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, and 1x antibiotics/antifungals. This will be used to keep tissues in before digestion. Place stock and aliquot in 37 &#186;C water bath.</li>
	<li>Prepare five milliliters of Type III collagenase solution at 5mg/ml dissolved in HBSS (+Ca, +Mg) for each type of fat pad being isolated, up to five mice. For example, when isolating SQ and VIS from a single genotype, make 10 ml, if wild-type and mutant SQ and VIS, make 20 ml. Adjust pH to 7.4 and sterile filter. Aliquot into 50 ml tubes. Set aside away from light.</li>
	<li>Use 70% ethanol to disinfect surgical field. Wipe down and cover with a blue pad. Spray some ethanol on the blue pad.</li>
	<li>Use the 22 G needles to pin down an absorbent pad to the styrofoam board and trim with scissors. Spray the pad with ethanol then set the board on the blue pad and cover with another blue pad until beginning dissection.</li>
	<li>Fill two 50 ml tubes with ethanol and place in a stand adjacent to the surgical field. Place the scissors and forceps in the ethanol.</li>
	<li>Fill another 50 ml tube with PBS plus 1x anti-anti for rinsing ethanol off surgical tools.</li>
	<li>In the hood, label 60 mm dishes for each tissue type and fill with culture media warmed to 37 &#186;C. Place the plates adjacent to the surgical area.</li>
</ol>
2. Isolation of Subcutaneous (SQ) Fat Pads
<ol>
	<li>Euthanize mouse with an overdose of isoflurane and pneumothorax.</li>
	<li>Gently spray mouse with 70% ethanol and lay supine. Pin the paws to the foam board with 22 G needles.</li>
	<li>Make a small cut in the skin of the lower abdomen. Holding the top of the cut with the forceps, take the scissors and separate the skin from the peritoneum.</li>
	<li>Once the skin is separated from the peritoneum from the abdomen to the thorax, reflect the skin away from the peritoneum towards the head by making lateral cuts in the skin along the side of the body.</li>
	<li>Reflect the remaining skin holding the inguinal fat away from the body and pin down to the board with 22 G needles.</li>
	<li>Switch to fine scissors and forceps. Grab the inguinal fat pad with the forceps at the origin near the peritoneum and snip away between the skin and inguinal fat progressing towards the groin avoiding contaminating the SQ with skin.</li>
	<li>Place the insolated inguinal fat pad in the labeled 60 mm dish.</li>
</ol>
3. Isolation of Visceral (VIS) Fat Pads
<ol>
	<li>In a similar fashion to step 2.5, cut the peritoneum open to expose the visceral fat pads and gut.</li>
	<li>Move the gut away toward the thorax.</li>
	<li>Grab the VIS fat pad at the distal end and gently pull up. Dissect out the VIS fat pad while being careful to exclude epididymal (or uterine, if using female mice) tissue.</li>
	<li>Place in labeled dish and repeat step 3.3 for the remaining VIS pad.</li>
</ol>
4. Collagenase Digestion of Fat Pads
<ol>
	<li>Move 60 mm dishes to the tissue culture hood and aspirate off media.</li>
	<li>Mince the fat pads with curved scissors then add to collagenase solution. Be sure to rinse the scissors and forceps in between tissue types with ethanol and PBS.</li>
	<li>Shake at 300 rpm and 37 &#186;C for 10-20 min until tissues are digested. It is acceptable if some pieces of SQ are still visible. This will be addressed in a later step.</li>
	<li>Add 25 ml of culture media to the collagenase solution to stop the collagenase treatment, and pipette up and down 10 times with a 10 ml serological pipette to disperse the digested tissues.</li>
	<li>Strain cells using a 100 &#181;m cell strainer. Centrifuge for 10 min at 300 x g.</li>
	<li>Carefully decant off media not to disturb the pellet and add 5 ml of sterile water to the pellet and gently pipette to suspend cells. Wait 2 min to lyse erythrocytes.</li>
	<li>Add 25 ml of media with 10% FBS and strain cells through a new 100 &#181;m cell strainer.</li>
	<li>Centrifuge for 10 min at 300 x g. Decant media and resuspend pellet in 1 ml of culture media.</li>
	<li>Count cells with a hemocytometer using 1:1 trypan blue.</li>
	<li>Plate 1 x 106 cells in one well on a 6-well plate.</li>
</ol>
5. Magnetic Cell Separation
<ol>
	<li>After about 4 to 6 hr of cell adhesion, rinse cells twice with HBSS (-Ca, -Mg). Dissociate adherent cells using 0.05% trypsin. Dilute trypsin cell suspension in 1 ml separation buffer and spin for 5 min at 300 x g.</li>
	<li>Aspirate supernatant. Resuspend cells in 500 &#181;l buffer. Remove a 10 &#181;l aliquot and count cells using a hemocytometer.</li>
	<li>Spin cells for 5 min at 300 x g.</li>
	<li>Aspirate supernatant completely. Resuspend cells in 90 &#181;l buffer. Add 10 &#181;l anti-Sca1-FITC primary antibody. Mix well and incubate for 10 min at 4 &#186;C in the dark.</li>
	<li>Wash cells with 1 ml buffer and centrifuge cells for 5 min at 300 x g. Remove supernatant completely.</li>
	<li>Resuspend cells in 80 &#181;l buffer. Add 20 &#181;l anti-FITC microbeads. Mix well and incubate for 15 min at 4 &#186;C in the dark.</li>
	<li>Wash cells with 1 ml buffer and centrifuge cells for 5 min at 300 x g.</li>
	<li>Remove supernatant and resuspend pellet in 500 &#181;l buffer.</li>
	<li>Place column in the magnetic holder and rinse column with 500 &#181;l buffer.</li>
	<li>Add cell suspension to column. Avoid bubbles while pipetting.</li>
	<li>Collect the unlabeled Sca1- cells and wash column 3 times with 500 &#181;l buffer. Only add new buffer when the reservoir is empty. Avoid bubbles while pipetting.</li>
	<li>Remove column from the magnetic holder and place into a 15 ml conical tube.</li>
	<li>Add 1 ml buffer to column and elute Sca1+ by pushing supplied plunger through the column reservoir.</li>
	<li>Spin down cell fractions for 5 min at 300 x g. Resuspend cells in 1 ml culture media.</li>
	<li>Remove a 10 &#181;l aliquot from the labeled and unlabeled fractions and count cells using a hemocytometer.</li>
	<li>Plate each cell fraction in 1 well of a 6-well plate.</li>
</ol>
6. Verification of Immunomagnetic Separation of Sca1high ACSs with Flow Cytometry
<ol>
	<li>Rinse cells twice with HBSS (-Ca, -Mg) and dissociate cells using 0.05% trypsin.</li>
	<li>Centrifuge cells at 300 x g for 5 min. Resuspend cells in 1 ml culture media and count using a hemocytometer.</li>
	<li>Obtain &#62;106 cells in 1 ml culture media and centrifuge at 300 x g for 5 min.</li>
	<li>Remove supernatant and repeat step 6.3 two more times.</li>
	<li>Resuspend cells with 1 ml of 2% goat serum + 2% BSA and block for 30 min at RT.</li>
	<li>Centrifuge cells at 300 x g for 5 min.</li>
	<li>Remove blocking solution.</li>
	<li>Add rat IgG2a Alexa Fluor 647 (0.25 &#181;g, 1:400) or anti-Sca1 Alexa Fluor 647 (0.25 &#181;g, 1:400), in 100 &#181;l of PBS with 2% goat serum and 2% BSA + PBS at 4 &#176;C for 30 min in the dark.</li>
	<li>Add 1 ml of cold PBS to cells. Centrifuge 300 x g for 5 min at 4 &#176;C .</li>
	<li>Remove supernatant and repeat step 6.9 two more times.</li>
	<li>Resuspend cells in 1 ml PBS. Pass cell suspensions through a 100 &#181;m cell strainer in preparation for flow cytometric analysis.</li>
</ol>

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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. 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The authors have nothing to disclose.

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 the surface antigen is well-characterized, the use of immunomagnetic separation increases the number of cells to be analyzed without relying on the use of FACS equipment15, which is still not readily accessible to many biological researchers at small institutes without core facilities.
There are some critical aspects of the procedure that must be observed to ensure a successful outcome. The procurement of adipose-derived stem cells requires the surgical isolation of mouse adipose tissue depots. Therefore, the speed and accuracy of tissue retrieval is imperative to yield a high number of viable cells. Additionally, the maintenance of clean surgical fields and instruments are vital to the outcome of the procedure by preventing microbial contamination of cell and tissue samples. Dissected adipose tissues must be enzymatically dissociated in a type II collagenase solution. ECM composition differs between SQ and VIS fat pads10,16, where VIS contain less collagens than SQ. Therefore, the duration of VIS fat pad digestion requires approximately half the amount of time as SQ. It is acceptable to stop tissue digestion after the stated incubation period even if small particles of tissue are still present in the collagenase solution. These pieces may be mechanically dissociated with pipetting once culture media has been added to inhibit collagenase activity. While it is possible to proceed directly to immunomagnetic sorting following SVF isolation, our group seeds 1 x 106 unsorted cells on plastic plates for about 4 to 6 hr before continuing with cell sorting. Despite multiple filtration steps during SVF isolation, debris will still be present within the cell suspension. Plating the cells before proceeding to immunomagnetic cell separation provides an opportunity to wash debris and unattached cells away, thus allowing magnetic sorting to only be applied to adherent cells that contain adipogenic progenitor cells.
While Sca1 is not found in human genome, the identification and validation of alternative cell surface antigens expressed on fat depot-dependent human adipose stem cells17, when coupled with this cell separation technique, may help us define the biology of human adipose stem cells.

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) Sca1high cells display stretched, fibroblast-like cell shape, whereas VIS Sca1low cells demonstrate epithelioid shape. Sca1high cells isolated from Sca1-GFP mice are easily identified as GFP-positive cells in tissue culture (Figure 1B). When these cells were assessed with flow cytometry, most of the GFP positive cells were confirmed to express Sca1 proteins on the cell surface as detected with Anti-Sca1 antibody (Figure 1C). Sca1high cells derived from inguinal fat pad maintain increased capacity of adipocyte differentiation whereas eWAT-derived Sca1high cells are more difficult to differentiate into adipocyte with conventional adipogenic mix10.
Fat Depot-dependent Gene Expression of Sca1highASCs (Figure 2).
Genome-wide transcriptome analyses with RNA sequencing demonstrated the enrichment of genes related to extracellular matrix proteins and modifiers (GO:0031012, GO:0005578) in those Sca1high ASCs10. Coupled with real-time PCR analyses, we were able to demonstrate the differential expression of the collagenolytic MMPs (MMP2, MMP8, MMP13, MMP14) between iWAT- and eWAT-derived Sca1high ASCs. When fluorescein-labeled type I collagen gels were used to assess pericellular degradation activity, we observed the markedly increased collagen remodeling activity mediated by VIS Sca1high ASCs10.
<img alt="Figure 1" src="/files/ftp_upload/53890/53890fig1.jpg" /> 
Figure 1: Immunomagnetic Separation of Murine Sca1high ASCs from Different Fat Depots. (A) Sca1high and Sca1low ASCs isolated from SQ (iWAT) and VIS (eWAT). Scale = 100 &#181;m. (B) Sca1-GFP cells isolated from iWAT of Sca-GFP mice. Scale = 100 &#181;m. (C) Cell surface expression of Sca1 in Sca1-GFP-positive cells assessed with flow cytometry. (left) control rat IgG (right) anti-Sca1 antibody. X-axis, GFP intensity. Y-axis, Alexa-Fluor 647. Panel A shown previously in Tokunaga, M. et al., (2014). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53890/53890fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53890/53890fig2.jpg" /> 
Figure 2: Fat Depot-Dependent Expression of Collagenolytic MMPs, TIMPs, and Collagens. (A) Differential gene expression of ECMs and ECM modifiers in Sca1high ASCs isolated from different fat depots. (B) Increased collagen degradation activity of VIS (eWAT-derived) Sca1high ASCs. Degradation of collagen is shown as the disappearance of fluorescent signals (arrows and arrowheads). Inset is an enlarged image of focused collagen degradation mediated by individual cell of SQ ASCs. Cells were cultured for 72 hr. Data shown previously in Tokunaga, M. et al., (2014). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53890/53890fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

immunomagnetic-separation-fat-depot-specific-sca1high-adipose-derived

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.8K Views.  Biointerfaces Institute, University of Michigan. 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.

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

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 process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

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. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

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 therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these 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 differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

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 body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

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 vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

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 microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

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 order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

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

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

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 disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

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

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

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

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

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