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

Summary

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.

Abstract

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 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 integrinα6^hi/EGFR^lo 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.

Introduction

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 repair^1,2,3. This multifaceted response occurs in several sequential steps which remain poorly understood.

A major obstacle in studying the intricate proce....

Protocol

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 "research not involving human subjects", 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.

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Affymetrix platform	Affymetrix		For microarray experiments
Affymetrix’s HuGene-2_0-st library file	Affymetrix		Process
Agilent 2100 Bioanalyzer	Agilent		For microarray experiments
All Prep DNA/RNA Mini Kit	Qiagen	80204	Used for RNA isolation
Analysis Console Software version 3.0.0.466			analyze cell type specific transcriptional responses using one-way between-subject analysis of variance
BD FACSAria II flow cytometer	Beckman		For flow cytometry
Console Software version 3.0.0.466/Expression console Software	Affymetrix/Thermo Fisher Scientific		For confirming data quality
Cytokeratin 14	Santa Cruz Biotechnology	sc-53253	1:200 dilution
Dispase	Sigma-Aldrich	D4818	For cell media
FITC-conjugated anti-integrinα6	Abcam	ab30496	For FACS analysis
GeneChip Command Console 4.0 software	Affymetrix/Thermo Fisher Scientific		For confirming data quality
GeneChip Fluidics Stations 450 (Affymetrix/Thermo Fisher Scientific)	Affymetrix/Thermo Fisher Scientific		For washing and staining of hybridized arrays
GeneChip HuGene 2.0 ST Arrays	Affymetrix/Thermo Fisher Scientific		For hybridization and amplifycation of total RNA
GeneChip Hybridization Oven 640	Thermo Fisher Scientific		For hybridization and amplifycation of total RNA | Amplify labeled samples
GeneChip Hybridization Wash, and Stain Kit (Affymetrix/Thermo Fisher Scientific).	Affymetrix/Thermo Fisher Scientific		For washing and staining of hybridized arrays
GeneChip Scanner 3000 7G system	Affymetrix/Thermo Fisher Scientific		Scanning hybridized arrays
GeneChip WT PLUS Reagent Kit	Affymetrix/Thermo Fisher Scientific		For amplifycation of biotinylating total RNA
Human Basic Fibroblast Growth Factor (hFGF basic/FGF2)	Cell Signaling Technology	8910	For cell media
Human Epidermal Growth Factor (hEGF)	Cell Signaling Technology	8916	For cell media
Human Insulin	Millipore Sigma	9011-M	For cell media
iQ SYBR Green Supermix (Bio-Rad)	Bio-Rad	1708880	Used for RT-qPCR
iScript cDNA Synthesis Kit	Bio-Rad	1708890	Used for RT-qPCR
KSFM	ThermoFisher Scientific	17005041	Supplemented with 1% Penicillin/Streptomycin, 20 ng/ml EGF, 10 ng/ml basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 µg/ml insulin
KSFM-scm	ThermoFisher Scientific	17005042	Supplemented with 1% Penicillin/Streptomycin, 20 ng/ml EGF, 10 ng/ml basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 µg/ml insulin
MCDB 153-LB basal medium	Sigma-Aldrich	M7403	MCDB 153-LB basal media w/ HEPES buffer
NEST Scientific 1-Well Cell Culture Chamber Slide, BLACK Walls on Glass Slide, 6/PK, 12/CS	Stellar Scientific	NST230111	For immunostaining
P63	Thermo Scientific	703809	1:200 dilution
PE-conjugated anti-EGFR ( San Jose, CA; catalog number )	BD Pharmingen	555997	For FACS analysis
pMSCV-IRES-EGFP plasmid vector	Addgene	20672	For transfection
Promega TransFast kit	Promega	E2431	For transfection
Qiagen RNeasy Plus Micro Kit	Qiagen		For microarray experiments
Thermo Scientific™ Sterile Single Use Vacuum Filter Units	Thermo Scientific	09-740-63D	For cell media
Zeiss Axionvert 135 fluorescence microscope	Zeiss		Use with Axiovision Rel. 4.5 software