Fisher Scientific</td>
			<td></td>
			<td>For washing and staining of hybridized arrays</td>
		</tr>
		<tr>
			<td>GeneChip Scanner 3000 7G system</td>
			<td>Affymetrix/Thermo Fisher Scientific</td>
			<td></td>
			<td>Scanning hybridized arrays</td>
		</tr>
		<tr>
			<td>GeneChip WT PLUS Reagent Kit</td>
			<td>Affymetrix/Thermo Fisher Scientific</td>
			<td></td>
			<td>For amplifycation of biotinylating total RNA</td>
		</tr>
		<tr>
			<td>Human Basic Fibroblast Growth Factor (hFGF basic/FGF2)</td>
			<td>Cell Signaling Technology</td>
			<td>8910</td>
			<td>For cell media</td>
		</tr>
		<tr>
			<td>Human Epidermal Growth Factor (hEGF)</td>
			<td>Cell Signaling Technology</td>
			<td>8916</td>
			<td>For cell media</td>
		</tr>
		<tr>
			<td>Human Insulin</td>
			<td>Millipore Sigma</td>
			<td>9011-M</td>
			<td>For cell media</td>
		</tr>
		<tr>
			<td>iQ SYBR Green Supermix (Bio-Rad)</td>
			<td>Bio-Rad</td>
			<td>1708880</td>
			<td>Used for RT-qPCR</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>1708890</td>
			<td>Used for RT-qPCR</td>
		</tr>
		<tr>
			<td>KSFM</td>
			<td>ThermoFisher Scientific</td>
			<td>17005041</td>
			<td>Supplemented with 1% Penicillin/Streptomycin, 20 ng/ml EGF, 10 ng/ml 
			basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 &#181;g/ml insulin</td>
		</tr>
		<tr>
			<td>KSFM-scm</td>
			<td>ThermoFisher Scientific</td>
			<td>17005042</td>
			<td>Supplemented with 1% Penicillin/Streptomycin, 20 ng/ml EGF, 10 ng/ml 
			basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 &#181;g/ml insulin</td>
		</tr>
		<tr>
			<td>MCDB 153-LB basal medium</td>
			<td>Sigma-Aldrich</td>
			<td>M7403</td>
			<td>MCDB 153-LB basal media w/ HEPES buffer</td>
		</tr>
		<tr>
			<td>NEST Scientific 1-Well Cell Culture Chamber Slide, BLACK Walls on Glass Slide, 6/PK, 12/CS</td>
			<td>Stellar Scientific</td>
			<td>NST230111</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>P63</td>
			<td>Thermo Scientific</td>
			<td>703809</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>PE-conjugated anti-EGFR ( San Jose, CA; catalog number )</td>
			<td>BD Pharmingen</td>
			<td>555997</td>
			<td>For FACS analysis</td>
		</tr>
		<tr>
			<td>pMSCV-IRES-EGFP plasmid vector</td>
			<td>Addgene</td>
			<td>20672</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>Promega TransFast kit</td>
			<td>Promega</td>
			<td>E2431</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>Qiagen RNeasy Plus Micro Kit</td>
			<td>Qiagen</td>
			<td></td>
			<td>For microarray experiments</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#8482; Sterile Single Use Vacuum Filter Units</td>
			<td>Thermo Scientific</td>
			<td>09-740-63D</td>
			<td>For cell media</td>
		</tr>
		<tr>
			<td>Zeiss Axionvert 135 fluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Use with Axiovision Rel. 4.5 software</td>
		</tr>
	</tbody>
</table>

establishing a high throughput epidermal spheroid culture system to model keratinocyte stem cell plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

During the skin epidermosphere assay, NHKc cultures are seeded in agarose-coated wells of a 96-well plate (Figure 1A). Spheroid-forming cells should self-aggregate within 48h. Autonomous spheroid formation can be assessed as early as 24 h using a standard inverted phase-contrast microscope. skin epidermosphere formation and re-plating assay model various phases of epidermal tissue regeneration (Figure 1B). Figure 2 shows high resolu...

Watch this Scientific Journal Video about Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity at JoVE.com

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

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