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>
	<li>Chun, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pericellular+collagenase+directs+the+3-dimensional+development+of+white+adipose+tissue.">A pericellular collagenase directs the 3-dimensional development of white adipose tissue.</a> Cell. 125 (3), 577-591 (2006).</li><li>Chun, T. H., 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=Genetic+link+between+obesity+and+MMP14-dependent+adipogenic+collagen+turnover.">Genetic link between obesity and MMP14-dependent adipogenic collagen turnover.</a> Diabetes. 59 (10), 2484-2494 (2010).</li><li>Chun, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peri-adipocyte+ECM+remodeling+in+obesity+and+adipose+tissue+fibrosis.">Peri-adipocyte ECM remodeling in obesity and adipose tissue fibrosis.</a> Adipocyte. 1 (2), 89-95 (2012).</li><li>Sun, K., Tordjman, J., Clement, K., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrosis+and+adipose+tissue+dysfunction.">Fibrosis and adipose tissue dysfunction.</a> Cell Metab. 18 (4), 470-477 (2013).</li><li>Spangrude, G., Heimfeld, S., Weissman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+Characterization+of+Mouse+Hematopoietic+Stem+Cells.">Purification and Characterization of Mouse Hematopoietic Stem Cells.</a> Science. 241, 58-62 (1988).</li><li>Welm, B. 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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J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ready-to-use,+storable+and+reconstituted+type+I+collagen+from+rat+tail+tendon+for+tissue+engineering+applications.">Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications.</a> Nat Protoc. 1 (6), 2753-2758 (2006).</li><li>Ma, X., Robin, C., Ottersbach, K., Dzierzak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ly-6A+(Sca-1)+GFP+Transgene+is+Expressed+in+all+Adult+Mouse+Hematopoietic+Stem+Cells.">The Ly-6A (Sca-1) GFP Transgene is Expressed in all Adult Mouse Hematopoietic Stem Cells.</a> Stem Cells. 20 (6), 514-521 (2002).</li><li>Berry, R., Rodeheffer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+adipocyte+cellular+lineage+in+vivo.">Characterization of the adipocyte cellular lineage in vivo.</a> Nat Cell Biol. 15 (3), 302-308 (2013).</li><li>Jeffery, E., Church, C. D., Holtrup, B., Colman, L., Rodeheffer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+depot-specific+activation+of+adipocyte+precursor+cells+at+the+onset+of+obesity.">Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity.</a> Nat Cell Biol. 17 (4), 376-385 (2015).</li><li>Mori, S., Kiuchi, S., Ouchi, A., Hase, T., Murase, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristic+Expression+of+Extracellular+Matrix+in+Subcutaneous+Adipose+Tissue+Development+and+Adipogenesis;+Comparison+with+Visceral+Adipose+Tissue.">Characteristic Expression of Extracellular Matrix in Subcutaneous Adipose Tissue Development and Adipogenesis; Comparison with Visceral Adipose Tissue.</a> Int J Biol Sci. 10 (8), 825-833 (2014).</li><li>Ong, W. 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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 ...

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