Name: isolation, proliferation and differentiation of rhesus macaque adipose-derived stem cells
Uploaded: 2021-05-26T12:30:00
Description: Watch this Scientific Journal Video about Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells at JoVE.com

The authors would like to thank Curtis Vande Stouwe for his technical assistance. The research underlying development of the protocols was supported by grants from the National Institute on Alcohol Abuse and Alcoholism (5P60AA009803-25, 5T32AA007577-20 and 1F31AA028459-01).

All obtained tissues and procedures were approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Sciences Center and were performed in accordance with the guidelines of the National Institute of Health (NIH publication No. 85-12, revised 1996).
1. Preparation of buffers and solutions
<ol>
	<li>Prepare sterile 5-phosphate-buffered saline wash buffer (5-PBS) solution by adding 5% penicillin/streptomycin (pen/strep) and 0.25 &#956;g/mL of fungal inhi...

<ol>
	<li>Fain, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+interleukins+and+other+inflammatory+cytokines+by+human+adipose+tissue+is+enhanced+in+obesity+and+primarily+due+to+the+nonfat+cells.">Release of interleukins and other inflammatory cytokines by human adipose tissue is enhanced in obesity and primarily due to the nonfat cells.</a> Vitamins and Hormones. 74, 443-477 (2006).</li><li>Ibrahim, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+and+visceral+adipose+tissue:+structural+and+functional+differences.">Subcutaneous and visceral adipose tissue: structural and functional differences.</a> Obesity Reviews. 11 (1), 11-18 (2010).</li><li>Wang, P., Mariman, E., Renes, J., Keijer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretory+function+of+adipocytes+in+the+physiology+of+white+adipose+tissue.">The secretory function of adipocytes in the physiology of white adipose tissue.</a> Journal of Cellular Physiology. 216 (1), 3-13 (2008).</li><li>Zuk, P. A., 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=Human+adipose+tissue+is+a+source+of+multipotent+stem+cells.">Human adipose tissue is a source of multipotent stem cells.</a> Molecular Biology of the Cell. 13 (12), 4279-4295 (2002).</li><li>Via, A. G., Frizziero, A., Oliva, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+properties+of+mesenchymal+Stem+Cells+from+different+sources.">Biological properties of mesenchymal Stem Cells from different sources.</a> Muscles, Ligaments and Tendons Journal. 2 (3), 154-162 (2012).</li><li>Horwitz, E. 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=Clarification+of+the+nomenclature+for+MSC:+The+International+Society+for+Cellular+Therapy+position+statement.">Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement.</a> Cytotherapy. 7 (5), 393-395 (2005).</li><li>Zuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells+in+tissue+regeneration:+A+Review.">Adipose-derived stem cells in tissue regeneration: A Review.</a> International Scholarly Research Notices Stem Cells. 2013, 713959 (2013).</li><li>Smith, U., Kahn, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+regulates+insulin+sensitivity:+role+of+adipogenesis,+de+novo+lipogenesis+and+novel+lipids.">Adipose tissue regulates insulin sensitivity: role of adipogenesis, de novo lipogenesis and novel lipids.</a> Journal of Internal Medicine. 280 (5), 465-475 (2016).</li><li>Laclaustra, M., Corella, D., Ordovas, 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=Metabolic+syndrome+pathophysiology:+the+role+of+adipose+tissue.">Metabolic syndrome pathophysiology: the role of adipose tissue.</a> Nutrition, Metabolism &amp; Cardiovascular Disease. 17 (2), 125-139 (2007).</li><li>Grundy, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+and+metabolic+syndrome:+too+much,+too+little+or+neither.">Adipose tissue and metabolic syndrome: too much, too little or neither.</a> European Journal of Clinical Investigation. 45 (11), 1209-1217 (2015).</li><li>Neeland, I. J., 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=Dysfunctional+adiposity+and+the+risk+of+prediabetes+and+type+2+diabetes+in+obese+adults.">Dysfunctional adiposity and the risk of prediabetes and type 2 diabetes in obese adults.</a> Journal of the American Medical Association. 308 (11), 1150-1159 (2012).</li><li>Kahn, C. R., Wang, G., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+adipose+tissue+and+adipocyte+function+in+the+pathogenesis+of+metabolic+syndrome.">Altered adipose tissue and adipocyte function in the pathogenesis of metabolic syndrome.</a> Journal of Clinical Investigation. 129 (10), 3990-4000 (2019).</li><li>Sarjeant, K., Stephens, 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=Adipogenesis.">Adipogenesis.</a> Cold Spring Harbor Perspectives in Biology. 4 (9), 008417 (2012).</li><li>Ford, S. 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=Differential+contribution+of+chronic+binge+alcohol+and+antiretroviral+therapy+to+metabolic+dysregulation+in+SIV-infected+male+macaques.">Differential contribution of chronic binge alcohol and antiretroviral therapy to metabolic dysregulation in SIV-infected male macaques.</a> American Journal of Physiology - Endocrinology and Metabolism. 315 (5), 892-903 (2018).</li><li>Gagliardi, C., Bunnell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rhesus+adipose-derived+stem+cells.">Isolation and culture of rhesus adipose-derived stem cells.</a> Methods in Molecular Biology. 702, 3-16 (2011).</li><li>Bunnell, B. A., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+isolation,+expansion+and+differentiation.">Adipose-derived stem cells: isolation, expansion and differentiation.</a> Methods. 45 (2), 115-120 (2008).</li><li>Bourin, P., 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=Stromal+cells+from+the+adipose+tissue-derived+stromal+vascular+fraction+and+culture+expanded+adipose+tissue-derived+stromal/stem+cells:+a+joint+statement+of+the+International+Federation+for+Adipose+Therapeutics+and+Science+(IFATS)+and+the+International+Society+for+Cellular+Therapy+(ISCT).">Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT).</a> Cytotherapy. 15 (6), 641-648 (2013).</li><li>Wang, Q. A., Scherer, P. E., Gupta, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methodologies+for+the+study+of+adipose+biology:+insights+gained+and+opportunities+ahead.">Improved methodologies for the study of adipose biology: insights gained and opportunities ahead.</a> Journal of Lipid Research. 55 (4), 605-624 (2014).</li></ol>

ADSC isolation, proliferation and differentiation protocols are straight-forward and reproducible, but they require careful technique to ensure adequate isolation, healthy expansion, and efficient differentiation. A sterile working environment is critical for all cell culture experiments. Bacteria or fungi may be introduced into cell cultures through contaminated tools, media or work environment. Fungal contamination is indicated by spore growth in the culture, while bacterial contamination is indicated by the presence o...

Adipose tissue is comprised of a heterogeneous mixture of cells, predominantly mature adipocytes and a stromal vascular fraction including fibroblasts, immune cells and adipose-derived stem cells (ADSCs)1,2,3. Primary ADSCs can be isolated directly from white adipose tissue and stimulated to differentiate into adipocytes, cartilage or bone cells4. ADSCs exhibit classical stem cell characteristics such as maintenance of multipotency in vitro and self-renewal; and are adherent to plastic in culture5,6. ADSCs are of important interest for the use in regenerative medicine due to their multipotency and ability to be easily harvested in large quantities using non-invasive techniques7. Adipogenic differentiation of ADSCs produces cells that functionally mimic mature adipocytes including lipid accumulation, insulin-stimulated glucose uptake, lipolysis, and adipokine secretion8. Their resemblance to mature adipocytes has led to the widespread use of ADSCs for physiological investigation of cellular characteristics and metabolic function of adipocytes. There is increasing evidence&#160;supporting the idea that the development of metabolic dysfunction and disorders originates at the cellular or tissue level9,10,11,12. Optimal ADSC differentiation is required for sufficient adipose tissue expansion, proper adipocyte function, and effective metabolic regulation13.
Protocols described in this manuscript are straightforward techniques utilizing standard laboratory equipment and basic reagents. The manuscript first describes the protocol for the isolation of primary ADSCs from fresh adipose tissue using mechanical and enzymatic digestion. Next, the protocol for proliferation and passaging of ADSCs in stromal medium is described. Lastly, the protocol for adipogenic differentiation of ADSCs is described. Following differentiation, these cells can be used for studies to better understand adipocyte metabolism and mechanisms of dysfunction. The protocols for confirmation of adipogenic differentiation and lipid droplet detection using Oil Red O and boron-dipyrromethene (BODIPY) staining are also described. The details of these protocols focused on primary ADSCs isolated from fresh omental adipose tissue of rhesus macaques. We and others have used this protocol to successfully isolate ADSCs from rhesus macaque subcutaneous and omental adipose tissues depots14,15. For the same amount of tissue used, we have observed that subcutaneous adipose tissue is more dense, tougher and yields less cells from digestion compared to omental adipose tissue. This protocol has also been used to isolate ADSCs from human adipose samples16.

<table>
	<tbody>
		<tr>
			<td>0.4 % trypan blue</td>
			<td>Thermo-Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5-ml microcentrifuge tube</td>
			<td>Dot Scientific</td>
			<td>707-FTG</td>
			<td></td>
		</tr>
		<tr>
			<td>100 % isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>PX1838-P</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mm cell culture dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I7018</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL plastic conical tube</td>
			<td>Fisher Scientific</td>
			<td>50-465-232</td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#181;m cell strainer</td>
			<td>Corning</td>
			<td>CLS431751</td>
			<td></td>
		</tr>
		<tr>
			<td>a-MEM</td>
			<td>Thermo-Fisher</td>
			<td>12561056</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds Wrap</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY</td>
			<td>Thermo-Fisher</td>
			<td>D3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>05470</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type I</td>
			<td>Thermo-Fisher</td>
			<td>17100017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone-Water Soluble</td>
			<td>Sigma-Aldrich</td>
			<td>D2915</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide, DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>Thermo-Fisher</td>
			<td>15230162</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, USDA-approved</td>
			<td>Sigma-Aldrich</td>
			<td>F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungizone/Amphotericin B (250 ug/mL)</td>
			<td>Thermo-Fisher</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Thermo-Fisher</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer with cover slip</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Nikon</td>
			<td>DIAPHOT-TMD</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Thermo-Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory rocker, 0.5 to 1.0 Hz</td>
			<td>Reliable Scientific</td>
			<td>Model 55 Rocking</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral buffered formalin (10 %)</td>
			<td>Pharmco</td>
			<td>8BUFF-FORM</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma-Aldrich</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformeldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo-Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Thermo-Fisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell (RBC) lysis buffer</td>
			<td>Qiagen</td>
			<td>158904</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes, 2 to 25 mL</td>
			<td>Costar Stripettes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard humidified cell culture incubator, 37 &#176;C, 5 % CO2</td>
			<td>Sanyo</td>
			<td>MCO-17AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo-Fisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, proliferation and differentiation of rhesus macaque adipose-derived stem cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

The ADSCs isolated from rhesus macaque adipose tissue samples were seeded on culture plates and is shown in Figure 1. On the day of plating, cells are non-adherent and float in the culture dish as shown in Figure 1A. Within 72 h, ADSCs will become 80% confluent and are ready for adipocyte differentiation (Figure 1B). ADSCs exhibit strong adipogenic characteristics after chemical induction. After 14 days of differentiation, mature ad...

Watch this Scientific Journal Video about Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells at JoVE.com

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Adipose-derived stem cell isolation and proliferation produces a large number of stem cells that can be used for several downstream applications and experimental techniques. A major intended use for differentiated adipocytes in this protocol is metabolic assays such as insulin stimulated glucose uptake, lipogenesis, and stimulated lipolysis. To begin, create a sterile working environment by disinfecting the biosafety hood and all the tools.Pipette approximately 10 milliliters of five PBS buffer into four 100 millimeter cell culture dishes. Transfer a 50 gram adipose sample into one of the dishes and wash it four times by sequentially transferring it across the other dishes containing five PBS. Then transfer the washed adipose tissue into a clean 100 millimeter culture dish and thoroughly mince it using scissors or sterile forceps.Transfer a one to three centimeter section of the minced tissue to a 50 milliliter conical tube containing 13 milliliters of collagenase buffer. Rinse the remaining tissue from the culture dish with two milliliters of collagenase buffer to ensure a total volume of 15 milliliters. Thoroughly mix the sample using a 25 milliliter serological pipette and incubate the tube at 37 degrees Celsius on a rocker for 30 to 60 minutes.In order to neutralize the enzyme activity, add 10 milliliters of ADSC growth medium into the tube after incubation and mix it well with a pipette to separate any tissue aggregates. Transfer the liquid portion to a new sterile 50 milliliter conical tube, leaving the solid tissue. Wash the tissue three times using seven milliliters of two PBS and transfer the liquid to the new tube.Next, centrifuge the tube at 500 times G for five minutes and carefully remove as much supernatant as possible without disturbing the pellet. Resuspend the pellet in one milliliter of 1X red blood cell or RBC lysis buffer and incubate it at room temperature for 10 minutes. Then wash the cells twice by adding five milliliters of ADSC growth medium to the tube and centrifuging to remove the supernatant.After the last wash, resuspend the pellet in two milliliters of ADSC growth medium and filter it through a 70 micron cell strainer into a sterile 50 milliliter conical tube. Rinse the strainer with an additional two milliliters of the medium and transfer four milliliters of the suspension into a sterile 100 milliliter culture dish. Rinse the conical tube twice with three milliliters of medium to collect a total volume of 10 milliliters in the culture dish.Observe the collected cells under an inverted microscope at 10X magnification to check for floating cells. Incubate the cells for 24 hours in a carbon dioxide incubator at 37 degrees Celsius, 5%carbon dioxide and 100%relative humidity. To ensure sterility of the culture media, include a plate with media alone.After 24 hours, view the cells under the inverted microscope to check for cell adherence. Remove and replace the medium with warm medium every 48 hours until cells are 80 to 90%confluent. For proliferation, remove the growth medium from the confluent ADSC cells and wash them twice with two milliliters of sterile room temperature PBS.Aspirate the PBS and add two milliliters of 0.25%trypsin EDTA into each culture plate, covering the entire surface area of the plate. Incubate the plate for seven minutes at 37 degrees Celsius and 5%carbon dioxide, then mechanically dislodge the trypsinized cells by forceful pipetting. Add two milliliters of ADSC growth medium and gently mix the cells.Transfer the cells into a 50 milliliter conical tube, rinsing the dish with an additional two milliliters of medium to ensure maximal cell recovery from the plate. Next, centrifuge the tube at 500 times G for five minutes at room temperature and decant the supernatant to obtain the cell pellet. Resuspend the cell pellet in five to six milliliters of ADSC growth medium per million cells.Count the cells using a hemocytometer and plate them as described in the text manuscript. Once the cells reach 80 to 90%confluency, aspirate the growth medium and rinse the cells with sterile room temperature PBS, then add 10 milliliters of ADSC differentiation medium. Replace the medium every three days for approximately 14 to 21 days.Observe the cells for the presence of lipid droplets under the inverted microscope at 40X magnification. Mature adipocytes are generally observed after 14 days. On the day of plating, the ADSCs were non-adherent and floated in culture.The cells became 80%confluent after 72 hours and were ready for adipocyte differentiation. After 14 days of ADSC differentiation, mature adipocytes exhibited strong adipogenic characteristics which were observed under 40X magnification. On day 14, cells were stained and fixed with Oil Red O and BODIPY for lipid droplet visualization.Differentiated adipocytes could be observed under 20X magnification. When attempting this protocol, it is important to remember that a sterile working environment is critical for healthy isolation, adequate expansion, and efficient differentiation of adipose-derived stem cells. Following this procedure, these cells can be used for several metabolic assays such as glucose uptake, lipogenesis, and lipolysis which is a major focus of our research.This technique allows for the investigation of how chronic alcohol and SIV impact adipocyte metabolic capacity.

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Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization

Inside a biosafety cabinet, remove the murine adipose tissue with sterile forceps. Once excess PBS is removed, transfer the tissue to another petri dish and wash twice for five minutes each with 10 milliliters of PBS AA solution. Add 20 milliliters of prepared collagenase solution into a crystal beaker containing one square centimeter sized fragmented tissue and a magnetic bar.Incubate the sealed beaker at 37 degrees Celsius with continuous agitation. Filter the homogenate through a 40-mesh 0.38-millimeter stainless steel aperture Filter on a petri dish and collect the cell suspension in a sterile 50-milliliter conical tube. Carefully suspend the stromal vascular fraction in 20 milliliters of warm supplemented DMEM and centrifuge the suspension.After discarding the supernatant, gently wash the cells twice with 40 milliliters of non-supplemented DMEM medium. Suspend the palate in five milliliters of supplemented DMEM. Transfer the suspension to a 25-square centimeter T bottle and incubate.Wash cells three times with five milliliters of warm PBS AA and twice with non-supplemented DMEM. To remove any cell debris and non-murine cells add five milliliters of fresh, warm supplemented DMEM and change the medium every three to four days. Once the cells reach 90 to 100%confluency, add one milliliter of warm trypsin EDTA solution to the DMEM washed cells.Incubate for five to seven minutes at 37 degrees Celsius. Add four milliliters of supplemented DMEM to the detached cell monolayer and gently disaggregate the suspension. Suspend the cells in five milliliters of warm supplemented DMEM.After centrifuging and discarding the supernatant, gently mix the pellet in one milliliter of supplemented DMEM. Stain the cells with trypan blue and count them in a Neubauer chamber. Seed cells in T-75 flasks.To ensure colony formation and high proliferation, observe the morphology of the hematoxylin and eosin-stained cells under an optical microscope. hematoxylin and eosin staining reveal extensive cytoplasm with elongated prolongations and abundant intracellular microfilaments.

Immunofluorescent Detection of Adipose-Derived Stem Cell Expression Markers

Begin by washing murine adipose-derived stem cells in a six-well plate with two milliliters of wash buffer. Add one milliliter of blocking reagent after incubating and washing the cells thrice. Incubate the cells with slow agitation for 20 minutes.Add 200 microliters of the primary antibodies to each well and incubate overnight at four degrees Celsius. After washing the plates thrice as demonstrated previously, incubate with 200 microliters of the secondary antibodies. Counterstain the cell nuclei with 100 microliters of DRAQ7 for 20 minutes after washing the plates thrice.Repeat washing and mount slides. Visualize the mounted slides under epifluorescence confocal microscopy. Adipose-derived stem cells were 100%positive for CD9 surface markers in all subpassages tested.All cells expressed CD63 markers in the subpassages one, three and nine, while 49.3%of the cells expressed it in passage seven. CD34 markers were absent in passages one, seven, and nine, but visible in passage three.

Flow Cytometric Detection of Adipose-Derived Stem Cell Expression Markers

Begin by adjusting the concentration of thawed murine adipose-derived stem cell suspension using PBS with 5%fetal bovine serum. Aliquot 100 microliters of the suspension containing 100, 000 cells into a 1.5 milliliter centrifuge tube. Add appropriate amounts of anti-CD105 AF594 and anti-CD31 AF680 as per the manufacturer's instructions.Incubate for 20 minutes in the dark at four degrees Celsius. Remove excess stain by washing with PBS/FBS and centrifuge at 250 x g for five minutes. Resuspend the cells in 300 microliters of 1%formalin.Create a dot plot graph by clicking on the polygon symbol to select the subpopulation of interest and exclude the debris areas using FSC-A versus SSC-A gating. Next, click on the polygon symbol to generate a new dot plot and exclude the duplexes and multiples cells using SSC-H versus SSC-A followed by FSC-H and FSC-A. Use Y610-mCHERRY for the AF594 and the R660 APC-A detectors for the AF680 fluorophores, respectively.Click on the quadrant symbol and set each detector's threshold for autofluorescence cells. Select samples labeled with CD105 and positive population to be shown on Q1LR. Next, choose samples labeled with CD31 and positive population to be shown on Q1LR.Immunophenotyping showed that 98.7%of the cell population was positive for CD105, while only 5.88%expressed the CD31 marker.

Adipose-Derived Stem Cell Differentiation to the Adipogenic Lineage

Begin by seeding a six well plate with 10, 000 adipose-derived stem cells per square centimeter. Once cells become 60 to 80%confluent add the differentiation medium. On day 12, wash the cells trice with PBS and incubate them with 500 microliters of 4%formalin for one hour at room temperature.Once the cells are washed with distilled water, wash them with 60%isopropanol for five minutes and let them dry. Add 500 microliters of 0.5%oil red O staining dye diluted in 60%alcohol. After incubation at room temperature wash the wells twice with one milliliter of distilled water and observe the plate under an inverted microscope.Adipose-derived stem cells differentiated towards the adipogenic lineage showing reduced cell size and rounded shape within 48 hours. After seven days, lipid vesicles were visible, which were positive for oil red staining 12 days after differentiation.