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 the exposure of these cells to calcium. In order to ensure that cells are not exposed to calcium during the isolation process chelation is used to remove any residual calcium from the Fetal Bovine Serum used for preparation of staining buffer 29. The following protocol prepares approximately 1 L of Calcium-free Fetal Bovine Serum required for the preparation FACS staining buffer.
	<ol>
		<li>Add 400 g of dry chelating material, e.g., Chelex, into a 4 L beaker and add distilled H2O to a total volume 4 L. Cover and stir continuously using a magnetic stirrer.</li>
		<li>Adjust pH to 7.4 using 10 N HCl solution while stirring. Continue stirring for 20 min and re-adjust the pH as needed until pH remains stable for more than 20 min.</li>
		<li>Incubate beaker at 4 &#176;C overnight to allow the chelating material to form a compact pellet. Carefully aspirate H2O and add fresh distilled H2O to a total volume of 4 L.</li>
		<li>Repeat the pH adjustment as in step 1.1.2.</li>
		<li>Place the beaker at 4 &#176;C for 1 hr to allow the chelating material to form a compact pellet. Carefully aspirate H2O.</li>
		<li>Slowly add two 500 ml bottles of fetal bovine serum to the chelating material. Stir slowly at 4 &#176;C for 1 hr ensuring that the speed setting does not produce bubbles.</li>
		<li>Place the beaker at 4 &#176;C for 1 hr to allow the chelating material to form a compact pellet.</li>
		<li>Carefully transfer the serum into 1 L glass bottle and filter it through a bottle top filter under sterile conditions. Store the chelated serum at -20 &#176;C or use immediately.</li>
	</ol>
	</li>
	<li>Preparation of Staining Buffer
	<ol>
		<li>Prepare 100 ml of 3% chelated FBS in PBS without Ca2+ and Mg2+ and keep on ice.</li>
	</ol>
	</li>
</ol>
2. Isolation of Hair Follicles from Adult Epidermis
<ol>
	<li>Anesthetize mice using 5% isoflurane using an induction box. The protocol described here was optimized using 50-80 day old mice that are in the telogen phase of the HF cycle.</li>
	<li>Euthanize mice according to standard laboratory procedures. Here, euthanize mice using CO2 overdose in a euthanasia chamber.</li>
	<li>Shave all hair from the dorsal skin using electric clippers. Avoid head and limbs. Keep the clippers as close as possible to the skin. Avoid damaging the skin and subcutaneous layer.</li>
	<li>When all hair is removed, use 70% ethanol to remove any hair residue and to disinfect the dorsal skin.</li>
	<li>Place the mouse on a dissecting pad, and using forceps pull up the skin near the tail and make a small nick using scissors. Cut the entire dorsal skin in a posterior-anterior direction by carefully cutting away the skin and separating it from the fascia. Avoid damaging the subcutaneous layer.</li>
	<li>Place the skin on a dissecting mat, hair side facing down, and pin down two adjacent edges of the skin to fix in place. Gently scrape off the fat, using a blunt scalpel until the dermis is clear and neat. 
	Note: When scraping off the fat, use curved forceps to apply gentle pressure on the skin. Apply pressure on the skin using the curved side of forceps. This will prevent the skin from tearing.</li>
	<li>Place the skin, dermis side down, in 100 mm sterile culture dish, and straighten the skin to ensure that there are no folds. Add 10 ml of PBS without Ca2+ and Mg2+ (PBS--) to the culture dish and straighten the skin if needed.</li>
	<li>Aspirate the PBS and add 10 ml of 0.25% trypsin. Ensure that the skin is unfolded and is freely floating. Incubate one piece of skin per 10 ml of 0.25% trypsin.</li>
	<li>Incubate samples at 37 &#176;C for 30-120 min or at 4 &#176;C overnight.</li>
</ol>
3. Preparation of Single Cell Suspension from Hair Follicles
<ol>
	<li>Scrape all the hair off the skin using curved forceps and a scalpel, in an anterior to posterior direction. Hold the skin in place using the curved side of forceps and use the scalpel to scrape off the hair. 
	Note: Scrape off the hair into the trypsin solution added in step 2.8. Start at the tail and follow the direction of hair growth. Scraping against the direction of hair growth will result in substantial loss of HFs and reduction in cell yield. HFs tend to adhere to each together and form small clumps; in order to reduce the size of HF clumps try to scrape off a small area at a time.</li>
	<li>Transfer the hairless skin to new culture dish and add 10 ml of PBS-- to prevent it from drying. Inspect the skin and scrape off any remaining hair.</li>
	<li>Break down the HFs using a scalpel and forceps until a single HF suspension is obtained. Vigorously triturate the HF suspension using 10 ml pipette for a few minutes to break up all hair clumps. 
	Note: Perform all of the following steps on ice.</li>
	<li>Transfer the HF suspension in pre-labelled 50 ml tube.</li>
	<li>Aspirate the PBS-- from the skin and use it to rinse the cell culture dish that contained the HF suspension; transfer to the 50 ml tube.</li>
	<li>Washing
	<ol>
		<li>Filter the suspension by passing through a 70 &#181;m cell strainer fitted onto a 50 ml tube. Wash the initial 50 ml tube and the strainer with 10 ml of staining buffer (PBS-- with 3% chelated FBS).</li>
		<li>Filter the suspension by passing through 40 &#181;m cell strainer fitted onto a 50 ml tube. Wash the tube with 5-10 ml of staining buffer to ensure that all cells have been transferred to the new tube. Keep the suspension on ice until all animals have been processed.</li>
		<li>Centrifuge for 15 min at 300 x g at 4 &#176;C. Carefully aspirate supernatant without disturbing the pellet and resuspend pellet in 5 ml of staining buffer.</li>
		<li>Centrifuge for 5 min 300 x g at 4&#176;C. Carefully aspirate supernatant and resuspend the cell pellet in 800 &#181;l of staining buffer. 
		Note: The volumes specified above is enough for staining cells derived from two adult mice.</li>
	</ol>
	</li>
	<li>Transfer Suspension into Pre-labeled FACS Tubes.
	<ol>
		<li>For controls tubes, take 25-50 &#181;l of cell suspension (or any residual left from the cell suspension) and make up to a total volume of 300 &#181;l with staining buffer. 
		Note: Prepare control tubes (total volume 300 &#181;l) for unstained (no antibody and no DAPI); DAPI only; Integrin &#946;1; Integrin &#945;6; Sca-1; CD34.</li>
	</ol>
	</li>
	<li>Add the primary antibodies and DAPI (see Materials table for appropriate dilutions) at the desired concentrations to the appropriate tube. Gently flick the tubes to mix the cell suspension. Return on ice and cover with aluminum foil to protect from light.</li>
	<li>Incubate for 30 min on ice and gently flick the tubes every 10 min to ensure cells are kept in suspension.</li>
	<li>Wash cells by filling up each FACS tube with staining buffer (approximately 4 ml per tube); centrifuge for 5 min at 300 x g at 4 &#176;C.</li>
	<li>Carefully aspirate supernatant and resuspend the cell pellet in 800 &#181;l of staining buffer.</li>
	<li>Transfer cell suspension into FACS tube with cell strainer caps to ensure single cell suspension.</li>
</ol>
4. Flow Cytometry Analysis
<ol>
	<li>Proceed to cell sorting immediately using a flow cytometer with appropriate filters for signal detection. Use 405 nm violet (for measurements of DAPI), 488 nm blue (for measurements of FSC, SSC, PE, PE-Cy7 and FITC) and 633 nm red lasers (for excitation of APC) 
	Note: Ensure that the instrument is recording from detectors for all three fluorescent channels.</li>
	<li>Set fluorescence gates based on unstained samples and compensation for spectral overlap using single stained controls. 
	Note: Use the single stained controls for adjusting the compensation between channels in order to eliminate any overlap in the fluorescent signals. Compensation adjustment will depend on the FACS sorter used.</li>
	<li>Set up primary gates based on DAPI to exclude dead cells.</li>
	<li>Adjust the scatter plot (SSC-A vs FSC-A) to select for singlet events.</li>
	<li>Set up FSC and SSC height and width parameters to eliminate for doublets and discriminate for singlet events .</li>
	<li>Gate cells with high &#945;6 and &#946;1 expression and from this population gate cells with either CD34 high expression or Sca1 high expression.</li>
	<li>Sort cells into pre-labeled FACS tube. 
	Note: Two general populations of cells can be isolated; &#945;6+/&#946;1+/CD34+/Sca-1- and &#945;6+/&#946;1+/ Sca-1+/CD34-. Sorted cells should be kept on ice at all times until they are used downstream applications such as cell culture or RNA isolation</li>
</ol>

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The authors have nothing to declare

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 can be used to isolate up to four different HFSC populations based on the differential expression levels of the markers used. It can be used for enrichment and isolation of HFSCs for further molecular analysis, such as RNA-Seq or mass spectrometry but also for subsequent cell culture and expansion and in vivo grafting.
The preparation of a high quality, single cell suspension is an essential component of a successful FACS 29. Efficiency of this protocol also depends on the starting material used for HF isolation. In order to minimize the amount of non-epidermal tissue in the starting material care must be taken when shaving and when dissecting the dorsal skin. This improves the purity and the yield of epidermal cells. In order to ensure an optimal single cell suspension, HFs should be scraped off in a head to tail direction and in small amounts at a time ensuring that all hair clumps are broken up in smaller pieces using a scalpel and that a single HF suspension is obtained. Additionally, cells should be handle with care at all times and suspensions should be kept on ice to ensure cell viability. Finally, sterile techniques are critical for minimizing the risk of contamination especially if cells are to be used for cell culture.
In the protocol described here, isolation of different populations of cells is achieved by using four cell surface markers. A crucial step for successful cell sorting is the appropriate initial settings of the FACS, the appropriate compensation using single stained controls and the hierarchy of the gating parameters. Successful cell sorting also requires elimination of debris and effectively discriminating singlet events from doublets/aggregates. Singlets were distinguished from doublets based on FSC and SSC width and height parameters. The parameters for cell sorting need to be adjusted according to the sorter type but also to the fluorochrome conjugate used. Here, PE fluorescence was collected using a 585/42 filter, FITC and PE-Cy7 fluorescence were collected using a 530/30 and 780/60 filter, respectively and APC fluorescence was collected using a 780/60 filter.
Cell sorting is performed initially by gating for live cells based on low DAPI expression and forward scatter. Singlet events were initially selected by adjusting the scatter gate and doublets were eliminated by gating against forward singlets followed by scatter singlets. Two integrin antibodies, &#945;6 and &#946;1 were used to select for epidermal cells to guarantee enrichment for all bulge SCs. Blanpain et al., showed the presence of two populations of bulge HFSCs expressing either high or low levels of integrin &#945;6 but positive for CD342. Consequently, cells expressing both &#946;1high/&#945;6low and &#946;1high/&#945;6high should be selected prior to gating for CD34. Two populations were then selected based on expression of a marker specific to bulge SCs, CD34, and a marker that selects epidermal keratinocytes, Sca-1 23. Sca-1 co-localizes with integrin-&#945;6 in the infundibulum and the IFE but is not expressed in the HF bulge. In contrast, bulge HFSCs express CD34 and integrin-&#945;6 23, allowing the differential isolation of two distinct populations; HFSCs and epidermal keratinocytes.
The robust method discussed here has been used for a number of downstream applications, e.g., isolation of HFSCs for cell culture 23,28, grafting 23,24 and molecular analysis 17. Additionally, selecting a combination of specific markers or using different reporter mice allows the isolation of subpopulations within the HF structure, e.g., Lgr6 21, Lgr5 25, Lrig1 24 and permits direct comparison of molecular changes occurring in these cells. Furthermore, among other applications this method can be used to compare changes in HFSC number in wild type versus knockout animals, examining changes in the SC populations upon wound infliction 28 and is therefore a valuable tool in SC and skin research.

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.remotexs.ntu.edu.sg/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><tbody><tr><td>Isoflurane&amp;nbsp;</td><td>Primal Critical Care</td><td>&amp;nbsp;66794-017-10</td><td></td></tr><tr><td>Carbon dioxide</td><td>-</td><td>-</td><td></td></tr><tr><td>Electro Shaver</td><td>Oster&amp;nbsp;</td><td>Golden A5</td><td>Shaver from any other company could be used</td></tr><tr><td>70% ethanol</td><td>Gadot Lab</td><td>830000051</td><td>96% ehtanol diluted with distilled water&amp;nbsp;</td></tr><tr><td>Dissection mat</td><td></td><td></td><td>Dissection tools from any provider can be used</td></tr><tr><td>Forceps</td><td>Dumont</td><td>11251-10</td><td>Foreceps from any other company could be used</td></tr><tr><td>Scissors&amp;nbsp;</td><td>Dumont</td><td>14094-11</td><td>Scissors from any other company could be used</td></tr><tr><td>Needles/Pins</td><td>-</td><td>-</td><td></td></tr><tr><td>Scalpel</td><td>Albion</td><td>10</td><td>Ensure that the scalpel has a blunt end</td></tr><tr><td>Tissue culture dish 60 mm x 15 mm</td><td>Sigma-Aldrich</td><td>CLS430166</td><td></td></tr><tr><td>PBS</td><td>-</td><td></td><td>In house PBS without Calcium and Magnesium</td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Biological Industries</td><td>03-050-1A</td><td>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><td>Pipettes 10 ml</td><td>Sigma-Aldrich</td><td>Corning, 4488</td><td></td></tr><tr><td>Ice</td><td>-</td><td>-</td><td></td></tr><tr><td>50 ml sterile centrifuge tubes</td><td>Minplast Ein-shemer</td><td>35050-43</td><td></td></tr><tr><td>70 &amp;micro;m Cell strainer</td><td>Fisher</td><td>22362548</td><td></td></tr><tr><td>40 &amp;micro;m Cell strainer</td><td>Fisher</td><td>22362549</td><td></td></tr><tr><td>Staining buffer</td><td>-</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf 5804 R</td><td>5805 000.017</td><td></td></tr><tr><td>FACS tubes with Cell strainer caps&amp;nbsp;</td><td>Falcon&amp;nbsp;</td><td>352235</td><td></td></tr><tr><td>FACS tubes&amp;nbsp;</td><td>Falcon&amp;nbsp;</td><td>352063</td><td></td></tr><tr><td>Integrin &amp;beta;1</td><td>eBioscience</td><td>25-0291</td><td>1:400</td></tr><tr><td>Integrin &amp;alpha;6</td><td>eBioscience</td><td>15-0495</td><td>1:600</td></tr><tr><td>Sca I</td><td>eBioscience</td><td>11-5981</td><td>1:200</td></tr><tr><td>CD34</td><td>eBioscience</td><td>9011-0349</td><td>1:300</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>50 ng/ml</td></tr><tr><td>Dry Chelex</td><td>BioRad</td><td>142-2842</td><td></td></tr><tr><td>Beaker&amp;nbsp;</td><td>Pyrex</td><td>-</td><td></td></tr><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>-</td><td>-</td><td></td></tr><tr><td>Stir bar</td><td>-</td><td>-</td><td></td></tr><tr><td>NHCl</td><td>BioLab</td><td>1903059</td><td></td></tr><tr><td>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><td>1 L glass bottle</td><td>Ilmabor</td><td>Boro 3.3</td><td></td></tr><tr><td>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+/&#946;1+ population of skin epithelial cells. Cells were first gated according to the expression of integrin-&#945;6 and integrin-&#946;1, which is designated as P1 in Figure 3. The population of cells with positive for &#945;6/&#946;1expression was then gated according to expression of CD34 and Sca-1 cells. Two distinct populations of cells are seen in the CD34 vs Sca-1 plot. &#945;6+/&#946;1+/CD34+/ Sca-1- represents the HFSCs (designated as P2 in Figure 3) and &#945;6+/&#946;1+/Sca-1+ /CD34- representing epidermal keratinocytes (designated as P3 in Figure 3). Approximately 150-500 x 103 cells per animal of the &#945;6+&#946;1+ pool are CD34+ HFSCs.
<img alt="Figure 2" src="/files/ftp_upload/53931/53931fig2.jpg" /> 
Figure 2: HFSC and Keratinocyte Isolation. Dorsal skin was removed from the mice. The fat was scraped off and the skin was incubated in trypsin. HFs were scrapped off from the skin and the suspension was filtered through 70 &#181;m and 40 &#181;m cell strainers. Following labeling with surface antibodies cells were sorted using flow cytometry and two populations of cells were collected based on relative expression: CD34+/Sca-1- and CD34-/Sca-1+. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53931/53931fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53931/53931fig3.jpg" /> 
Figure 3: Profiling of Sorted Cells. FACS analysis of dorsal skins assessing the percentage of HFSCs isolated from mice. FACS-purified &#945;6+&#946;1+ cells were sorted for CD34+Sca-1- (pink, &#945;6+&#946;1+CD34+Sca-1-; blue, &#945;6+&#946;1+CD34-Sca-1+; orange, &#945;6+&#946;1+CD34-Sca-1-). Data shown is from four mice combined and sorted together. In wild type mice approximately 4-6% of the &#945;6+&#946;1+ pool (designated P1) are CD34+/Sca-1- HFSCs (P2). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53931/53931fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Stem Cell Biology And Renewal in Epithelial Tissue

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20.9K Views.  Technion Israel Institute of Technology. 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.

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

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Biology

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 ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

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.

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 ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Neuroscience

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...