This work was supported in part by a grant from NIH grant R01 GM087609 (to H.P.L.), a Gift from Ingrid Lai and Bill Shu in honor of Anthony Shu (to H.P.L.), NIH grant U01 HL099776 (to M.T.L.), the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation (to M.T.L., G.C.G. and H.P.L.). G.G.W. was supported by the Stanford School of Medicine, the Stanford Medical Scientist Training Program, and NIGMS training grant GM07365. Z.N.M. was supported by the Plastic Surgery Foundation Research Fellowship Grant and the Hagey Family Fund. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159, the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award, and the Transplant and Tissue Engineering Fellowship Award.

This protocol follows methods approved by the Stanford University Administrative Panel on Laboratory Animal Care.
1. Digestion of Murine Dermis
<ol>
	<li>Euthanize mice by cervical dislocation after anesthesia with an intraperitoneal injection of ketamine 100 mg/kg + xylazine 20 mg/kg + acepromazine 3 mg/kg. 
	Note: Various ages and backgrounds can be used.</li>
	<li>Shave and depilate the dorsal skin. Approximately 100,000 cells can be isolated from a piece of dorsal skin 60 mm x 100 mm.</li>
	<li>Submerge the mouse in 70% ethanol and place on a clean, sterile surface to dry.</li>
	<li>Immediately harvest dorsal mouse skin using sterile dissecting scissors. In female mice, avoid including the mammary tissue.</li>
	<li>Starting the base of the tail, use forceps to tent up the skin and make a transverse cut before dissecting along the supra-fascial plane.</li>
	<li>Carefully avoid including any subcutaneous fat while harvesting the skin. Examine the harvested skin for any subcutaneous fat and carefully scrape it off using the blunt edge of a scalpel.</li>
	<li>Rinse the harvested skin in betadine followed by 5x PBS washes on ice. 
	Note: It is important to keep the skin as close to sterile as possible to avoid contamination.</li>
	<li>Mince the skin using razor blades and dissecting scissors in a sterile dish until the sample is of a uniform consistency with 2-3 mm pieces.</li>
	<li>Prepare 50 ml conical tubes containing 20 ml collagenase IV at a concentration of 1 mg/ml in DMEM. Divide the dermis into the tubes on the basis of five mice per tube.</li>
	<li>Agitate samples vigorously while incubating at 37 &#176;C for 1 hr in either a water bath or oven.</li>
	<li>Remove samples from the incubator and pass through a 10 ml syringe without a needle 3-5x in a sterile hood.</li>
	<li>Place the samples back into the incubator at 37 &#176;C and shake vigorously for a further 30 min.</li>
	<li>In a sterile hood, pipette the samples up and down 3-5x using a 10 ml pipette. Pipette the sample through a 100 &#181;m filter into a new 50 ml conical tube.</li>
	<li>Pass 20 ml of 10% FBS DMEM through the same filter to maximize cell yield and bring the total volume to 40 ml. Centrifuge at 300 g for 8 min at 4 &#176;C.</li>
	<li>Remove the supernatant using a sterile glass pipette, taking great care to first remove the upper fat layer prior to remaining supernatant. 
	Note: This step is critical to reduce adipocyte contamination.</li>
	<li>Resuspend the pellets in 20 ml 10% FBS DMEM.</li>
	<li>Pass the cell/DMEM suspension through a 70 &#181;m filter.</li>
	<li>Rinse the filter with 10 ml 10% FBS DMEM and centrifuge the filtered suspension at 300 g for 8 min at 4 &#176;C.</li>
	<li>Remove the supernatant using a sterile glass pipette, again taking care to first remove any remaining fat layer.</li>
	<li>If there is significant RBC contamination (the pellet is visibly red), re-suspend the pellets in 20 ml ACK lysis buffer and incubate for 5 min at RT. Otherwise skip to step 24.</li>
	<li>Add an equal volume (20 ml) of FACS buffer (PBS, 10% FBS, 0.1% sodium azide), then mix, and keep aside a 5 ml aliquot as an unstained control. Centrifuge the remaining sample at 300 g for 8 min at 4 &#176;C.</li>
	<li>Remove supernatant and put pellet on ice. The cells may be frozen down at this point if FACS time is not available.</li>
</ol>
2. Isolation of Fibroblasts by FACS
<ol>
	<li>Make 500 &#181;l of lineage antibody incubation mix for each pellet. Do this by first adding 475 &#181;l of FACS buffer containing DNase (10 &#181;g/ml) to a tube, and then adding fluorophore-conjugated CD31 (1:100), CD45 (1:200), Tie2 (1:50), Ter-119 (1:200), and EpCAM (1:100) antibodies to achieve the respective dilution for each antibody.</li>
	<li>Re-suspend each pellet in 500 &#181;l of lineage antibody incubation mix and incubate this suspension on ice for 20 min.</li>
	<li>Add 5 ml FACS buffer containing DNase (10 &#181;g/ml) to the sample and gently mix. Centrifuge at 300 g for 8 min at 4 &#176;C.</li>
	<li>Remove the supernatant and wash the cell pellet with 5ml FACS buffer containing DNase (10 &#181;g/ml) and centrifuge using the same conditions as in step 26.</li>
	<li>Resuspend the pellet in 500 &#181;l FACS buffer containing DNase (10 &#181;g/ml) and put aside a 50 &#181;l aliquot as a viability dye control.</li>
	<li>Add viability dye of choice to the remaining sample in the concentration indicated for the chosen dye.</li>
	<li>Perform FACS analysis31 and sorting for Viability Dye-/CD31-/CD45-/Tie2-/Ter-119-/EpCAM- cells (see Figure 2A). Sort directly into FACS buffer.</li>
</ol>

<ol>
	<li>Sorrell, J. M., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts-a+diverse+population+at+the+center+of+it+all.">Fibroblasts-a diverse population at the center of it all.</a> International review of cell and molecular biology. 276, 161-214 (2009).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+fibrosis.">Cellular and molecular mechanisms of fibrosis.</a> The Journal of pathology. 214, 199-210 (2008).</li><li>Powell, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblasts.+I.+Paracrine+cells+important+in+health+and+disease.">Myofibroblasts. I. Paracrine cells important in health and disease.</a> The American journal of physiology. 277, C1-C9 (1999).</li><li>Wilson, M. S., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+fibrosis:+pathogenesis,+etiology+and+regulation.">Pulmonary fibrosis: pathogenesis, etiology and regulation.</a> Mucosal immunology. 2, 103-121 (2009).</li><li>Hinz, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myofibroblast:+one+function,+multiple+origins.">The myofibroblast: one function, multiple origins.</a> The American journal of pathology. 170, 1807-1816 (2007).</li><li>Li, B., Wang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+and+myofibroblasts+in+wound+healing:+force+generation+and+measurement.">Fibroblasts and myofibroblasts in wound healing: force generation and measurement.</a> J Tissue Viability. 20, 108-120 (2011).</li><li>Wong, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+in+oral+mucosa+results+in+reduced+scar+formation+as+compared+with+skin:+evidence+from+the+red+Duroc+pig+model+and+humans.">Wound healing in oral mucosa results in reduced scar formation as compared with skin: evidence from the red Duroc pig model and humans.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 17, 717-729 (2009).</li><li>Szpaderska, A. M., Zuckerman, J. D., DiPietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+injury+responses+in+oral+mucosal+and+cutaneous+wounds.">Differential injury responses in oral mucosal and cutaneous wounds.</a> Journal of dental research. 82, 621-626 (2003).</li><li>Hantash, B. M., Zhao, L., Knowles, J. A., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+and+fetal+wound+healing.">Adult and fetal wound healing.</a> Frontiers in bioscience : a journal and virtual library. 13, 51-61 (2008).</li><li>Buchanan, E. P., Longaker, M. T., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+skin+wound+healing.">Fetal skin wound healing.</a> Advances in clinical chemistry. 48, 137-161 (2009).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors:+wounds+that+do+not+heal.+Similarities+between+tumor+stroma+generation+and+wound+healing.">Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing.</a> The New England journal of medicine. 315, 1650-1659 (1986).</li><li>Dumont, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+fibroblasts+modulate+early+dissemination,+tumorigenesis,+and+metastasis+through+alteration+of+extracellular+matrix+characteristics.">Breast fibroblasts modulate early dissemination, tumorigenesis, and metastasis through alteration of extracellular matrix characteristics.</a> Neoplasia. 15, 249-262 (2013).</li><li>Servais, C., Erez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+sentinel+cells+to+inflammatory+culprits:+cancer-associated+fibroblasts+in+tumour-related+inflammation.">From sentinel cells to inflammatory culprits: cancer-associated fibroblasts in tumour-related inflammation.</a> The Journal of pathology. 229, 198-207 (2013).</li><li>Orimo, A., Weinberg, R. 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A., Grove, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skin+fibroblasts+derived+from+papillary+and+reticular+dermis:+differences+in+growth+potential+in+vitro.">Human skin fibroblasts derived from papillary and reticular dermis: differences in growth potential in vitro.</a> Science. 204, 526-527 (1979).</li><li>Schafer, I. A., Pandy, M., Ferguson, R., Davis, B. 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The authors have nothing to disclose.

The protocol described in this manuscript offers a means to isolate fibroblasts by FACS-based sorting, in comparison to existing methods, which either select for a subpopulation or require time in cell culture before subsequent analyses. The time required from harvesting of the skin to sorting of fibroblasts is approximately 6 hr; however, the number of mice used in the harvest will influence this estimate.
Several points in the protocol require particular care. The first is limiting adipocyte...

Fibroblasts are frequently defined morphologically as spindle-shaped cells that adhere to plastic substrates. Fibroblasts are the principle cell type responsible for synthesizing and remodeling the extracellular matrix in embryonic and adult organs1. Fibroblasts are thus critical to mammalian development and contribute substantially to the extracellular milieu that influences the behavior of neighboring cell types present in each tissue and organ.
Fibroblasts are also the principal cell type behind a diverse set of medical conditions that cause enormous clinical burden. Pathologic fibroblast activity impairs normal tissue function and includes tissue and organ fibrosis (such as the lung and liver), scarring following cutaneous wound healing, atherosclerosis, systemic sclerosis, and formation of atheromatous plaques after blood vessel injury2-5. Wound healing in particular, both acutely and chronically, involves deposition of scar tissue that neither resembles nor functions like the normal tissue surrounding it, and leads to significant morbidity across diverse pathologic states. Following injury, there is a transition of fibroblasts to myofibroblasts, which then secrete structural ECM components, exert paracrine effects on neighboring cell types, and restore mechanical stability by depositing scar tissue6.
In cutaneous tissues there exists significant variation in the quality of wound repair across developmental time and between anatomic sites. In the first two trimesters of life the fetus heals without scarring; however, from the third trimester on and throughout adulthood, humans heal with a scar. Site-specific, in addition to age-specific, differences in wound healing exist. Wounds in the oral cavity remodel with minimal scar formation7,8, while scar tissue deposition within cutaneous wounds is significant9. Controversy persists concerning the relative influence of the environment versus the intrinsic properties of local fibroblasts on the outcome of wound healing in regards to both age and location10,11. Given the significant differences in the healing of mouse oral vs. cutaneous dermis and earlier embryonic (E15) vs. later embryonic (E18) dermis, it is likely that intrinsic differences in the populations of fibroblasts at certain developmental ages and among various anatomic sites exist.
In 1986, Harold F. Dvorak posited tumors are wounds that do not heal12. Dvorak concluded that tumors behave like wounds in the body and induce their stroma by activating the wound healing response of the host. Numerous studies have since investigated the contribution of fibroblasts to the progression of carcinomas13-15, but as in the case of wound healing, the identity and embryonic origin of the fibroblasts that contribute to the stromal compartment of cutaneous carcinomas has not been adequately defined. The answer to this question bears medical relevance given recent studies exposing the tumor-associated fibroblast as a potentially effective target for anti-cancer therapy16.
Identifying and prospectively isolating the fibroblast lineages endowed with fibrogenic potential in vivo is an essential step towards effectively manipulating their response to injury across a wide range of acute and chronic disease states. In 1987, Cormack demonstrated two subpopulations of fibroblasts, one residing within the papillary and one within the reticular dermis17,18. A third subpopulation was found associated with hair follicles in the dermal papilla region of the follicle19,20. When cultured, these fibroblast subtypes exhibit differences in growth potential, morphology, and growth factor/cytokines profiles21-24.
To date, studies examining fibroblast heterogeneity have largely failed to adequately characterize developmental and functional diversity among fibroblasts in vivo. This is, in part, is a result of a reliance on cultured fibroblast populations and the homogenizing effect of cell culture or positive selection on the basis of a self surface receptor not expressed by all fibroblasts25. A recent study from our lab demonstrated a profound surface marker and transcriptional shift in cultured vs. uncultured fibroblasts isolated by the FACS-based isolation methodology presented in this manuscript26.
Subsequently, we identified a specific fibroblast lineage within the murine dorsal dermis and determined that this lineage, defined by embryonic expression of Engrailed-1, is primarily responsible for connective tissue deposition in the dorsal skin. The lineage functions during both acute and chronic forms of fibrosis including wound healing, cancer stroma formation, and radiation induced fibrosis27. The characterization of distinct fibroblast lineages has critical implications for therapies aimed at modulating fibrogenic behavior.
Rather than using existing protocols that rely upon in vitro manipulation to achieve cell isolation28,29, the harvest protocol (Figure 1) detailed here will help yield informative analyses of fibroblasts that more accurately capture phenotype and behavior in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="1000">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:369px;">Surgical Forceps</td>
			<td style="width:369px;">Kent Scientific</td>
			<td style="width:108px;">INS650916</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elastase</td>
			<td>Abcam</td>
			<td>ab95133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>A14430-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium-Chloride-Potassium (ACK) lysing buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD31</td>
			<td>BioLegend</td>
			<td>102421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45</td>
			<td>BioLegend</td>
			<td>103125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tie2</td>
			<td>BioLegend</td>
			<td>124005</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ter-119</td>
			<td>BioLegend</td>
			<td>116233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EpCAM (CD326)</td>
			<td>eBioscience</td>
			<td>48-5791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">propidium iodide (PI viability stain)</td>
			<td>BioLegend</td>
			<td>421301</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The validity of this approach (Figure 1) has been verified in a number of ways, which can be examined in detail in our recent publication27. These include immunocytochemistry of sorted cells and mass cell and single cell transcriptional analysis of freshly sorted cells. Sorting fibroblasts directly rather than relying on culture more accurately captures their in vivo phenotype. Using a lineage negative depletion approach (Figure 2A) rather than a positive selection approach avoids pre-sel...

Watch this Scientific Journal Video about Murine Dermal Fibroblast Isolation by FACS at JoVE.com

Murine Dermal Fibroblast Isolation by FACS

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 ...

murine-dermal-fibroblast-isolation-by-facs

Research

JoVE Journal

Developmental Biology

20.8K Views.  Stanford University School of Medicine. 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.

Video: Murine Dermal Fibroblast Isolation by FACS

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.