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 inhibitor to 1x PBS. Prepare sterile 2-PBS wash buffer (2-PBS) solution by adding 2% pen/strep and 0.25 &#956;g/mL of fungal inhibitor in 1x PBS. Wash buffers can be stored at 4 &#176;C for future use and must be used within 4 weeks. 
	NOTE: 5-PBS buffer is used for adipose tissue collection and initial washing to minimize possible contamination as tissue samples obtained at necropsy are collected in a non-sterile environment.</li>
	<li>Prepare sterile collagenase buffer (CB) by combining 0.075% collagenase Type I (125 units/mg activity), 2% pen/strep, and 0.25 &#956;g/mL of fungal inhibitor in Hank&#8217;s balanced salt solution (HBSS) with 1% Bovine Serum Albumin. CB must be used within 1 h.</li>
	<li>Prepare sterile ADSC growth medium using &#945;-MEM buffer. Combine 100 mL of fetal bovine serum (FBS), 5 mL of 200 mM L-glutamine solution, 500 &#956;L of 0.25 &#956;g/mL of fungal inhibitor and 10 mL of pen/strep solution in 394 mL of &#945;-MEM buffer. ADSC growth medium can be stored at 4 &#176;C and must be used within 4 weeks. 
	NOTE: Heat inactivation of FBS is not necessary.</li>
	<li>Prepare sterile ADSC differentiation medium by combining ADSC growth medium as prepared above and induction agents to achieve final concentration of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine and 50 &#956;M indomethacin.</li>
</ol>
2. ADSC isolation
<ol>
	<li>Tissue preparation and digestion
	<ol>
		<li>Pipette ~10 mL of 5-PBS buffer into four 100 mm cell culture dishes.</li>
		<li>Transfer ~50 g of adipose sample to one of the 100 mm dishes containing 5-PBS. Wash the collected adipose tissue sample four times by transferring it sequentially across the four culture dishes containing 5-PBS.</li>
		<li>Transfer adipose sample to a clean 100 mm culture dish and thoroughly mince adipose tissue using scissors or two sterile scalpels. Mincing allows for increasing the surface area for efficient and complete enzymatic digestion.</li>
		<li>Transfer the minced adipose tissue to 50 mL plastic conical tube containing 13 mL of CB (1&#8211;3 cm section of tissue per 15 mL of CB).</li>
		<li>Rinse the culture dish with 2 mL of CB and transfer the medium to the 50 mL plastic conical tube. 
		NOTE: At this point, there should be a total of 15 mL of CB with tissue in a 50 mL plastic conical tube.</li>
		<li>Pipette up and down several times using 25 mL serological pipette to facilitate mechanical tissue digestion.</li>
		<li>Incubate the 50 mL plastic conical tube containing tissue in CB on a rocker at medium speed at 37 &#176;C for 30&#8211;60 min.</li>
	</ol>
	</li>
	<li>ADSC plating for culture
	<ol>
		<li>Add 10 mL of ADSC growth medium to the 50 mL tube containing tissue to neutralize enzyme activity. Pipette up and down several times using 25 mL serological pipette to separate any adipose tissue aggregates.</li>
		<li>Transfer the liquid portion to a new sterile 50 mL conical tube leaving the solids behind. Wash the original 50 mL tube 3 times with ~7 mL of 2-PBS buffer and transfer the liquid portion to the 50 mL tube.</li>
		<li>Centrifuge at 500 x g for 5 min to obtain a cell pellet. Carefully remove as much supernatant as possible using a serological pipette without disturbing the cell pellet. Do not decant.</li>
		<li>Resuspend the cell pellet in 1 mL of 1x red blood cell (RBC) lysis buffer and incubate at room temperature for 10 min. Add 5 mL of ADSC growth medium to the tube and centrifuge at 500 x g for 5 min, then carefully remove and discard the supernatant.</li>
		<li>Add 5 mL of ADSC growth medium and centrifuge at 500 x g for 5 min to wash cell pellet. Discard the supernatant.</li>
		<li>Resuspend the pellet in 2 mL of ADSC growth medium and filter through a 70 &#956;m cell strainer into a new sterile 50 mL plastic conical tube.</li>
		<li>Rinse the cell strainer with an additional 2 mL of ADSC growth medium. Transfer 4 mL of the suspension containing ADSCs from the 50 mL conical tube to a sterile 100 mm culture dish.</li>
		<li>Wash the 50 mL conical tube 2 times with 3 mL of ADSC growth medium and transfer the liquid into the 100 mm culture dish containing ADSCs for a total of 10 mL of medium in culture dish.</li>
		<li>View cells under an inverted microscope at 10x magnification to check for floating cells in the media as shown in Figure 1A. Cells should be maintained in a CO2 incubator at 37 &#176;C at 5% CO2 and 100% relative humidity.</li>
		<li>After 24 h, view cells under an inverted microscope to check for cell adherence. Aspirate ADSC growth medium from the plate and replace with 10 mL of fresh, warm (37 &#176;C) media. Remove and replace media every 48 h until cells are 80%&#8211;90% confluent (Figure 1B). 
		NOTE: At least 10&#8211;20 % of cells will be adherent by 24 h. Check the cell culture for signs of contamination such as cell granularity, turbidity of the media, or growth of spores. Once cells are 80% confluent, they can be harvested for proliferation and cryopreservation or induced to differentiate. ADSCs can be expanded to 4&#8211;6 passages before losing their ability to efficiently proliferate or differentiate.</li>
	</ol>
	</li>
</ol>
3. ADSC proliferation
<ol>
	<li>Cell detachment
	<ol>
		<li>Remove ADSC growth medium using an aspirator/pipette. Rinse confluent ADSCs 2 times with 2 mL of sterile, room temperature PBS.</li>
		<li>Aspirate PBS and add 2 mL of 0.25 % trypsin-EDTA per 100 mm culture plate. Ensure that the entire surface area is covered with trypsin.</li>
		<li>Incubate plate for ~7 min at 37 &#176;C in 5% CO2 until ADSCs are detached. Remove plate from incubator and mechanically dislodge cells by forcefully pipetting the trypsin solution in the plate.</li>
		<li>Add 2 mL of ADSC growth medium to the dislodged cells and gently pipette to mix. Transfer cells to sterile 50 mL plastic conical tube. To ensure maximal cell recovery, rinse culture dish with another 2 mL of ADSC growth medium, and transfer medium to the conical tube containing detached cells.</li>
		<li>Centrifuge the 50 mL conical tube at 500 x g for 5 min at room temperature to obtain a cell pellet. Carefully decant supernatant without disturbing the cell pellet.</li>
		<li>Resuspend cell pellet in 5&#8211;6 mL of ADSC growth medium per 1 x 106 cells.</li>
	</ol>
	</li>
	<li>Cell count and plating
	<ol>
		<li>In a 1.5 mL microcentrifuge tube, dilute 10 &#956;L of cell solution from above and 10 &#956;L of 0.04% trypan blue solution (0.4% trypan blue diluted 1:10 in PBS) and count cells using a hemocytometer.</li>
		<li>Resuspend the desired number of ADSCs in growth medium. For 100 mm culture dish, plate ~3.0 x 105 cells in 10 mL ADSC growth media to allow for 80% confluence in 72 h or 100% confluency in 96 h.</li>
		<li>View the dish under an inverted microscope at 10x magnification prior to incubation to ensure presence of suspended cells. Maintain the cells at 37 &#176;C at 5% CO2 and 100% relative humidity.</li>
		<li>Every 48 h aspirate the growth medium and replace with warm (37 &#176;C) medium until cells are 80% to 90% confluent as shown in Figure 1B.</li>
		<li>Repeat steps from sections 3.1 and 3.2 for appropriate number of passages to obtain desired cell number. 
		NOTE: After passages 5 to 6, primary cells appear to undergo senescence; therefore, aim to obtain desired cell numbers by passage 4.</li>
	</ol>
	</li>
</ol>
4. ADSC adipogenic differentiation
<ol>
	<li>Aspirate ADSC growth medium from adhered ADSCs that have reached 80% to 90% confluence.</li>
	<li>Quickly rinse ADSC cell layer with sterile, room temperature PBS.</li>
	<li>Add ADSC differentiation medium. For 100 mm culture dish, add 10 mL of ADSC differentiation medium.</li>
	<li>Replace medium every 3 days for approximately 14&#8211;21 days. Mature adipocytes are generally observed after 14 days of differentiation.</li>
	<li>Examine cells under an inverted light microscope at 40x magnification to confirm the presence of lipid droplet as shown in Figure 2A.</li>
</ol>
5. Adipocyte detection
NOTE: This section will describe staining protocols used for lipid droplet detection in differentiated adipocytes, however, immunofluorescent staining for CD105, a mesenchymal stem cell marker, can also be used for ADSC confirmation.
<ol>
	<li>Oil Red O staining
	<ol>
		<li>Prepare 0.5% Oil Red O stock solution in isopropanol. For 20 mL of Oil Red O stock solution, dissolve 100 mg Oil Red O in 20 mL of isopropanol. Filter the solution through a sterile syringe filter with 0.2 &#956;m membrane.</li>
		<li>Carefully aspirate the differentiation medium and wash cells 2 times by adding PBS along the sides of the well to not disturb the cell monolayer.</li>
		<li>Carefully aspirate PBS from cells and add enough neutral buffered formalin (NBF, 10%) to cover cell layer. Incubate at room temperature for 30&#8211;60 min.</li>
		<li>During the formalin fixation step, prepare Oil Red O working solution by diluting 3 parts of Oil Red O stock solution with 2 parts of distilled water. Mix the solution and filter through a sterile syringe filter with 0.2 &#956;m membrane. Working solution is stable for up to 2 h.</li>
		<li>Carefully aspirate NBF and quickly wash (~ 15 s) the cell layer with distilled water. Add enough 60% isopropanol to cover the cell layer after aspirating water and incubate at room temperature for 5 min.</li>
		<li>Carefully aspirate isopropanol after 5 min incubation and add enough Oil Red O working solution to cover the cell layer and incubate at room temperature for 10&#8211;15 min.</li>
		<li>Carefully aspirate off the Oil Red O working solution and wash the cell layer several times with distilled water until the water becomes clear.</li>
		<li>Add PBS to the cell culture dish and examine cells under an inverted microscope for indication of differentiation and presence of lipid droplets (Figure 2B).</li>
	</ol>
	</li>
	<li>BODIPY Staining
	<ol>
		<li>Prepare 5 mM BODIPY stock solution by dissolving 1.3 g of BODIPY in 1 mL of DMSO. Prepare 2 &#956;g/mL BODIPY working solution by diluting the stock solution 1:25,000 times in PBS.</li>
		<li>Carefully aspirate the differentiation medium and wash cells 2 times by adding PBS along the sides of the well to not disturb the cell monolayer.</li>
		<li>Carefully aspirate PBS and add enough BODIPY working solution to cover the cell layer and incubate at 37 &#176;C for 30 min. 
		NOTE: From this point, protect cells from light by covering plate with foil.</li>
		<li>Carefully aspirate the BODIPY working solution and wash the cell layer 3 times with PBS.</li>
		<li>Fix cells by adding 4% paraformaldehyde and incubating at room temperature for 15 min.</li>
		<li>Carefully aspirate the fixation buffer and wash cells 2 times by adding PBS along the sides of the well to not disturb the cell monolayer.</li>
		<li>Add PBS to cell culture dish and examine cells under an inverted fluorescent microscope for evidence of differentiation and presence of lipid droplet (Figure 2C). Image cells using a FITC/EGFP/Bodipy Fl/Fluo3/DiO Chroma filter set. Excitation wavelength is at 480 nm with a band width of 40 nm and emission wavelength is at 535 nm with a bandwidth of 50 nm. A similar or appropriate filter set, and microscope can be used.</li>
	</ol>
	</li>
</ol>

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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-&amp;micro;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&#039; 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 &amp;deg;C, 5 % CO&lt;sub&gt;2&lt;/sub&gt;</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...

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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) ...

isolation-proliferation-differentiation-rhesus-macaque-adipose

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

2.0K Views.  Louisiana State University Health Sciences Center - New Orleans. 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.

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

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 ...

Developmental Biology

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. ...

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.

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.

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

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Medicine

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

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 ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

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 ...

Primary Culture of Adipose Tissue Derived Stem Cells from Mouse Epididymis

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

Author Spotlight: Advancing Treatment Approaches with Adipose-Derived Stem Cells

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...