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 laboratory personnel underwent laboratory biosafety training. All procedures were conducted in concordance to the safety and ethics standards of UofSC.
1. Isolation and culture of human keratinocytes from neonatal foreskin tissue
<ol>
	<li>Prepare wash medium by adding 0.1 M HEPES buffer to 500 mL of KSFM medium and adjust it to a pH of 7.2. Sterilize medium in a 500 mL vacuum filter (0.22 &#181;m pore size). MCDB 153-LB basal medium can also be used as an alternative wash solution in place of KSFM.</li>
	<li>In a laminar flow hood, wash neonatal foreskin with 5 mL of wash media in a 50 mL conical tube. Repeat twice.</li>
	<li>Transfer washed foreskin to a sterile Petri dish. Using a scalpel and forceps, scrape off adipose and loose connective tissues from the dermal layer. Rewash foreskin with 5 mL of wash media in a 50 mL conical tube.</li>
	<li>Place foreskin epidermis side up in a 6-well plate containing 2 mL of dispase enzyme diluted in wash media (50 U/mL).</li>
	<li>Transfer the plate to an incubator for 4 hours (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Remove foreskin from the incubator and, under sterile conditions, transfer it to a Petri dish. Using fine-tip forceps, separate the epidermis from dermis layer.</li>
	<li>Place epidermis into a 15 mL conical tube containing 2 mL of 0.25% Trypsin-EDTA. Using a 5 mL serological pipet, mechanically crush the floating epidermis. Incubate for 15 min at 37 &#176;C with periodic vortexing for 5 s every 5 min.</li>
	<li>After incubation, add 2 mL of soybean trypsin inhibitor and mix by pipetting to neutralize the trypsin.</li>
	<li>Centrifuge cell suspension for 2 min at 450 x g, and then for 8 min at 250 x g.</li>
	<li>Remove floating debris with forceps, being careful not to dislodge the cell pellet. Carefully aspirate supernatant from the cell pellet.</li>
	<li>Resuspend pellet in 12 mL of complete KSFM-scm medium and plate in a 10-cm dish. Incubate dish overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>On the following day, remove medium using an aspirating pipette and replace with 12 mL complete KSFM-scm. Repeat on days 4 and 7.</li>
	<li>To maintain optimal growth of normal human keratinocyte (NHKc) cultures, passage cells 1:5 on day 10 to prevent cells from achieving greater than 80% confluency.</li>
</ol>
2. Generating skin epidermosphere cultures in vitro
<ol>
	<li>Prepare a 5% agarose mixture by adding 2.5 g of agarose to 50 mL of 1x phosphate buffered saline (PBS), in a 50 mL glass bottle. Autoclave bottle under liquid cycle. Allow to cool down to room temperature.</li>
	<li>To prepare plates, place the cooled glass bottle containing agarose solution in a 1 L plastic beaker filled with 200 mL deionized water (dH2O). Melt agarose mixture in a research-grade microwave for up to 2 minutes, mixing the agar every 60 s by gently tilting the bottle side to side. 
	CAUTION: Melting agar in the microwave will cause the flask container to become extremely hot resulting in pressure buildup. Release pressure buildup every 30 s. Wear appropriate protective equipment to prevent injury.</li>
	<li>Add 3 mL of melted 5% agarose to 12 mL KSFM-scm prewarmed to 42 &#176;C for a final concentration of 1% agarose (wt/vol). 
	Optional: pre-warm the serological pipettes and pipette tips to prevent premature polymerization of the soft agar.</li>
	<li>Quickly pipette 200 &#181;L of the 1% agar mix into individual wells of a 96-well round-bottom plate using a multichannel pipettor (Figure 1A). Leave plate in sterile environment at 25 &#176;C for 4 h to allow agarose to fully polymerize. 
	NOTE: Experimentation can be stopped here and continued the next day. Polymerized agarose plates can be maintained at 37 &#176;C up to 24 h or at 4 &#176;C for up to 48 h when sealed with parafilm wrap. Warm plate to 37 &#176;C in a humidified incubator for at least 1 h before use.</li>
	<li>Passage spheroid-forming NHKc by aspirating media and washing cells in 2 mL of PBS. Aspirate PBS and add 2 mL of 0.25% Trypsin-EDTA to washed cells. Incubate for 5 min at 37 &#176;C or until all cells completely detach. Add 2 mL of Soybean Trypsin Inhibitor to plate and wash off cells from the plate into a 15 mL tube. Centrifuge at 250 x g for 5 min.</li>
	<li>Resuspend pellet in 1 mL of PBS. Quantify cell viability using trypan blue staining and a hemocytometer or an automated cell counter.</li>
	<li>Aliquot 2 x 104 NHKc in 100 &#181;L of KSFM-scm and seed into individual wells of previously prepared 96-well round-bottom plate. Incubate plate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Using an inverted phase contrast microscope, analyze seeded well for epidermosphere formation. 
	NOTE: Human epidermospheres from normal human keratinocytes can remain viable in 3D suspension culture for up to 96 h, although with considerable decrease in cell viability (Figure 2).</li>
</ol>
3. Epidermal spheroid re-plating assay
<ol>
	<li>24-48 h after 3D epidermosphere formation, add 4 mL of prewarmed KSFM-scm to a 6 cm dish. Use a wide bore 1 mL pipette tip to transfer a single spheroid to the plate, ensuring to not break it apart. Alternatively, widen a 1 mL pipette tip using a sterile razor blade to cut the tip.</li>
	<li>Incubate the plate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Analyze seeded spheroid for attachment to the bottom of the plate and observe for propagating cells using an inverted phase contrast microscope (Figure 3). Feed cells every 96 h by gently removing 2 mL of the media inside the plate and slowly adding 2 mL fresh KSFM-scm media to the plate.</li>
	<li>Passage spheroid-derived (SD) NHKc once they reach 70-80% confluency and continue with assay of choice. Monitor SD-NHKc frequently to prevent cells from reaching full confluency in culture, as this results in premature differentiation and cell growth arrest (Figure 3E-F).</li>
	<li>The epidermal spheroid replating assay models keratinocyte-mediated wound repair by capturing each of the key sequential phases of epidermal regenerative plasticity: a) homeostatic maintenance, b) differentiation halt/reversal c) stress lineage mobility, and d) tissue restoration (Figure 1B; Table 2). This assay can also be used to produce cell populations for organotypic raft cultures or to model HPV-mediated neoplasia as demonstrated in our previous work 11,12.</li>
</ol>
4. 3D fluorescence cell tracking
<ol>
	<li>In a 6 cm dish, transfect spheroid-forming 2D monolayer NHKc cultures with 1 &#181;g of pMSCV-IRES-EGFP plasmid vector carrying the enhanced green fluorescent protein (eGFP) gene. Incubate plate overnight (37 &#176;C, 5% CO2, 95% humidity) microscope (Figure 4A). 
	NOTE: It is important that transfection is completed in serum-free antibiotic-free conditions.</li>
	<li>Without removing transfection mix, add 2 mL of prewarmed KSFM-scm to cells. Incubate plate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Aspirate all transfection media from plate and feed cells with 3 mL of prewarmed KSFM-scm. Assess for presence of EGFP-expressing cells under the FITC channel of a fluorescent microscope.</li>
	<li>Passage and seed 2 x 104 EGFP-transfected cells into individual wells of a previously prepared 96-well round-bottom plate. Incubate plate overnight (37 &#176;C, 5% CO2, 95% humidity). Visualize and monitor EGFPpos spheroid cell movement under the FITC channel of a fluorescence microscope (Figure 4B-C).</li>
	<li>24 h after plating, add 3 mL of prewarmed KSFM-scm to a 6 cm dish. Use a wide bore 1 mL pipette tip to transfer a single spheroid to the plate, ensuring to not break it apart. Incubate plate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Observe and analyze propagating SD-NHKcEGFP using a fluorescence microscope. Feed cells every 96 h by gently removing 2 mL the media inside the plate and slowly adding 2 mL fresh KSFM-scm media to the plate.</li>
	<li>Harvest SD-NHKCEGFP for FACS isolation or assays of choice.</li>
</ol>
5. Characterization of spheroid-derived (SD) sub-populations by FACS
<ol>
	<li>Passage SD-NHKc and corresponding autologous 2D monolayer cultures by aspirating old media and washing cells in 2 mL of PBS. Remove PBS and add 2 mL of 0.25% Trypsin-EDTA to washed cells. Incubate at 37 &#176;C for 5 min or until all cells completely detach.</li>
	<li>Add 2 mL of Soybean Trypsin Inhibitor to plate and transfer cells into a 15 mL tube. Centrifuge at 250 x g for 5 min.</li>
	<li>Resuspend pellets in 1 mL of PBS. Quantify cell viability using trypan blue and an automated cell counter of hemocytometer.</li>
	<li>Aliquot 100 &#181;L containing 0.1-4 x 106 NHKc cells to 1.5 mL microcentrifuge tubes. Place tube on ice in a dark environment, created by turning off bright lights within the laminar hood.</li>
	<li>Add 2 &#181;L of FITC-conjugated anti-integrin&#945;6 and 2 &#181;L of PE-conjugated anti-EGFR to tubes to achieve a 1:50 dilutions. Prepare a tube with no antibodies added to serve as the unstained control. Incubate tubes on ice in dark or at 4 &#176;C for 30 min. The use of beads or a skin SCC line can serve as positive control, as skin SCC cells express elevated levels of integrin&#945;6 and EGFR.</li>
	<li>Perform flow cytometry analysis using flow cytometer of choice containing with appropriate lasers.</li>
	<li>Use the negative and positive controls to establish gates. Sort the subpopulation of integrin&#945;6hi/EGFRlo cells; these are the epidermal stem cell fraction. Integrin&#945;6hi/EGFRhi cells are the proliferative progenitor cell fraction. The integrin&#945;6lo/EGFRhi cells are the committed progenitor cell fraction (Figure 4D). Sorting FACS tube(s) should contain at least 1 mL of ice-cold KSFM-scm.</li>
	<li>Test for proliferative capacity of sorted cell subpopulations by transferring the content of each respective sorting tube into a 15 mL conical tube containing 10x the volume of sorted cells in PBS. NOTE: It is important to remove all cells attached to the rim by pipetting the wall of the sorting FACS tube(s) several times, as keratinocytes can often adhere there.</li>
	<li>Centrifuge 15 mL tubes at 250 x g for 5 min. Remove supernatant and resuspend pellet in 12 mL of KSFM-scm. Transfer the resuspended cells into a 6 cm plate and incubate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>The next day, remove media in plates and add 8 mL pre-warmed KSFM-scm. Incubate at 37 &#176;C, 5% CO2, 95% humidity. Re-feed cells with 12 mL medium every 3 days until 70-80% confluent (Figure 4E).</li>
</ol>
6. Immunofluorescence and staining of epidermospheres for basal stem cell markers
<ol>
	<li>Transfer epidermospheres onto coverslips coated with poly-lysine. Allow SD-NHKc to propagate until 75% confluent.</li>
	<li>Wash cells twice in PBS (4 &#176;C) for 5 min each.</li>
	<li>Fix cells with 4% paraformaldehyde (PFA) for 20 min at room temperature.</li>
	<li>Permeabilize cells with 0.5% Triton X-100 in 1% glycine. Block using 0.5% BSA and 5% goat serum for 30 min at room temperature.</li>
	<li>Incubate samples with antibodies against P63 (1:200) and cytokeratin 14 (1:200) in blocking solution overnight at 4 &#176;C.</li>
	<li>Wash three times with PBS (4 &#176;C) containing Tween 20 (PBST), followed by incubation with FITC-and Alexa 568- conjugated secondary antibodies (1:1000 dilution).</li>
	<li>Stain nuclei with 4&#39;, 6-diamidino-2-phenylindole (DAPI) (1:5000 dilution) before mounting cells.</li>
	<li>Observe cells using a fluorescent-capable microscope with FITC and PE laser lines (Figure 4F).</li>
</ol>
7. Transcriptional analysis of epidermosphere cultures
<ol>
	<li>Set up triplicate plates of low passage NHKc cultures to isolate RNA can be from monolayer, spheroid, and spheroid-derived cultures from the same autologous cell line.</li>
	<li>Pool 3-5 corresponding epidermospheres into a 1.5 mL microcentrifuge tube. Separately harvest autologous SD-NHKc and 2D monolayer cultures.</li>
	<li>Isolate total RNA from all three groups.</li>
	<li>Perform reverse transcription with 1 &#181;g of total RNA.</li>
	<li>Using cDNA, perform real-time PCR, using GAPDH as an internal control (Table 1).</li>
	<li>Validate amplicon product size by agarose gel electrophoresis (2% v/v). 
	NOTE: For transcriptomic-wide profiling of epidermosphere cultures using whole-genome microarray analysis, isolate total RNA from mass cultures of a single NHKc donor and corresponding SD-NHKc from the same donor, in six replicates each.</li>
	<li>Assess RNA quality using a bioanalyzer to achieve RNA Integrity Numbers (RIN) ranging from at least 9.0 to 9.1.</li>
	<li>Perform microarray experiments by amplifying and biotinylating total RNA samples.</li>
	<li>Reverse transcribe 100 ng of total RNA per sample into ds-cDNA using NNN random primers containing a T7 RNA polymerase promoter sequence.</li>
	<li>Add T7 RNA polymerase to cDNA samples to amplify RNA, then copy RNA to ss-cDNA. Degrade excess RNA by using RNase H.</li>
	<li>Fragment sscDNA molecules and label with biotin.</li>
	<li>Amplify labeled samples for 16 h at 45 &#176;C.</li>
	<li>Hybridize samples using a hybridization oven and a wash and stain kit.</li>
	<li>Wash and stain hybridized arrays.</li>
	<li>Scan arrays using a system and computer workstation.</li>
	<li>Following completion of array scans, import probe cell intensity (CEL) files into expression console software and process at the gene-level using library file and Robust Multichip Analysis (RMA) algorithm to generate CHP files.</li>
	<li>After confirming data quality within Expression Console, import CHP files containing log2 expression signals for each probe into a transcriptome analysis software to analyze cell type specific transcriptional responses using one-way between-subject analysis of variance.</li>
</ol>
8. Assessing SD-NHKc colony-forming efficiency
<ol>
	<li>Aspirate media from plates when cells reach 70-80% confluency. Wash cells three times in PBS (4 &#176;C) for 5 min each.</li>
	<li>Fix cells with 3 mL of 100% methanol (4 &#176;C) for 15 min. Wash three times in PBS for 5 min each.</li>
	<li>Stain cells in 3 mL of 10% Giemsa for 30 min. Wash three times in PBS for 5 min each. Allow cells to air dry overnight.</li>
	<li>Analyze colony formation the following day and quantify the number of colonies obtained</li>
</ol>
9. Determine SD-NHKc population doublings
<ol>
	<li>Passage SD-NHKc and corresponding autologous 2D monolayer cultures by aspirating old media and washing cells in 2mL PBS. Remove PBS and add 2 mL 0.25% Trypsin-EDTA to washed cells. Incubate for 5 min or until all cells completely detach (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Add 2 mL of Soybean Trypsin Inhibitor to plate and transfer cells into a 15 mL tube.</li>
	<li>Centrifuge at 250 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend pellet in 12 mL of KSFM-scm.</li>
	<li>Seed cells at low density 1-2 x 104 NHKc into individual 10 cm dishes and incubate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Feed plates with 8 mL KSMF-scm the following day. Feed every 4 days until at least 25% confluent, then feed every 2 days.</li>
	<li>Serially passage cells 1:5 in 60 cm dishes until cell proliferative capacity is exhausted. Quantify cell viability by trypan blue staining. Determine population doublings at each passage using the formula: log(N/N0) / log2, where N represents the total cell number obtained at each passage and N0 represents the number of cells plated at the beginning of the experiment.</li>
</ol>

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The authors do not have financial relationships to disclose.

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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JoVE Journal

Developmental Biology

3.6K Views.  University of South Carolina School of Medicine. 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.

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

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

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

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