Name: immunomagnetic separation of fat depot-specific sca1high adipose-derived stem cells (ascs)
Uploaded: 2016-08-11T14:00:00
Description: Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

This work is supported by NIH DK095137 (to THC). We thank the current and former lab members who contributed to the development and sophistication of the described methods.

Ethics Statement: The University of Michigan Committee on Use and Care of Animals (UCUCA) has approved all methods and protocols in accordance with the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council). Mice are maintained in a University of Michigan vivarium and are given free access to food and water and kept on a 12 hr dark/light cycle.
1. Preparations
<ol>
	<li>Prepare primary culture media with DMEM, 10% FBS, 1x P/S/G,...

<ol>
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E., 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=Sca-1(pos)+cells+in+the+mouse+mammary+gland+represent+an+enriched+progenitor+cell+population.">Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population.</a> Dev Biol. 245 (1), 42-56 (2002).</li><li>Gesta, S., Tseng, Y. H., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+origin+of+fat:+tracking+obesity+to+its+source.">Developmental origin of fat: tracking obesity to its source.</a> Cell. 131 (2), 242-256 (2007).</li><li>Tang, W., Zeve, D., Suh, J. M., Bosnakovski, D., Kyba, M., Hammer, R. E., Tallquist, M. D., Graff, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White+fat+progenitor+cells+reside+in+the+adipose+vasculature.">White fat progenitor cells reside in the adipose vasculature.</a> Science. 322, 583-586 (2008).</li><li>Rodeheffer, M. S., Birsoy, K., Friedman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+white+adipocyte+progenitor+cells+in+vivo.">Identification of white adipocyte progenitor cells in vivo.</a> Cell. 135 (2), 240-249 (2008).</li><li>Tokunaga, M., 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=Fat+depot-specific+gene+signature+and+ECM+remodeling+of+Sca1(high)+adipose-derived+stem+cells.">Fat depot-specific gene signature and ECM remodeling of Sca1(high) adipose-derived stem cells.</a> Matrix Biol. 36, 28-38 (2014).</li><li>Chun, T. 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Herein we demonstrate the isolation and immunomagnetic cell separation of murine ASCs from different fat pads and their use for in vitro experiments. The presented method is effective for the quick isolation of large number of Sca1-positive ASCs, which is advantageous over the technically complex and expensive FACS-mediated isolation of ASCs9,14. Unlike FACS, immunomagnetic cell separation does not allow the use of multiple antigen for the identification of a target cell population. Nonetheless, if th...

Stem cell antigen 1 (Sca1, or Ly6A/E) was first identified as a cell surface marker expressed by hematopoietic and mesenchymal stem cells5,6. The stromal vascular fraction (SVF) of adipose tissue obtained from mouse fat depots is a heterogeneous population of cells comprising of fibroblasts, macrophages, vascular endothelial cells, neuronal cells, and adipocyte progenitor cells7. Adipocyte progenitor cells, or adipose-derived stem cells (ASCs) are non-lipid-laden cells that reside in the collagen-rich perivascular extracellular matrix (ECM)8. Approximately 50% of the SVF consist of ASCs, which are characterized as lineage-negative (Lin-) and CD29+: CD34+: Sca1+ 9. Most of these cells are Sca1+: CD24- adipocyte progenitors, which are capable of adipocyte differentiation in vitro; however, only a fraction of cells (0.08% of SVF) constitutes Sca1+: CD24+ cells that are fully capable of proliferating and differentiating into adipocytes in the in vivo conditions9. Despite the potential caveat of using Sca1+ SVF without discriminating CD24+ cells from CD24- cells, isolating Sca1+ ASCs from fat depots using immunomagnetic cell separation is an efficient and practical approach to determine the cell-autonomous phenotype of primary adipocyte progenitor cells.
In the field of obesity and diabetes, tissue fibrosis and inflammation play a critical role in the development and maintenance of type-2 diabetes3. Recently, Tokunaga et al. showed that Sca1high cells isolated from inguinal (or subcutaneous, SQ) and perigonadal (or visceral, VIS) C57BL6/J fat depots exhibit different gene signatures and ECM remodeling in vitro10. MMP14 (MT1-MMP), a prototypical member of the membrane-type matrix metalloproteinase (MMP) family mediates the development of white adipose tissue (WAT) through its collagenolytic activity1.
Examples of experiments that may be conducted with the cells isolated and enriched through the following protocol include three-dimensional culture, differentiation studies, collagen degradation assays, and RNA sequencing10,11. Degradation assays should be conducted with acid-extracted collagen to ensure the preservation of telopeptide11,12. The following protocol will demonstrate the methods to isolate primary vascular stromal cells from different fat depots and enrich adipocyte progenitor cells using immunomagnetic cell separation. The validity of the cell sorting will be assessed with flow cytometry and through using Sca1-GFP mice that express GFP in Sca1+ cells, driven by a Sca1 promoter13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Type 3 Collagenase</td>
			<td>Worthington Biochemical</td>
			<td>LS004182</td>
			<td>Tissue digestion</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>High-glucose culture medium</td>
		</tr>
		<tr>
			<td>Pen/Strep/Glutamine (100x)</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td>Media antibiotic</td>
		</tr>
		<tr>
			<td>Anti-anti (100x)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>Media antifungal</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x, pH 7.4)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (0.05%)</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD Bioscience</td>
			<td>352360</td>
			<td>100-&#956;m cell strainer</td>
		</tr>
		<tr>
			<td>60mm plates</td>
			<td>BD Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14001-12</td>
			<td>Large</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14091-11</td>
			<td>Fine, curved tip</td>
		</tr>
		<tr>
			<td>Large Forceps</td>
			<td>FST</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25G 5/8&#8221; needles</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>22G 1.5&#8221; needles</td>
			<td>BD</td>
			<td>305159</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>BD Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>BD Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separation columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Anti-Sca1 microbead kit (FITC)</td>
			<td rowspan="3">Miltenyi Biotec</td>
			<td rowspan="3">130-092-529</td>
			<td rowspan="3">FITC-anti-Sca1 1&#186;Ab and anti-FITC microbeads 2&#186;Ab</td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>AutoMACS running buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue chux pads</td>
			<td>Fisher</td>
			<td>276-12424</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent pads</td>
			<td>Fisher</td>
			<td>19-165-621</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam board</td>
			<td></td>
			<td></td>
			<td>Use from 50ml tubes</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>R2a21</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a anti-mouse Sca1 Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>MSCA21</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a R-PE</td>
			<td>Invitrogen</td>
			<td>R2a04</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a anti-mouse F4/80 R-PE</td>
			<td>Invitrogen</td>
			<td>MF48004</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-bottom tube</td>
			<td>BD Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (&#8211;Ca, &#8211;Mg)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (+Ca, +Mg)</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td>For collagenase solution</td>
		</tr>
		<tr>
			<td>Type I collagen (2.7 mg/ml in 37mm acetic acid</td>
			<td>Prepare in house12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x MEM</td>
			<td>Gibco</td>
			<td>11430-030</td>
			<td></td>
		</tr>
		<tr>
			<td>1M HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>0.34N NaOH</td>
			<td>Prepare in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Corning</td>
			<td>2870-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 carboxylic acid, succinimidyl ester, mixed isomers</td>
			<td>Invitrogen</td>
			<td>A-20004</td>
			<td></td>
		</tr>
		<tr>
			<td>0.89M NaHCO3&#160;</td>
			<td>Gibco</td>
			<td>25080-094</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunomagnetic separation of fat depot-specific sca1high adipose-derived stem cells (ascs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Enrichment of Sca1high ASCs from Different Fat Pads.
The vascular stromal cells isolated from SQ fat display fibroblast-like, stretched cell shape regardless of Sca1 expression level (Figure 1A). On the other hand, VIS (eWAT-derived) Sca1high and Sca1low cells demonstrate distinct difference in their cell shape. Like SQ (iWAT-derived) Sca1high cells, VIS (eWAT-derived)...

Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

The overall goal of this procedure is to isolate Sca1 positive mesenchymal stem cells from various mouse fat pads. The main advantage of this technique is that by using the immunomagnetic beads-based cell sorting adipose-derived stem cells from different fat depots can be effectibely enriched for subsequent experimentations. Generally, investigators new to this method will struggle because the strength and duration of the collagenase-mediated tissue digestion should be optimized to procure an adequate number of cells for sorting.To isolate the subcutaneous fat pads, begin by placing the mouse on a foam dissecting board in the supine position and spraying the animal with 70%ethanol. After securing the paws to the board, make a small incision in the skin of the lower abdomen. Then, grasping the top of the cut with forceps, use scissors to separate the skin and the peritoneum from the abdomen to the thorax.Making lateral cuts in the skin, along the side of the body, reflect the skin away from the peritoneum towards the head. Then, pull back the remaining skin, holding the inguinal fat away from the body, and use 22 gauge needles to pin the skin to the board. Next, use the forceps to grab one inguinal fat pad at its origin near the peritoneum, and use fine scissors to snip between the skin and inguinal fat, progressing toward the groin, and avoiding contaminating the subcutaneous fat pad with skin.Then, place the isolated inguinal fat pad in an SQ-labeled 60 millimeter dish containing 37 degrees Celsius culture medium, and harvest the inguinal fat pad from the other side of the animal in the same way. To isolate the visceral fat pads, cut the peritoneum to expose the visceral fat pads and gut. Then move the gut aside, towards the thorax.Grasping the visceral fat pad at the distal end, gently pull up on the fat tissue, and dissect it out, taking care to exclude the epididymal tissue. Then place the fat pad in a VIS-labeled 60 millimeter dish containing 37 degrees Celsius culture medium. When all of the fat pads have been collected, transfer the dishes to a tissue culture hood, and aspirate the medium.Use curved scissors to mince the pads. Then, add the fat pieces to individual 50 milliliter tubes containing freshly prepared collagenase. Next, shake the tubes at 300 RPM and 37 degrees Celsius for 10 to 20 minutes, until the tissues are digested.Some pieces of subcutaneous fat pad tissue may still be visible. Add 20 milliliters of culture medium to the collagenase solution to stop the digestion, and use a 10 milliliter serological pipette to aspirate and dispense the tissue slurry 10 times to fully dissociate the digested tissues. Filter the cells through 100 micron cell strainers, and centrifuge the tissues.Then carefully decant the medium, taking care not to disturb the pellets, and gently resuspend both groups of cells in five milliliters of sterile water. Wait two minutes for the erythrocytes to be lysed. Then arrest the reaction in each tube with 25 milliliters of medium supplemented with FBS.Filter both adipocyte populations through new 100 micron cell strainers, spin them down again, and then resuspend the pellets in one to three milliliters of medium. After counting by trypan blue exclusion, separately plate one times ten to the sixth subcutaneous fat pad cells and visceral cells into individual wells of a six-well cell culture plate. After trypsinizing the cells, dilute the trypsin with one milliliter of separation buffer, and then centrifuge the culture plates.We suspend the cells in 500 microliters per tube of separation buffer, and centrifuge the cells again. Now, resuspend the cells in 90 microliters per tube of separation buffer, and mix 10 microliters of anti-Sca1 FITC primary antibody into the cell suspension. After 10 minutes of incubation at four degrees Celsius in the dark, wash the cells with one milliliter of separation buffer per tube.After spinning, cells are resuspended in 80 microliters of separation buffer per cell suspension. Then mix 20 microliters of Anti-FITC Microbeads into each well, and place the plates at four degrees Celsius, protected from light for 15 minutes. At the end of the incubation, wash the cells in one milliliter of separation buffer, and resuspend the cells in 500 microliters of fresh buffer per tube.Now, place a column in the magnetic holder, and rinse the column with 500 microliters of separaton buffer. Transfer the cells from the subcutaneous fat pad tissue culture onto the column, taking care to avoid bubbles, and collect the unlabeled Sca1 negative cells in a 15 milliliter conical tube. When all of the cells have run through, wash the column three times with 500 microliters of separation buffer.Then transfer the column from the magnet into a new 15 milliliter conical tube, and plunge one milliliter of fresh separation buffer through the column to elute the Sca1 positive cells. Finally, spin down both Sca1 positive and negative cell fractions, and resuspend the pellets in one milliliter of culture medium. Then count the number of cells in each fraction, and plate the fractions in individuals wells of a new six-well cell culture plate.Vascular stromal cells isolated from subcutaneous fat display a fibroblast-like, stretched cell shape regardless of their level of Sca1 expression. Like subcutaneous inguinal fat-derived Sca1 high cells, visceral epididymal fat-derived Sca1 high cells display a stretched fibroblast-like cell shape. Whereas VIS Sca1 low cells exhibit an epithelioid shape.Sca1 high cells isolated from isolated Sca1-GFP mice are easily identified as GFP positive cells in tissue culture. Indeed, when these cells are assessed by flow cytometry, most of the GFP positive cells are confirmed to express Sca1 proteins on their cell surfaces, as detected by anti-Sca1 antibody. Coupled with real-time PCR analyses, the differential expression of the collagenolytic matrix metalloproteinases between inguinal and epididymal fat-derived Sca1 high adipose-derived stem cells can be demonstrated.Further, when fluorescein labeled type I collagen gels are used to assess pericellular degradation activity, a markedly increased collagen remodeling activity mediated by visceral Sca1 high adipose-derived stem cells is observed. Once mastered, the fat pad cell isolation can be completed in two hours if it is performed properly. While attempting this procedure, it's important to remember to maintain a clean and organized surgical field to ensure efficient fat pad isolation.Following this procedure, other methods, like a collagen degradation assay, can be performed to answer additional questions about adipose-derived stem cell-mediated tissue remodeling. After its development, this technique paved the way for researchers in the field of obesity and matrix biology to explore the role of adipose-derived stem cells in adipose tissue remodeling and function in mice and humans. After watching this video, you should have a good understanding of how to procure and enrich primary Sca1 positive cells from adipose tissue depots.

Research

JoVE Journal

Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

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

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

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

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

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

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