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...

<ol>
	<li>Patel, G. K., Wilson, C. H., Harding, K. G., Finlay, A. Y., Bowden, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerous+keratinocyte+subtypes+involved+in+wound+re-epithelialization.">Numerous keratinocyte subtypes involved in wound re-epithelialization.</a> Journal of Investigative Dermatology. 126 (2), 497-502 (2006).</li><li>Dekoninck, S., Blanpain, C. <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+dynamics,+migration+and+plasticity+during+wound+healing.">Stem cell dynamics, migration and plasticity during wound healing.</a> Nature Cell Biology. 21 (1), 18-24 (2019).</li><li>Byrd, K. 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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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphere+formation+increases+the+ability+of+cultured+human+dermal+papilla+cells+to+induce+hair+follicles+from+mouse+epidermal+cells+in+a+reconstitution+assay.">Sphere formation increases the ability of cultured human dermal papilla cells to induce hair follicles from mouse epidermal cells in a reconstitution assay.</a> Journal of Investigative Dermatology. 132 (1), 237-239 (2012).</li><li>Woappi, Y., Hosseinipour, M., Creek, K. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+multipotent+skin-derived+precursors+from+human+skin.">Isolation and characterization of multipotent skin-derived precursors from human skin.</a> Stem Cells. 23 (6), 727-737 (2005).</li><li>Li, F., 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=Loss+of+the+Epigenetic+Mark+5-hmC+in+Psoriasis:+Implications+for+Epidermal+Stem+Cell+Dysregulation.">Loss of the Epigenetic Mark 5-hmC in Psoriasis: Implications for Epidermal Stem Cell Dysregulation.</a> Journal of Investigative Dermatology. , (2020).</li><li>Kuo, C. T., 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=Three-dimensional+spheroid+culture+targeting+versatile+tissue+bioassays+using+a+PDMS-based+hanging+drop+array.">Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.</a> Scientific Reports. , (2017).</li><li>Woappi, Y., Ezeka, G., Vercellino, J., Bloos, S. M., Creek, K. 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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 ...

This protocol presents a novel method for the cultivation and maintenance of epidermal sphere cultures, and the strategy to closely investigate them using the spheroid replating assay. Begin by preparing wash medium as described in the text manuscript. In a laminar flow hood, wash neonatal foreskin twice with five milliliters of wash media in a five milliliter conical tube, then transfer the washed foreskin to a sterile Petri dish.Using a scalpel and forceps, scrape off adipose tissue and loose connective tissues from the dermal layer, then rewash the foreskin with wash media. Place the foreskin epidermis side up in a six-well plate containing two milliliters of Dispase enzyme diluted in wash media. Transfer the plate to an incubator for four hours.After incubation, transfer the foreskin to a Petri dish and use fine-tip forceps to separate the epidermis from the dermis layer. Place the epidermis into a 15 milliliter conical tube containing two milliliters of 0.25%Trypsin-EDTA. Crush the floating epidermis using a five milliliter serological pipette and incubate it for 15 minutes at 37 degrees Celsius with periodic vortexing for five seconds every five minutes.After incubation, add two milliliters of soybean trypsin inhibitor and mix it by pipetting to neutralize the trypsin, then centrifuge the cell suspension. Resuspend the pellet in 12 milliliters of complete KSFM-SCM medium and plate the cells in a 10 centimeter dish to incubate overnight at 37 degrees Celsius and 5%carbon dioxide. On the next day, remove the medium using an aspirating pipette and replace it with 12 milliliters of complete KSFM-SCM.Repeat this on days four and seven. Prepare 5%agarose mixture by adding 2.5 grams of agarose to 50 milliliters of PBS in a 100 milliliter glass bottle. Autoclave the bottle onto the liquid cycle and allow it to cool to room temperature.To prepare plates, place the cooled glass containing agarose solution in a one liter beaker filled with 200 milliliters of deionized water. Melt the agarose mixture in a research-grade microwave for up to two minutes, mixing the agarose every 60 seconds by gently tilting the bottle side to side. Add three milliliters of melted 5%agarose to 12 milliliters of prewarmed KSFM-SCM at 42 degrees Celsius for a final concentration of 1%agarose.Add 200 microliters of the 1%agar mix to each well of a 96-well plate using a multi-channel pipetter. Then leave the plate in a sterile environment at 25 degrees Celsius for four hours. Passage spheroid-forming normal human keratinocytes by aspirating media and washing cells in two milliliters of PBS.Aspirate PBS and add two milliliters of 0.25%Trypsin-EDTA to the washed cells. Then incubate for five minutes at 37 degrees Celsius. After the incubation, add two milliliters of soybean trypsin inhibitor to the plate and wash the cells from the plate into a 15 milliliter tube.Centrifuge at 250 times G for five minutes. Resuspend the cell pellet in one milliliter of PBS and quantify the cells using Trypan Blue staining. Aliquot 20, 000 normal human keratinocytes in 100 microliters of KSFM-SCM and seed the cells in each well of the previously prepared 96-well plate.Then incubate the plate overnight. Using an inverted phase contrast microscope, analyze the seeded wells for epidermis sphere formation. 24 to 48 hours after 3D epidermis sphere formation, add four milliliters of prewarmed KSFM-SCM at 37 degrees Celsius to a six centimeter dish.Using a wide bore one milliliter pipette tip, transfer a single spheroid to the plate and incubate the plate overnight. Analyze the seeded spheroid for attaching or propagating cells using an inverted phase contrast microscope. Feed cells every 96 hours by removing the media and adding two milliliters of fresh KSFM-SCM media to the plate.Passage 70 to 80%confluent spheroid-derived normal human keratinocytes and 2D monolayer cultures as previously demonstrated. Then quantify cell viability using Trypan Blue and an automated cell counter. Aliquot 100 microliters containing 100, 000 to 4 million normal human keratinocytes into 1.5 milliliter microcentrifuge tubes.Place the tubes on ice in a dark environment by turning off the bright lights within the laminar hood. Add two microliters of FITC-conjugated anti-integrin alpha six and two microliters of PE-conjugated anti-EGFR to the tubes to achieve a one to 50 dilution. Prepare a tube with no antibodies added to serve as the unstained control.Incubate the tubes on ice in the dark for 30 minutes. Perform flow cytometry analysis with appropriate lasers. Use the negative and positive controls to establish gates.Sort the subpopulation of epidermal stem cell fraction, the proliferative progenitor cell fraction, and committed progenitor cell fraction. Test for proliferative capacity of sorted cell subpopulations by transferring the content of each respective sorting tube into a 15 milliliter conical tube containing 10 times the volume of sorted cells in PBS. Centrifuge the tubes at 250 times G for five minutes.Remove the supernatant and resuspend the pellet in four milliliters of KSFM-SCM. Transfer the resuspended cells to a six centimeter plate and incubate overnight at 37 degrees Celsius in a 5%carbon dioxide incubator with 95%humidity. On the next day, remove media from the plates and add four milliliters of prewarmed KSFM-SCM at 37 degrees Celsius every three days until 70 to 80%confluency is reached.Autonomous epidermal spheroid-forming ability of normal human keratinocytes in 3D culture was assessed using the skin epidermis sphere assay. Non-spherical cell aggregation was not considered as adequate epidermis sphere formation. Dense sphere-shaped aggregation was considered a hallmark of spontaneous spheroid formation.It was necessary to use more than two times 10 to the four cells to ensure proper spontaneous aggregation. Planting of epidermis spheres in 2D culture resulted in the proliferation of a small-sized viable normal human keratinocytes. It's important to maintain these cultures below 100%confluency as this can considerably impair their growth and stem cell state in culture.The process of epidermal spheroid formation was functionally tracked at the single cell level by transfected cells with a fluorescent reporter. Spheroid-derived normal human keratinocytes subpopulation are integrin alpha six high and EGF are low cells. The cells generally make up about 25%of cultures.Characterization of this stem-like keratinocyte subpopulation was achieved by immunostaining analysis of basal cytokeratin 14 and tumor protein 63. The spheroid replating assay is an effective strategy for epidermal stem cell enrichment from neonatal human skin. This system can be employed as a high-throughput cell-based model to investigate cutaneous diseases such as wound healing and cancer.

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Developmental Biology

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.

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

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 ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...