We gratefully acknowledge assistance in FACS-sorting by B&#228;rbel Reininger and Wolfgang Bauer. This work was supported by grants to B. M. L. from the Austrian Science Fund (FWF: V 525-B28), and the Federation of European Biochemical Societies (FEBS, Follow-Up Research Fund). S. F. is recipient of a DOC Fellowship of the Austrian Academy of Sciences (OeAW). We gratefully acknowledge excellent support from the core facilities at the Medical University of Vienna. We thank Bernhard Gesslbauer, Christine Radtke and Martin Vierhapper for providing human skin material.

Human skin was obtained from Caucasian men and women aged 26 to 61 years (sun-protected skin; abdominal corrections, breast reductions). Written informed patient consent was obtained before tissue collection in accordance with the Declaration of Helsinki, and with approval from the Vienna Medical University Institutional Review Board.
NOTE: We provide a protocol for the isolation of functionally distinct fibroblast subpopulations either from full thickness dermis (please refer to section 1) or after sectioning human skin dermis into its different layers (skip section 1 and directly refer to section 2).
1. Preparation of Full Thickness Dermis
<ol>
	<li>Place a 10 cm x 10 cm human skin piece (e.g., from abdominal corrections or breast reductions) with the epidermis facing downwards on thick filter paper.</li>
	<li>Hold the epidermis tightly on its edges with forceps and scrape off the subcutaneous fat layer with a scalpel. Then slice the tissue into 5 mm wide strips before putting them into a Petri dish. 
	NOTE: This facilitates penetration of Dispase II (referred to as dissociating enzyme henceforth, see section 3) and is used if the epidermis needs to be separated from the entire dermis. For this skip section 2 and directly refer to section 3. To avoid contamination, keep the tissue sterile after surgical removal or clean it thoroughly with ethanol or iodine solution. If at all, ethanol treatment of the skin surface causes only minor cell death. Work under sterile conditions in a tissue culture flow hood.</li>
</ol>
2. Sectioning of Human Skin Dermis into Papillary and Reticular Layers with a Dermatome
<ol>
	<li>Place a 10 cm x 10 cm skin piece (e.g., from abdominal corrections or breast reductions) on thick filter paper, with the epidermis facing upwards. Hold the skin tightly on its edges with forceps.</li>
	<li>Slice the skin with an electric dermatome, adjusted to a cutting thickness/depth of 300 &#181;m, by sliding the head of the dermatome away from oneself, with the blade being at a 90&#176; angle in relation to the surface of the skin.</li>
	<li>Take the first layer consisting of epidermis and papillary dermis (300 &#181;m thick, &#8220;Dermal layer 1&#8221;, Figure 1 and Figure 2) and put it in a Petri dish. Proceed immediately to section 3 or add 1x PBS to keep the tissue from drying out. 
	NOTE: To avoid contamination, keep tissue sterile after surgical removal or clean it thoroughly with ethanol or iodine solution. Work under sterile conditions in a tissue culture flow hood. 
	CAUTION: Handle dermatome carefully since the blades are very sharp.</li>
	<li>Repeat step 2.2 adjusting the dermatome to a cutting thickness of 700 &#181;m and slice the remaining dermis. Place the upper slice which is defined as the upper reticular dermis (&#8220;Dermal layer 2&#8221;, Figure 2) into a separate Petri dish. Proceed immediately to section 4 or add 1x PBS to keep the tissue from drying out.</li>
	<li>Scrape away the subcutaneous fat layer with a scalpel from the residual lower reticular dermal layer (&#62;1,000 &#181;m) and discard it. Collect the lower reticular dermis (&#8220;Dermal layer 3&#8221;, Figure 2) in another Petri dish. Proceed immediately to section 4 or add 1x PBS to keep the tissue from drying out. 
	NOTE: Alternatively, instead of scraping the fat off with a scalpel, remove the fat layer with scissors. It may help to put the reticular dermis on ice prior to scraping the fat away.</li>
</ol>
3. Separation of Epidermis and Dermis
<ol>
	<li>Prepare a 3 U/mL dissociating enzyme (i.e., Dispase II) solution in sterile 1x PBS. Place the 5 mm skin stripes (from section 1), or the epidermis/papillary dermis (from step 2.2), with the epidermis facing upward in a 10 cm Petri dish with 10 mL of 3 U/mL dissociating enzyme solution. Incubate the Petri dish at 37 &#176;C for 1 h.
	<ol>
		<li>The protocol may be paused here. Incubate epidermis/papillary dermis in protease in the refrigerator at 4 &#176;C overnight instead. As needed store the other layers (Dermal layer 1, 2 and 3) overnight immersed in 1x PBS in 50 mL tubes at 4 &#176;C. 
		CAUTION: Work carefully with protease, it may irritate skin and eyes upon contact.</li>
	</ol>
	</li>
	<li>After incubation, transfer the epidermis/papillary dermis to the dry lid of the Petri dish and separate the epidermis from the upper dermis (Dermal layer 1) with two forceps each holding the edge of either the epidermis or dermis.</li>
</ol>
4. Enzymatic Digestion of Dermal Tissue
<ol>
	<li>Mince each dermal layer (Dermal layer 1, 2 and 3) separately with scissors/scalpel as thoroughly as possible; the smaller the pieces, the better the tissue digestion. 
	NOTE: The toughness of the dermis can vary depending on the body part it originates from (e.g., abdominal or upper arm skin).</li>
	<li>Prepare the digestion mix by combining 1.25 mg/mL collagenase I, 0.5 mg/mL collagenase II, 0.5 mg/mL collagenase IV and 0.1 mg/mL hyaluronidase in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with L-glutamine (see Table of Materials), 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (P/S). 
	CAUTION: Work carefully with collagenases as these may irritate skin and eyes upon contact.</li>
	<li>Put each of the minced tissues with 10 mL of the prepared digestion mix into a 50 mL tube and incubate in a 37 &#176;C warm water bath for 1 h under permanent agitation. During digestion invert the tubes several times. 
	NOTE: One should consider adjusting the digestion volume according to the size of the minced skin piece. Do not overload 10 mL digestion mix with too much tissue otherwise the cell yield will be minimal. Less than one third of tissue within the total volume is recommended (1:2 w/v).</li>
</ol>
5. Preparation of Single Cell Suspension and Erythrocyte Lysis
<ol>
	<li>Stop enzymatic tissue digestion by adding 10 mL fibroblast medium (DMEM with L-glutamine, 10% FCS and 1% P/S) to the digested tissue. Work on ice from here on.</li>
	<li>Pour the contents of each tube through a regular sterile stainless tea strainer and collect cell suspension in a clean Petri dish. Wash the strainer with medium and mash undigested tissue pieces with the edge of a syringe-plunger.</li>
	<li>Afterwards, pipette collected cell suspension through a 70 &#181;m cell strainer into a 50 mL tube. Rinse the cell strainer with medium and collect flow through into the same tube.</li>
	<li>Centrifuge tubes at 500 x g at 4 &#176;C for 10 min. Remove and discard supernatant and wash cell pellet with 5 mL fibroblast medium. Repeat the centrifugation step.</li>
	<li>Remove and discard the supernatant and resuspend the pellet in 1 mL self-made ACK erythrocyte lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA; pH 7.2&#8210;7.4). Leave cells in lysis buffer for approximately 1 min at ambient temperature (20&#8210;22 &#176;C).</li>
	<li>Stop lysis by adding 9 mL of 1x PBS with 10% FCS and centrifuge the tubes again at 500 x g at 4 &#176;C for 5 min. Discard the supernatant. 
	CAUTION: The incubation time in erythrocyte lysis may be adjusted if the lysis seems incomplete (red pellet). However, do not leave the cells in erythrocyte lysis buffer for too long to avoid lysis of fibroblasts and immune cells.</li>
</ol>
6. Blocking and Staining Fibroblasts for FACS
<ol>
	<li>Prepare 5 &#181;L of human Fc-receptor blocking solution in 100 &#181;L of 1x PBS with 10% FCS and resuspend cells in 100 &#181;L of human Fc blocker. Incubate on ice for 10 min.</li>
	<li>Design a FACS staining panel to stain human dermal fibroblasts. Mix anti-human FAP APC (1:20), anti-human CD90 AF700 (1:30), anti-human E-cadherin PE (1:20), anti-human CD31 FITC (1:30), anti-human CD45 Pacific blue (1:20), anti-human ITGA6 PE (1:20), anti-human CD235ab Pacific blue (1:1,000) and anti-human CD106 Pacific blue (1:100) in 1x PBS with 10% FCS.</li>
	<li>After Fc receptor blocking, spin cells down at 500 x g at 4 &#176;C for 3 min. Remove and discard the supernatant and resuspend cells in 100 &#181;L of antibody mix. Incubate cells in the dark at 4 &#176;C for 20 min.
	<ol>
		<li>Prior to staining, remove 20 &#181;L of a sample with a high cell number for unstained and single stain FACS controls.</li>
	</ol>
	</li>
	<li>Wash the stained cells and FACS controls twice with 500 &#181;L 1x PBS with 10% FCS and centrifuge at 500 x g at 4 &#176;C for 3 min. In the meantime, add 4&#8242;,6-diamidino-2-phenylindole (DAPI) to 1x PBS with 10% FCS to make a final concentration of 1 &#181;g/mL.</li>
	<li>Remove and discard supernatant, resuspend cells in 200 &#181;L DAPI solution and filter each cell suspension through a 70 &#181;m cell strainer into a 5 mL FACS tube. 
	NOTE: If FACS is going to be performed with delay, add DAPI and filter shortly before recording.</li>
</ol>
7. Gating Strategy for Human dermal Fibroblast Subset Isolation and FACS
<ol>
	<li>Record flow cytometry controls, set correct voltage settings and perform the appropriate compensation. Gate for single cells and live (DAPI-), Pacific blue, PE and FITC negative cells (CD45-, CD31-, CD235ab-, CD106-, ITGA6- and E-cadherin-) to get three fibroblast populations: FAP+CD90-, FAP+CD90+ and FAP-CD90+ cells. See Figure 3.</li>
	<li>Sort three fibroblast subpopulations into separate 1.5 mL screw cap microcentrifuge tubes filled either with 350 &#181;L lysis buffer for RNA isolation or filled with 350 &#181;L fibroblast growth medium (see the Table of Materials) for cell culture. Invert tubes immediately after the sort and either put lysis buffer tubes into liquid nitrogen for snap freezing or put medium tubes on ice. 
	CAUTION: Prepare lysis buffer with 0.1% b-Mercaptoethanol in fume hood and avoid inhalation.</li>
</ol>
8. Culturing of Fibroblasts and Adipogenesis Assay
<ol>
	<li>Following FACS, spin cells down at 500 x g for 3 min at 4 &#176;C and plate equal cell numbers (5,000&#8210;10,000 cells/well) in fibroblast growth medium (see the Table of Materials) in 48-well cell culture dishes. Leave cells to grow until they reach 70% confluency.</li>
	<li>Add 5 &#181;g/mL insulin, 5 &#181;M troglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 1 &#181;M dexametasone to fibroblast growth medium to prepare adipocyte differentiation medium.</li>
	<li>Replace medium of the cultured cells with adipocyte differentiation medium and let cells differentiate for 14 days. Exchange medium after day 2 with reduced adipogenesis medium containing 5 &#181;g/mL insulin and 5 &#181;M troglitazone. Replenish medium every 3rd or 4th day.</li>
	<li>Fix cells with 4% PFA for 20 min at ambient temperature (20&#8210;22 &#176;C) at differentiation day 14. Wash wells with 60% isopropanol and let it evaporate completely. Stain cells with 5 mM Oil Red O (filter prior to use) for 20 min. Wash stained cells four times with distilled water. Proceed to perform microscopy. 
	CAUTION: PFA is toxic and harmful. Handle carefully. 
	NOTE: The protocol can be paused here. Fixed cells can be stored in 1x PBS at 4 &#176;C until Oil Red O staining is performed. Cover dish with paraffin film prior to storage to avoid evaporation.</li>
	<li>Image cells immersed in water with an inverted microscope at 10x magnification with transmitted light.</li>
</ol>

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The authors have nothing to disclose.

In this article, we describe a method for the isolation of papillary and reticular fibroblasts from human skin. CD90 has been widely used for the identification or isolation of dermal fibroblasts18,20,21. However, we have demonstrated that besides CD90+ fibroblasts, human dermis also harbors a CD90- fibroblast population expressing FAP16, which has been established as a marker for activated fibroblasts and cancer-associated fibroblasts (CAFs)22,23,24,25. Importantly, we were able to identify three fibroblast subpopulations FAP+CD90-, FAP+CD90+ and FAP-CD90+ in skin biopsies from all healthy human donors. We therefore conclude that FAP is not only a marker for activated fibroblasts or CAFs but also normal tissue fibroblasts.
Of note, the FAP-CD90- cell population remaining after application of the above-described exclusion- and gating strategy does not contain fibroblasts, since these cells do not proliferate in fibroblast cultivation medium in vitro, but most likely a mixed cell population including lymphatic cells and pericytes amongst others16.
The cell yield obtained by use of the above-described protocol can vary depending on the body-part that the skin piece used for the isolation originates from. Dermis from different body parts differs regarding its structure, thickness as well as collagen composition. For example, skin from the face or the upper arm is much thinner than skin from the belly or the thigh, which also frequently display a thicker subcutaneous fat layer. Additionally, age and sex of the skin donors may further not only impact the tissue dissociation efficiency, but may also affect the distribution of the three fibroblast subpopulations (Figure 3) when isolated from full thickness skin. This results from the fact that the papillary dermis shrinks and that total fibroblast numbers decrease with age11,26,27,28. Furthermore, the cell pellet from papillary dermis will probably be larger than from reticular dermis, since the upper dermis is more densely populated by fibroblasts than the reticular dermis. Besides, the lower dermis is also tougher and more densely packed with collagen, making it harder to dissociate the tissue and to release the fibroblasts. Of note, the cell pellet may appear very red, which is why red blood cell lysis is recommended.
In addition to the identification of three subpopulations in intact human skin, we also show that in dermatomed skin, each fibroblast subset is enriched either in papillary or reticular dermis16. Precise slicing of the skin with the dermatome is critical to obtain a proper enrichment of each subpopulation from different dermal layers. Since the papillary dermis is very thin, the dermatomed slice representing it should not exceed a thickness of 300 &#181;m. Upper reticular and lower reticular fibroblasts both represent the reticular lineage and display similar functions and gene signatures, therefore, one could also consider not separating them.
Importantly, all three fibroblasts populations are found throughout the dermis and are not exclusively present in one layer, which is why explant cultures from papillary or reticular dermis result in mixed fibroblast cultures. However, FAP+CD90- papillary fibroblast are most abundant in the papillary dermis and follow a gradient from superficial to lower dermal layers while FAP+CD90+ and FAP-CD90+ fibroblasts follow an inverse gradient from the lower to the superficial layers16. Furthermore, the majority of CD90+ fibroblasts of the papillary dermis are almost exclusively found surrounding blood vessels and express the perivascular fibroblast marker CD14629, and thus probably exhibit different functions than the remaining CD146- reticular fibroblasts16. CD146 could be used as an additional marker in the gating strategy to exclude this population.
Following the dissociation of dermal layers, the isolated cells are stained with a specially designed antibody cocktail containing various antibodies for the exclusion of immune cells, endothelial and lymphatic cells, epidermal cells, erythrocytes and MSCs to obtain pure fibroblast populations. Of note, choosing a marker for the identification and exclusion of MSCs may be tricky because of the high number of published MSC markers30,31. Since MSCs express CD90 like fibroblasts, additional MSC markers such as CD105 or CD271 could prove useful for their identification. However, MSCs only represent a very low percentage of all dermal cells and since CD90+ fibroblasts display typical morphological features of fibroblasts upon sorting, one could argue that the exclusion of MSCs by use of distinct cell surface markers might be unnecessary.
Importantly, we analyzed FAP and CD90 gene expression after keeping the cells in culture for 7-14 days after sorting (data not shown) and found that expression of both markers is upregulated in the respective sorted single positive (FAP+CD90- or FAP-CD90+) cells16. We therefore emphasize that the above-described marker sets and protocol permit the isolation of primary fibroblast subsets directly from the tissue but not from previously cultured mixed fibroblast populations.
Nevertheless, we demonstrate that the functionality of all three subpopulations is retained in cell culture regardless of the alteration of cell surface marker expression, since fibroblasts sorted as FAP+CD90- papillary fibroblasts do not acquire the ability to undergo adipogenesis after a longer period of culture, while fibroblasts sorted as FAP+CD90+ or FAP-CD90+ reticular fibroblasts maintain their ability to differentiate into adipocytes 16. Importantly, we also found that papillary and reticular-specific genes are still expressed to a higher extent in FAP+CD90- and CD90+ respectively.
In conclusion, we have established a protocol for the isolation of functionally distinct fibroblast subsets via FACS that for the first time permits the isolation and analysis of pure and na&#239;ve fibroblast subpopulations from human skin dermis. This method establishes a major advancement to the commonly used fibroblast explant culture isolation protocol from upper and lower dermis as (i) an opposing gradient of papillary and reticular fibroblasts exists from the skin surface to the hypodermis and (ii) fibroblasts change their gene signature in vitro.

As the main cellular component of connective tissue, fibroblasts are primarily responsible for the deposition of collagen and elastic fibers that ultimately form the extracellular matrix1, and thus, their role in tissue homeostasis, regeneration and disease has been underestimated for a long time. However, fibroblasts have recently come under the spotlight of researchers, not only because they represent a prominent cellular source for induced pluripotent stem cells2 but also because of their high plasticity and implication in the pathogenesis of a wide number of diseases such as organ fibrosis3,4,5 or cancer6,7.
Human skin is composed of a multi-layered epithelium, the epidermis, and its underlying connective tissue, the dermis, which can be histologically subdivided into the upper papillary and the lower reticular dermis and is mainly composed of fibroblasts and extracellular matrix8 and the hypodermis. According to their location within the tissue, dermal fibroblasts have roughly been classified into papillary and reticular fibroblasts1.
Importantly, recent data indicate that these dermal fibroblast subpopulations are not only histologically distinguishable, but also that their function is considerably diverse. In mouse skin, papillary and reticular fibroblasts arise from two distinct lineages during embryogenesis9. Several lines of evidence suggest that the two lineages exert different roles not only in tissue homeostasis, hair follicle morphogenesis, wound repair and fibrosis7,9,10, but they also respond to different signals from neoplastic epidermal stem cells11, suggesting diverging roles in cancer pathogenesis. Conveniently, both lineages express a distinct set of mutually exclusive cellular markers in adult mouse skin, thus enabling the isolation of pure dermal fibroblast populations and subsequent extensive analysis of their specific functions in vitro9,11.
Correspondingly, at least two distinct fibroblast subsets with distinct morphology and functions, including diverging proliferation rates, tissue remodeling capacities12,13, as well as the ability to support the growth of epidermal stem cells in vitro, have been described for human skin dermis14,15. However, most of the published studies on human dermal fibroblasts have been conducted using mixed fibroblast populations isolated from explant cultures from dermatomed skin, since specific cell surface marker sets enabling the isolation of pure human papillary or reticular fibroblast subpopulations in analogy to mouse dermis were yet to be established.
We have recently demonstrated, that human skin papillary and reticular fibroblasts are characterized by specific cell surface markers that enable the isolation of the respective subpopulations via fluorescence-activated cell sorting (FACS)16: FAP+CD90- fibroblasts represent papillary fibroblasts primarily located in the upper dermis, presenting higher proliferation rates, a distinct gene signature but no adipogenic potential. FAP+CD90+ and FAP-CD90+ fibroblasts belong to the reticular lineage of the lower dermal compartments, which are less proliferative but readily undergo adipogenesis&#8212;a hallmark for reticular fibroblasts. This method enables to extensively study these distinct fibroblast subpopulations not only in regard to their specific functions under physiological conditions but also in the context of the pathogenesis of cutaneous diseases including skin cancer.
However, since fibroblasts alter their cell surface marker expression in two-dimensional in vitro culture16,17,19, the application of our protocol is limited to the isolation of primary fibroblasts from human dermis and does not permit the identification of papillary or reticular fibroblasts in mixed cell culture populations. Importantly, although the expression of cell surface markers changes in vitro, we have demonstrated that the fibroblast subsets isolated according to the protocol described below retain their specific functionality when cultivated16, thus enabling in vitro studies of subset-specific properties under physiological or pathological conditions.
In conclusion, we developed a protocol for the isolation of distinct fibroblast subsets via FACS that for the first time permits the isolation of pure fibroblast populations from human skin dermis in a na&#239;ve state.

<table>
	<tbody>
		<tr>
			<td>Material:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-isobutyl-1-methylxanthine</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma</td>
			<td>9718</td>
			<td>used for selfmade ACK Lysis Buffer</td>
		</tr>
		<tr>
			<td>Amniomax-C100 (1x) Basal Medium</td>
			<td>Gibco</td>
			<td>17001-074</td>
			<td>used for fibroblast growth medium</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Scharlau</td>
			<td>ME0095</td>
			<td>! CAUTION</td>
		</tr>
		<tr>
			<td>C100 Supplement</td>
			<td>Thermo Scientific</td>
			<td>12556015</td>
			<td>used for fibroblast growth medium</td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Thermo Scientific</td>
			<td>17100017</td>
			<td>! CAUTION</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Gibco</td>
			<td>17101015</td>
			<td>! CAUTION</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma</td>
			<td>C5138</td>
			<td>! CAUTION</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D8893</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td>! CAUTION</td>
		</tr>
		<tr>
			<td>Dulbecco&#8216;s Modified Eagle Medium + GlutaMAX</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>Sigma</td>
			<td>E1644</td>
			<td>used for selfmade ACK Lysis Buffer</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (heat inactivated)</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H4272</td>
			<td>! CAUTION</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Merck</td>
			<td>1,096,341,011</td>
			<td></td>
		</tr>
		<tr>
			<td>OilRed O</td>
			<td>Sigma</td>
			<td>O-0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyd</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>! CAUTION Cancerogenic. Skin and eye irritant. Handle with care.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline without Ca++ &#38; Mg++</td>
			<td>Lonza</td>
			<td>BE17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium bicarbonate</td>
			<td>Sigma</td>
			<td>237205</td>
			<td>used for selfmade ACK Lysis Buffer</td>
		</tr>
		<tr>
			<td>PureLink RNA MicroKit</td>
			<td>Invitrogen</td>
			<td>12183-016</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III First-Strand Synthesis SuperMix for qRT-PCR</td>
			<td>Invitrogen</td>
			<td>11752</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman 2xUniversal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4324018</td>
			<td>! CAUTION Skin and eye irritant.</td>
		</tr>
		<tr>
			<td>Troglitazone</td>
			<td>Sigma</td>
			<td>T2573</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Flow cytometry Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti human CD31-AF488</td>
			<td>Biolegend</td>
			<td>303109</td>
			<td>Dilution: 1:30, Lot: B213986, RRID: AB_493075</td>
		</tr>
		<tr>
			<td>anti human CD45-Pacific blue</td>
			<td>Biolegend</td>
			<td>304029</td>
			<td>Dilution: 1:20, Lot: B218608, RRID: AB_2174123</td>
		</tr>
		<tr>
			<td>anit human CD49f/ITGA6-PE</td>
			<td>Serotec</td>
			<td>MCA699PE</td>
			<td>Dilution: 1:20, Lot: 0109, RRID: AB_566833</td>
		</tr>
		<tr>
			<td>anti human CD90/Thy-1-AF700</td>
			<td>Biolegend</td>
			<td>328120</td>
			<td>Dilution: 1:30, Lot: B217250, RRID: AB_2203302</td>
		</tr>
		<tr>
			<td>anti human CD106-AF421</td>
			<td>BD</td>
			<td>744309</td>
			<td>Dilution: 1:100, Lot: 7170537, RRID: x</td>
		</tr>
		<tr>
			<td>anti human CD235ab-Pacific blue</td>
			<td>Biolegend</td>
			<td>306611</td>
			<td>Dilution: 1:1000, Lot: B224563, RRID: AB_2248153</td>
		</tr>
		<tr>
			<td>anti-human E-cadherin-PE</td>
			<td>Biolegend</td>
			<td>147304</td>
			<td>Dilution: 1:20, Lot: B197481, RRID: AB_2563040</td>
		</tr>
		<tr>
			<td>anti human FAP-APC</td>
			<td>R&#38;D</td>
			<td>FAB3715A</td>
			<td>Dilution: 1:20, Lot: AEHI0117111, RRID: x</td>
		</tr>
		<tr>
			<td>Human TruStain FcX</td>
			<td>Biolegend</td>
			<td>422302</td>
			<td>Dilution: 1:20, Lot: B235080, RRID: x</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.4 mL micronic tubes</td>
			<td>Thermo Scientific</td>
			<td>4140</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL screw cap micro tube</td>
			<td>Sarstedt</td>
			<td>72,692,405</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round Bottom Tube with cell strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well cell culture cluster</td>
			<td>Costar</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene canonical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer nylon</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Aesculap Acculan 3Ti Dermatome</td>
			<td>VWR</td>
			<td>AESCGA670</td>
			<td></td>
		</tr>
		<tr>
			<td>Aesculap Dermatome blades</td>
			<td>VWR</td>
			<td>AESCGB228R</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96well</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary cell culture dish</td>
			<td>Corning</td>
			<td>353803</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>F.S.T</td>
			<td>10022-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Tea strainer</td>
			<td>x</td>
			<td>x</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Fibroblast growth medium:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AmnioMAX basal medium with AmnioMAX C-100 Supplement, 10 % FCS and 1 % P/S</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of papillary and reticular fibroblasts from human skin by fluorescence-activated cell sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

An overview of the main steps for processing skin tissue to obtain a single cell suspension is shown in Figure 1, displaying the dermatoming of the skin (Figure 1A), different dermal layers (Figure 1B), removal of the subcutaneous fat layer (Figure 1C) and the separation of the epidermis and papillary dermis (Figure 1D), as well as the different steps of the manual and enzymatic tissue dissociation (Figure 1E,F). A scheme of the three dermal layers is provided in Figure 2.
Figure 3 displays the gating strategy of a flow cytometry staining panel for the analysis of different fibroblast subsets from human skin. Additional cell surface markers that are not expressed on fibroblasts permit the exclusion of various other cells present in the skin such as immune cells, epidermal cells, mesenchymal stem cells (MSCs), red blood cells or endothelial cells to attain maximal purity in the isolated populations. It is not critical to use the identical FACS panel used in Figure 2 for the identification of these fibroblast subpopulations, however this is our recommendation. One may use antibodies labelled with different fluorescent dyes or alter the combination of exclusion markers.
Of note, this protocol enables the identification of three fibroblast populations in human skin which present with different intradermal localization, gene expression profiles and also functions (Figure 4). FAP+CD90- are enriched in the papillary dermis whereas FAP+CD90+ and FAP-CD90+ are more abundant in the reticular dermis (Figure 4A). All three subpopulations exhibit the typical fibroblast morphology upon sorting in cell culture (Figure 4B). Interestingly, they differ regarding their ability to differentiate into adipocytes which is a hallmark for reticular fibroblasts. Combining these results with gene expression profiling via real-time polymerase chain reaction (RT-PCR)16 (Figure 4C), showing that FAP+CD90- cells express high levels of markers commonly attributed to papillary fibroblasts such as CD26, NTN and PDPN while CD90+ cells express the known reticular markers such as CD36, ACTA2 and PPAR&#947; at high levels, we conclude that FAP+CD90- cells belong to the papillary and CD90+ cells belong to the reticular lineage.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59372/59372fig1.jpg" /> 
Figure 1: Isolation of dermal single cell suspension from intact human skin. (A) Skin is sliced with an electric dermatome into papillary and reticular dermis. (B) Papillary dermis with adjacent epidermis is 300 &#181;m thick (top) while upper reticular dermis is 700 &#181;m thick (bottom). (C) Subcutaneous fat layer is scraped-off of lower reticular dermis with a scalpel. (D) After incubation of papillary dermis in dissociation enzyme solution at 37 &#176;C for 1 h, the epidermis can easily be removed with forceps. (E) Different dermal layers are minced with scissors and transferred into an enzyme digestion mix consisting of collagenase I, II and IV and hyaluronidase. (F) After 1 h of dissociation at 37 &#176;C, tissue digestion is stopped and the suspension is poured through a tea strainer to remove undigested skin pieces. (G) Cell suspension is filtered once more through a 70 &#181;m cell strainer and centrifuged at 500 x g at 4 &#176;C for 10 min. (H) After centrifugation, the cell pellet is washed with 1x PBS with 10% FCS and is ready for FACS staining. <a href="https://www.jove.com/files/ftp_upload/59372/59372fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59372/59372fig2.jpg" /> 
Figure 2: Sectioning of human skin dermis into papillary and reticular layers with a dermatome. Scheme showing the three dermal layers obtained by slicing full thickness skin into papillary dermis (including epidermis; 0&#8210;300 &#181;m; dermal layer 1), upper reticular (300&#8210;1,000 &#181;m; dermal layer 2) and lower reticular dermis (&#62;1,000 &#181;m; dermal layer 3) with a dermatome. <a href="https://www.jove.com/files/ftp_upload/59372/59372fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59372/59372fig3.jpg" /> 
Figure 3: FACS gating strategy for human dermal fibroblast subpopulations. (A&#8210;E) First, cells are gated on single (B) and viable (DAPI-) cells (C). Immune cells (CD45+), mesenchymal stem cells (CD106+), red blood cells (CD235ab+) are excluded (C) and Pacific blue negative cells are gated further on E-cadherin (Ecad) and ITGA6 double negative cells (PE channel, D). E-cadherin and ITGA6 are markers expressed on epidermal cells. Next, CD31-FITC positive cells (endothelial and lymphatic cells) are excluded (D) resulting in three fibroblast populations expressing either one or both of the two cell surface fibroblast markers FAP and CD90: FAP+CD90-, FAP+CD90+ and FAP-CD90+ (E). <a href="https://www.jove.com/files/ftp_upload/59372/59372fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59372/59372fig4.jpg" /> 
Figure 4: FAP+CD90-, FAP+CD90+ and FAP-CD90+ fibroblasts differ in dermal localization, gene expression and functionality. (A) Representative FACS plots of gated fibroblasts isolated from papillary, upper reticular and lower reticular dermis. Gating strategy is explained in Figure 3. FAP+CD90- fibroblasts are enriched in the papillary dermis (19.2%) compared to lower reticular dermis (0.83%). FAP+CD90+ cells can be found throughout the dermis but their highest abundance is in the upper reticular dermis (40.7%). FAP-CD90+ are enriched in the lower reticular dermis (64.4%). (B) Of note, all three sorted subpopulations display typical fibroblast morphology upon culture for 7 days (left). Interestingly, they behave differently in an adipogenesis assay. After 14 days in culture, FAP+CD90- do not differentiate into adipocytes while FAP+CD90+ and FAP-CD90+ readily undergo adipogenesis (right; Oil Red O stains lipid-bearing cells red). Scale bars = 1,000 &#181;m. (C) Directly sorted FAP+CD90- fibroblasts express the papillary fibroblast markers CD26, NTN1 and PDPN, while FAP-CD90+ cells express CD36, ACTA2 and PPAR&#947;, known to be expressed by the reticular lineage. * p &#8804; 0.05; *** p &#8804; 0.0005 compared to FAP+/CD90- cells; # p &#8804; 0.05; ### p &#8804; 0.0005 compared to FAP-/CD90+ cells. <a href="https://www.jove.com/files/ftp_upload/59372/59372fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting at JoVE.com

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

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15.5K Views.  Medical University of Vienna. This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Video: Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and salamanders. Individual LBCs can be up to 1 mm in length and they reside in a gigantic nucleus, itself up to 0.5 mm in diameter. The large size of the chromosomes permits unparalleled observations of active genes by light optical microscopy, but at the same time special techniques are required for isolating the nucleus, removing the nuclear envelope, and spreading the chromosomes on a microscope slide. The oocyte nucleus, also called the germinal vesicle (GV), is isolated in a medium that allows partial gelling of the nuclear actin and preserves the delicate structure of the LBCs. This step is carried out manually under a dissecting microscope using jeweler&#39;s forceps. Next, the nuclear envelope is removed, again manually with jeweler&#39;s forceps. The nuclear contents are quickly transferred to a medium that disperses the actin gel and allows the undamaged LBCs to settle onto a microscope slide. At this point the LBCs and other nuclear organelles can be viewed by phase contrast or differential interference contrast microscopy, although finer details are obscured by Brownian motion. For high resolution microscopical observation or molecular analysis, the whole preparation is centrifuged to attach the delicate LBCs firmly to the slide. A brief fixation in paraformaldehyde is then followed by immunofluorescent staining or in situ hybridization. LBCs are in a transcriptionally active state and their enormous size permits molecular analysis at the individual gene level using confocal or super-resolution microscopy.

We present simple techniques for isolating giant transcriptionally active lampbrush chromosomes from living oocytes of frogs and salamanders. We describe how to observe these chromosomes &#34;alive&#34; by phase contrast or differential interference contrast, and how to fix them for fluorescent in situ hybridization or immunofluorescent staining.

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and ...

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL).

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct&#160;in vivo&#160;CRISPR&#160;screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice.

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...