Name: isolation and culture of adult epithelial stem cells from human skin
Uploaded: 2011-03-31T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Adult Epithelial Stem Cells from Human Skin at JoVE.com

This work is funded by NIH/ NCI grant R01CA-118916

1. Extract Epithelial Stem Cells from Human Skin
<ol>
<li>Before starting the procedure of isolating epithelial stem cells one needs to prepare the respective medias and reagents (see table1).</li>
<li>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&deg;C. Incubation for 2-4 hr at 37&deg;C is also effective. Skin pieces should be a maximum width of 1 cm to allow for enzyme to penetrate.</li>
<li>Transfer the ski...

<ol>
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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...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11995</td>
<td>&nbsp;</td>
<tr>
<td>Hams F12</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11765</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Insulin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>12585</td>
<td>&nbsp;</td>
<tr>
<td>T3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T-2752</td>
<td>&nbsp;</td>
<tr>
<td>Transferrin</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10652202001</td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-4001</td>
<td>&nbsp;</td>
<tr>
<td>Cholera Toxin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C8052</td>
<td>&nbsp;</td>
<tr>
<td>Epidermal growth factor (EGF)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E-9644</td>
<td>&nbsp;</td>
<tr>
<td>Adenine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9795</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin(10X)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15090</td>
<td>&nbsp;</td>
<tr>
<td>VERSENE</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15040</td>
<td>&nbsp;</td>
<tr>
<td>G418 Sulfate</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-234-CR</td>
<td>&nbsp;</td>
<tr>
<td>Hanks&#x2019; Balanced Salt solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6648</td>
<td>&nbsp;</td>
<tr>
<td>1X PBS</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>21-040-CV</td>
<td>&nbsp;</td>
<tr>
<td>Mitomycin C</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10107409001</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140122</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17105</td>
<td>&nbsp;</td>
<tr>
<td>Crystal Violet</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>C581-25</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			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 &mu;g/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]
		</div>

isolation and culture of adult epithelial stem cells from human skin

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.

Watch this Scientific Journal Video about Isolation and Culture of Adult Epithelial Stem Cells from Human Skin at JoVE.com

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

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.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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