Name: isolating hair follicle stem cells and epidermal keratinocytes from dorsal mouse skin
Uploaded: 2016-04-29T15:00:00
Description: Watch this Scientific Journal Video about Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin at JoVE.com

This work was supported in part by NIH grant RO1GM60124 (to H.S.). H.S. is an Investigator with the Howard Hughes Medical Institute. Y.F. is supported by the Deloro Career Advancement Chair and The German Israeli Foundation (I-2381-412.13/2015). D.S. is supported by the Coleman-Cohen post-doctoral fellowship.

This study was performed in strict accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals of Israel&#39;s Ministry of Health. All of the animals were handled according to the approved institutional animal care protocol IL-02302-2015 of the Technion Israel Institute of Technology.
1. Experimental Preparation
<ol>
	<li>Preparation of chelated Fetal Bovine Serum 
	Note: Epithelial cells are very sensitive to calcium so it is crucial to control ...

<ol>
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A., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+between+stem+cells,+niche,+and+progeny+in+the+hair+follicle.">Dynamics between stem cells, niche, and progeny in the hair follicle.</a> Cell. 144, 92-105 (2011).</li><li>Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tortoise+and+the+hair:+slow-cycling+cells+in+the+stem+cell+race.">The tortoise and the hair: slow-cycling cells in the stem cell race.</a> Cell. 137, 811-819 (2009).</li><li>Plikus, M. V., Chuong, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+hair+cycle+domain+patterns+and+regenerative+hair+waves+in+living+rodents.">Complex hair cycle domain patterns and regenerative hair waves in living rodents.</a> J Invest Dermatol. 128, 1071-1080 (2008).</li><li>Ito, M., Kizawa, K., Hamada, K., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+stem+cells+in+the+lower+bulge+form+the+secondary+germ,+a+biochemically+distinct+but+functionally+equivalent+progenitor+cell+population,+at+the+termination+of+catagen.">Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen.</a> Differentiation. 72, 548-557 (2004).</li><li>Cotsarelis, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-retaining+cells+reside+in+the+bulge+area+of+pilosebaceous+unit:+implications+for+follicular+stem+cells,+hair+cycle,+and+skin+carcinogenesis.">Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.</a> Cell. 61, 1329-1337 (1990).</li><li>Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+stem+cells:+a+folliculocentric+view.">Epithelial stem cells: a folliculocentric view.</a> J Invest Dermatol. 126, 1459-1468 (2006).</li><li>Trempus, C. S., 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=Enrichment+for+living+murine+keratinocytes+from+the+hair+follicle+bulge+with+the+cell+surface+marker+CD34.">Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34.</a> J Invest Dermatol. 120, 501-511 (2003).</li><li>Morris, R. J., 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=Capturing+and+profiling+adult+hair+follicle+stem+cells.">Capturing and profiling adult hair follicle stem cells.</a> Nat Biotechnol. 22, 411-417 (2004).</li><li>Lyle, S., 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=The+C8/144B+monoclonal+antibody+recognizes+cytokeratin+15+and+defines+the+location+of+human+hair+follicle+stem+cells.">The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.</a> J Cell Sci. 111 (Pt 21), 3179-3188 (1998).</li><li>Liu, Y., Lyle, S., Yang, Z., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratin+15+promoter+targets+putative+epithelial+stem+cells+in+the+hair+follicle+bulge.">Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge.</a> J Invest Dermatol. 121, 963-968 (2003).</li><li>Vidal, V. P., 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=Sox9+is+essential+for+outer+root+sheath+differentiation+and+the+formation+of+the+hair+stem+cell+compartment.">Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment.</a> Curr Biol. 15, 1340-1351 (2005).</li><li>Snippert, H. J., 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=Lgr6+marks+stem+cells+in+the+hair+follicle+that+generate+all+cell+lineages+of+the+skin.">Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin.</a> Science. 327, 1385-1389 (2010).</li><li>Nijhof, J. G., 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=The+cell-surface+marker+MTS24+identifies+a+novel+population+of+follicular+keratinocytes+with+characteristics+of+progenitor+cells.">The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells.</a> Development. 133, 3027-3037 (2006).</li><li>Jensen, U. B., 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=A+distinct+population+of+clonogenic+and+multipotent+murine+follicular+keratinocytes+residing+in+the+upper+isthmus.">A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus.</a> J Cell Sci. 121, 609-617 (2008).</li><li>Jensen, K. B., 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=Lrig1+expression+defines+a+distinct+multipotent+stem+cell+population+in+mammalian+epidermis.">Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.</a> Cell Stem Cell. 4, 427-439 (2009).</li><li>Jaks, V., 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=Lgr5+marks+cycling,+yet+long-lived,+hair+follicle+stem+cells.">Lgr5 marks cycling, yet long-lived, hair follicle stem cells.</a> Nat Genet. 40, 1291-1299 (2008).</li><li>Horsley, V., 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=Blimp1+defines+a+progenitor+population+that+governs+cellular+input+to+the+sebaceous+gland.">Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland.</a> Cell. 126, 597-609 (2006).</li><li>Goldstein, J., Horsley, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Home+sweet+home:+skin+stem+cell+niches.">Home sweet home: skin stem cell niches.</a> Cell Mol Life Sci. 69, 2573-2582 (2012).</li><li>Fuchs, Y., 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=Sept4/ARTS+regulates+stem+cell+apoptosis+and+skin+regeneration.">Sept4/ARTS regulates stem cell apoptosis and skin regeneration.</a> Science. 341, 286-289 (2013).</li><li>Nowak, J. 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The protocol described here is well established for isolating HFSCs from the dorsal skin of adult mice but can be equally applied for isolation of other populations within the HF structure, based on the selection of markers 2,16,23,28,29. This method is notably advantageous over other methods of cell isolation, such as tissue dissociation, in that a specific cell type can be selected and harvested from a mixture of heterogeneous cell populations. Furthermore, the method described here is fast and reliable and ...

Adult stem cells (SCs) are essential for maintaining tissue homeostasis by replacing dying cells and repairing damaged tissues upon injury. These SCs are defined by their ability to undergo continual self-renewal and to differentiate into various cell lineages 1-3. The best studied systems, which are dependent upon adult SCs for their replenishment, include the hematopoietic system, the intestine and the skin 1,2,4.
During embryogenesis, the skin begins as a single layer of epidermal cells. Morphogenesis of the hair follicle (HF) starts when mesenchymal cells populate the skin and form an underlying collagenous dermis 5. Specialized mesenchymal cells, that later constitute the dermal papilla (DP), organize directly beneath the epidermal layer and stimulate the epithelium to form hair placodes that begin to grow downwards 6. Highly proliferating matrix cells, situated at the bottom of the HF, envelope these mesenchymal cells and form the hair bulb, while the inner layer begins to differentiate into concentric cylinders to form the hair shaft (HS) and the surrounding inner root sheath (IRS) 2,3.
In postnatal life the skin epidermis is comprised of three compartments: the interfollicular epidermis (IFE), the sebaceous gland (SG) and the HF. In contrast to the IFE and SG which are in a constant state of homeostasis, the HF is a dynamic mini-organ which undergoes continuous cycles of growth (anagen), destruction (catagen) and rest (telogen) 4,7. The hair follicle stem cells (HFSCs) that fuel this perpetual cycle, reside in a specialized niche within the HF known as the bulge 4. During anagen the HFSCs exit the bulge, following activation signals from the DP, begin proliferating and descend downward thus creating a long linear trail of cells known as the outer root sheath (ORS) 8-10. The matrix cells, that surround the DP at the base of the HF, rapidly cycle and migrate upward undergoing terminal differentiation thus generating the HS and the IRS 10 (Figure 1). The duration of anagen determines the length of the hair and is dependent on the proliferative and differentiation capacity of the matrix cells 6. When the HF enters catagen, the transit-amplifying matrix cells in the bulb cease to proliferate, undergo apoptosis and regress entirely while pulling the DP upward until it reaches the non-cycling part of the HF 8,11. During this retraction the HF forms a temporary structure known as the epithelial strand, which is characteristic of catagen, and contains many apoptotic cells. In mice, catagen lasts between 3-4 days and is highly synchronized in the first hair cycle. When the HF reaches telogen all HFSCs become quiescent. The distinct stages of the HF cycle are also characterized by changes in the color of the mouse&#39;s skin owing to melanin production. The skin changes from black during anagen to dark grey during catagen to pink during telogen 6,7,12,13.
<img alt="Figure 1" src="/files/ftp_upload/53931/53931fig1.jpg" /> 
Figure 1: The Hair Follicle Cycle. The HF is composed of a permanent upper part and a lower constantly remodeling, cycling portion that undergoes continuous cycles of rapid growth (anagen), destruction (catagen) and a relative quiescence phase or rest (telogen). <a href="https://www.jove.com/files/ftp_upload/53931/53931fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The SCs maintaining the HF were initially identified using chase experiments, with tritiated thymidine, that revealed a population of slow cycling label retaining cells (LRC) that resided in the permanent region of the HF just below the SG 14. Advances in HFSC characterization revealed a small number of markers that can be used to identify and isolate specific SCs from the HF niche 15. Perhaps the best marker for enrichment of HFSCs is CD34, a cell surface marker also identified as a hematopoietic SC marker in humans 16. Within this CD34+ populations two distinct populations have also been isolated based on &#945;6 integrin expression 2. Another marker is keratin 15 (K15) which is highly expressed in the bulge region, co-localizes with CD34 expression and a K15 promoter is used for targeting and isolating HFSCs in transgenic animals 15,17-19. In the past decade several other distinct populations of HFSCs and progenitor cells have also been reported to reside within the HF 17,20-27.
An additional exciting feature of HFSCs is their contribution to skin repair. Under normal conditions HFSCs replenish the HF and do not take part in IFE homeostasis. However, in response to wounding, these cells exit their SC niche and aid in repopulating the IFE 9. We have recently demonstrated that mice deleted for the pro-apoptotic Sept4/ARTS gene display an increased number of CD34, K15 and Sox9+ HFSCs, which demonstrate a resistance to apoptosis. HFSCs were isolated from Sept4/ARTS-/- dorsal skins utilizing fluorescence activated cell sorting (FACS) and there was more than a two fold increase in the number of CD34+ and K15+ HFSCs. These Sept4/ARTS-/- HFSCs were expanded in vitro and not only gave rise to more colonies but were also able to withstand harsher conditions as compared to controls 28.
As a result of having an increased number of HFSCs, Sept4/ARTS-/- mice healed significantly faster in response to skin excision injuries. Strikingly, Sept4/ARTS-/- mice displayed a large number of regenerated HFs from the wound bed, and significantly smaller scars. Furthermore, mice deleted for XIAP (X-linked inhibitor of apoptosis), the biochemical target of ARTS, demonstrated impaired healing 28.
Our results and work performed in other laboratories have shown that HFSCs serve as an ideal model for studying the biology and function of adult SCs. Here, we describe the methodology for the enrichment and isolation of HFSCs and epidermal keratinocytes based on the expression of four markers: integrin &#945;6; integrin &#946;1; Sca-1 (a marker for epidermal keratinocytes) and CD34. Similar isolation of K15+ HFSCs can also be performed using the K15-GFP reporter mouse 19.

<table border="0" cellpadding="0" cellspacing="0" width="1027">
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		<tr height="32">
			<td height="32" style="height:32px;width:295px;">Isoflurane&#160;</td>
			<td style="width:247px;">Primal Critical Care</td>
			<td style="width:144px;">&#160;66794-017-10</td>
			<td style="width:341px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Carbon dioxide</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Electro Shaver</td>
			<td>Oster&#160;</td>
			<td>Golden A5</td>
			<td>Shaver from any other company could be used</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">70% ethanol</td>
			<td>Gadot Lab</td>
			<td>830000051</td>
			<td>96% ehtanol diluted with distilled water&#160;</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Dissection mat</td>
			<td></td>
			<td></td>
			<td>Dissection tools from any provider can be used</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Forceps</td>
			<td>Dumont</td>
			<td>11251-10</td>
			<td>Foreceps from any other company could be used</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Scissors&#160;</td>
			<td>Dumont</td>
			<td>14094-11</td>
			<td>Scissors from any other company could be used</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Needles/Pins</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Scalpel</td>
			<td>Albion</td>
			<td>10</td>
			<td>Ensure that the scalpel has a blunt end</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Tissue culture dish 60mm x 15mm</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430166</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">PBS</td>
			<td>-</td>
			<td></td>
			<td>In-house PBS without Calcium and Magnesium</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;">0.25% Trypsin/EDTA</td>
			<td>Biological Industries</td>
			<td>03-050-1A</td>
			<td style="width:341px;">Trypsin obtained from a different company might have a different activity and duration of the trypsin digest has to be adjusted accordingly</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Pipettes 10ml</td>
			<td>Sigma-Aldrich</td>
			<td>Corning, 4488</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Ice</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">50 ml sterie centrifuge tubes</td>
			<td>Minplast Ein-shemer</td>
			<td style="width:144px;">35050-43</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">70&#181;M Cell strainer</td>
			<td>Fisher</td>
			<td>22362548</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">40&#181;M Cell strainer</td>
			<td>Fisher</td>
			<td>22362549</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Staining buffer</td>
			<td>-</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Centrifuge</td>
			<td>Eppendorf 5804 R</td>
			<td>5805 000.017</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">FACS tubes with Cell strainer caps&#160;</td>
			<td>Falcon&#160;</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">FACS tubes&#160;</td>
			<td>Falcon&#160;</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Integrin &#946;1</td>
			<td>eBioscience</td>
			<td>25-0291</td>
			<td>1:400</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Integrin &#945;6</td>
			<td>eBioscience</td>
			<td>15-0495</td>
			<td>1:600</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Sca I</td>
			<td>eBioscience</td>
			<td>11-5981</td>
			<td>1:200</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">CD34</td>
			<td>eBioscience</td>
			<td>9011-0349</td>
			<td>1:300</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>50ng/ml</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Dry Chelex</td>
			<td>BioRad</td>
			<td>142-2842</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Beaker&#160;</td>
			<td>Pyrex</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Distilled H2O&#160;</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Stir bar</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">NHCl</td>
			<td>BioLab</td>
			<td>1903059</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Fetal bovine serum (FBS)</td>
			<td>Beit Haemek Biological Industries</td>
			<td>400718</td>
			<td>FBS obtained from a different company can be used</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">1L glass bottle</td>
			<td>Ilmabor</td>
			<td>Boro 3.3</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Bottle top filter</td>
			<td>Autofil</td>
			<td>1102-RLS</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This protocol describes in detail the enrichment and isolation of two types of populations: bulge SCs and epidermal keratinocytes. Figure 2 illustrates the major steps of the protocol. Utilizing skin removed from the dorsal back of 8 week old mice, we enriched bulge SCs using the CD34 marker, which is only expressed in HFSCs; and Sca-1, which labels epidermal keratinocytes. Figure 3 shows different patterns of CD34 and Sca-1 expression within the &#945;6<...

Watch this Scientific Journal Video about Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin at JoVE.com

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

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 ...

The overall goal of this protocol is to isolate different populations of stem cells and epidermal keratinocytes from the dorsal mouse skin. This protocol allows the user to simultaneously isolate multiple cell populations from the dorsal mouse skin. The main advantages of this technique are that it is fast, reliable, and can be used to isolate specific cell types of interest from a mixture of heterogeneous skin cell populations.Demonstrating the procedure will be Dr.Yahav Yosefzon from my laboratory. Begin by using electric clippers to shave all of the dorsal skin hair from a 50 to 80 day old mouse in the telogen phase of the hair follicle cycle, keeping the clippers as close to the skin as possible, without damaging the skin and subcutaneous layer. Next, use 70%ethanol to disinfect the skin and to remove any hair residue.Place the mouse onto a dissecting pad. Then use forceps to lift the skin near the tail, and nick the skin with scissors. To harvest the entire dorsal skin, carefully cut the tissue in a posterior-anterior direction, separating the skin from the fascia.When all of the skin has been removed, pin the two adjacent edges of the tissue onto a dissecting mat, hair side down. Then, using curved forceps, apply light pressure to keep the skin from tearing and gently scrape away the fat with a blunted scalpel until the dermis is clear and neat. When all of the fat has been removed, place the skin dermis side down in a 100 millimeter sterile culture dish, and straighten out the tissue until there are no folds.Wash the skin with 10 milliliters of PBS without calcium and magnesium, straightening the skin again as necessary. Then remove the PBS and incubate the tissue in 10 milliliters of 0.25%trypsin, taking care that the skin is unfolded and freely floating. At the end of the incubation, using curved forceps to hold the skin in place, scrape all of the hair from the skin, starting at the tail and following the direction of hair growth.As the follicles tend to form small clumps, scrape small areas at a time to reduce the size of the clumps as much as possible. When all of the hair follicles have been collected, transfer the hairless skin to a new culture dish and hydrate the tissue with 10 milliliters of PBS without calcium and magnesium. Confirm that all of the hair follicles have been removed.Then use a scalpel and forceps to break down the follicles until a single hair follicle suspension is obtained. Next, vigorously triturate the hair follicle solution with a 10 milliliter pipette for a few minutes to break up all of the clumps. Then transfer the cells into 50 milliliter tubes on ice, pre-labeled with the animal and sample numbers.Now aspirate the PBS from the skin and use it to rinse the cell culture dish that contained the hair follicle suspension. Pool the wash with the hair follicle cells and filter the cell suspension through a 70 micron cell strainer into a new 50 milliliter tube. Wash the first 50 milliliter tube and the strainer with 10 milliliters of staining buffer.Then filter the suspension through a 40 micron strainer into a new 50 milliliter tube, and wash the second tube with five to 10 milliliters of fresh staining buffer. Next, spin down the cells two times, washing the cells in five milliliters of fresh straining buffer during the second centrifugation. After the second spin, re-suspend the pellet in 800 microliters of fresh staining buffer.Then transfer 25 to 50 microliters of cells to the unstained and DAPI control tubes, and add enough staining buffer to bring the total volume in each control tube up to 300 microliters. Now add the primary antibodies and DAPI to the appropriate sample tubes and mix the cells with gentle flicking. Label the cells for 30 minutes on ice in the dark with flicking every 10 minutes to keep the cells in suspension.At the end of the incubation, wash the cells with approximately four milliliters of staining buffer per tube, and re-suspend the pellets in 800 microliters of staining buffer. Then filter the cells into fax tubes with cell strainer caps to ensure a single-cell suspension during their analysis. In these graphs, different patterns of CD34 and Sca-1 cell surface expression within the alpha six positive beta one positive population of skin epithelial cells are shown.The cells were first gated according to their expression of integrin alpha six and integrin beta one. The integrin alpha six positive integrin beta one positive cells were then gated according to their CD34 and Sca-1 expression. Two distinct populations of cells were observed.The alpha six beta one CD34 positive Sca-1 negative cells, which represent the hair follicle stem cells, and the alpha six beta one Sca-1 positive CD34 negative cells, which represent the epidermal keratinocytes. Typically, 1.5 to five times 10 to the fifth alpha six beta one positive cells per animal or CD34 positive hair follicle stem cells. Once mastered, this technique can be completed in approximately eight hours, if properly performed.After watching this video, you should have a good understanding of how to dissociate the mouse dorsal skin and to isolate hair follicle stem cells and epidermal keratinocytes.

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Stem Cell Biology And Renewal in Epithelial Tissue

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