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. H., Inoue, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+adipocyte+differentiation+and+peri-adipocyte+collagen+turnover.">3-D adipocyte differentiation and peri-adipocyte collagen turnover.</a> Methods Enzymol. 538, 15-34 (2014).</li><li>Rajan, N., Habermehl, J., Cote, M. F., Doillon, C. 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. K., 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=Identification+of+Specific+Cell-Surface+Markers+of+Adipose-Derived+Stem+Cells+from+Subcutaneous+and+Visceral+Fat+Depots.">Identification of Specific Cell-Surface Markers of Adipose-Derived Stem Cells from Subcutaneous and Visceral Fat Depots.</a> Stem Cell Reports. 2 (2), 171-179 (2014).</li></ol>

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.

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

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 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 ...