Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Summary

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Abstract

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.

Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo ^1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress ^4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue ⁶. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles ^7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality ¹⁰.

Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias ^11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies ^14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)^16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

Protocol

1. Extract Epithelial Stem Cells from Human Skin

1. Before starting the procedure of isolating epithelial stem cells one needs to prepare the respective medias and reagents (see table1).
2. Fresh adult human scalp skin from facelift procedures or punch biopsy is collected, then incubate in DMEM / 10% FBS / Dispase (4 mg/ mL) overnight at 4°C. Incubation for 2-4 hr at 37°C is also effective. Skin pieces should be a maximum width of 1 cm to allow for enzyme to penetrate.
3. Transfer the skin into a sterilized Petri dish, pull off each hair from the skin by grasping the hair shaft near the skin surface and pulling firmly and smoothly. Select foll....

Discussion

The cell extraction and culture methods described are surprisingly facile and reproducible. We have generated epithelial stem cell cultures from dozens of individuals across a broad age range, including patients with inherited skin defects¹⁸. It is best to begin the process on the day of tissue harvest, however cells will remain viable in media on ice for several days, facilitating overnight shipping if needed. Discarded facelift skin yields hundreds of viable follicles for cell extraction as single cell suspe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	GIBCO, by Life Technologies	11995
Hams F12	GIBCO, by Life Technologies	11765
Fetal Bovine Serum (FBS)	GIBCO, by Life Technologies	16000
Insulin	GIBCO, by Life Technologies	12585
T3	Sigma-Aldrich	T-2752
Transferrin	Roche Group	10652202001
Hydrocortisone	Sigma-Aldrich	H-4001
Cholera Toxin	Sigma-Aldrich	C8052
Epidermal growth factor (EGF)	Sigma-Aldrich	E-9644
Adenine	Sigma-Aldrich	A9795
Trypsin(10X)	GIBCO, by Life Technologies	15090
VERSENE	GIBCO, by Life Technologies	15040
G418 Sulfate	Cellgro	30-234-CR
Hanks’ Balanced Salt solution	Sigma-Aldrich	H6648
1X PBS	Cellgro	21-040-CV
Mitomycin C	Roche Group	10107409001
Penicillin/streptomycin	Invitrogen	15140122
Dispase	Invitrogen	17105
Crystal Violet	Fisher Scientific	C581-25
Keratinocyte media (KCM) [DMEM and Ham s F12 (GIBCO, 3:1), adenine (Sigma, 180 mM), 10% fetal bovine serum (GIBCO), cholera toxin (ICN, 0.1 nM), penicillin/streptomycin (GIBCO, 100 U/ml and 100 mg/ml, respectively), hydrocortisone (Sigma, 0.4 mg/ml, 1.1 mM), T/T3 (transferrin, GIBCO, 5 μg/ml, 649 nM; and triiodo-l-thyronine, Sigma, 2 nM), insulin (Sigma, 5 mg/ml, 862 nM), and EGF (Sigma, 10 ng/ml, 1.6 nM), pH 7.2]
[DMEM and Ham s F12 (GIBCO, 3:1), adenine (Sigma, 180 mM), 10% fetal bovine serum (GIBCO), cholera toxin (ICN, 0.1 nM), penicillin/streptomycin (GIBCO, 100 U/ml and 100 mg/ml, respectively), hydrocortisone (Sigma, 0.4 mg/ml, 1.1 mM), T/T3 (transferrin, GIBCO, 5 μg/ml, 649 nM; and triiodo-l-thyronine, Sigma, 2 nM), insulin (Sigma, 5 mg/ml, 862 nM), and EGF (Sigma, 10 ng/ml, 1.6 nM), pH 7.2]