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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Unit 1

Stem Cell Biology And Renewal in Epithelial Tissue

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Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...