The authors wish to acknowledge Becky Marquez, the clinical coordinator at the Sentara Bariatric Center, for her assistance with the process of patient screening and consenting. This research was supported by R15HL114062 to Anca D. Dobrian.

The Institutional Review Board Committee at Eastern Virginia Medical School approved the research and collection of human adipose tissue samples used in the study. Informed written consent was collected from the patients.
1. Preparation of Buffers, Media, and Instruments
<ol>
	<li>Prepare a Krebs Ringer Bicarbonate-Buffered Solution (KRBBS): 135 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium sulfate, 0.4 mM potassium phosphate dibasic, 5.5 mM glucose, 1 mM adenosine, 0.01% antibiotic/antimycotic mix (50 &#181;g/mL of penicillin, 50 &#181;g/mL of streptomycin, 30 &#181;g/mL of gentamicin, 15 ng/mL of amphotericin) and 10 mM HEPES (pH = 7.4). This solution is prepared fresh each time for the tissue collection and digestion.</li>
	<li>Prepare a fresh collagenase solution: 1 mg/mL of collagenase, type 1, in KRBSS; make 3 mL/g of fat. Pre-warm the collagenase solution at 37 &#176;C in a water bath prior to the tissue digestion.</li>
	<li>Prepare a CD34 Cell Isolation Buffer: 2% fetal bovine serum (FBS) and 1 mM EDTA in sterile phosphate-buffered saline (PBS).</li>
	<li>Prepare Adipogenesis Media: DMEM/F12, 5% FBS, 50 &#181;g/mL of penicillin/streptomycin, 1.25 &#181;L/mL of human insulin (5 &#181;g/mL or 144 mU/mL), and 0.36 &#181;L/mL of 2.8 mM rosiglitazone (1 &#181;M).</li>
	<li>Prepare a 0.3% Oil Red O (ORO) stock solution (weight/volume) by dissolving 0.3 g of ORO into 100 mL of isopropanol.</li>
</ol>
2. Adipose Stromal Vascular Fraction Isolation
NOTE: The study included a cross-sectional cohort of morbidly obese type 2 diabetic (T2D) and non-diabetic subjects, aged 18&#8211;65 years, undergoing bariatric surgery at the Sentara Metabolic and Weight Loss Surgery Center (Sentara Medical Group, Norfolk, VA). Exclusion criteria included an autoimmune disease including type 1 diabetes mellitus, conditions requiring chronic immunosuppressive therapy, anti-inflammatory medications, thiazolinendiones, active tobacco use, chronic or acute infections, or a history of malignancy treated within the last 12 months. T2D was defined as a fasting plasma glucose of 126 mg/dL or greater, a glucose of 200 mg/dL or greater after a 2 h glucose tolerance test, or the use of antidiabetic medications.
<ol>
	<li>Collect human omental (OM) and subcutaneous (SC) adipose tissue (AT) from human subjects undergoing bariatric surgery.</li>
	<li>Keep the OM and SC AT in separate vials containing Hank&#8217;s Buffered Salt Solution with 50 &#181;g/mL of penicillin/streptomycin at room temperature immediately after tissue extraction.</li>
	<li>Carefully clean and remove fibrotic and cauterized sections of the tissue by using 70% ethanol swabbed scissors and tweezers when the sample arrives at the lab after the tissue extraction.</li>
	<li>Weigh the adipose tissue and partition it into 5 g (or less) aliquots for the collagenase digestion.</li>
	<li>Finely mince 5 g of the AT in 5 mL of a collagenase solution in a scintillation vial at room temperature, using two pairs of scissors.</li>
	<li>Add an additional 10 mL of collagenase solution to the minced tissue for 15 mL total.</li>
	<li>Digest the samples for 1 h, at 37 &#176;C, in a water bath with continuous shaking.</li>
	<li>Cut the tip off a 20 mL syringe to make a blunt end.</li>
	<li>Cut a 9 cm x 9 cm square from a 250 &#181;m mesh and push it halfway into a 50 mL conical tube using the blunt end syringe.</li>
	<li>Pour the samples into the 20 mL blunt end syringe and filter through the 250 &#181;m nylon mesh into the 50 mL conical tube to separate adipocytes and stromal vascular cells from undigested tissue. 
	NOTE: Do not use more than 5 g of AT per 50 mL conical tube, as the mesh will get clogged due to excess fibrotic undigested material.</li>
	<li>Wash out the scintillation vial with 10 mL of KRBBS and pour it through the filter to collect residual cells.</li>
	<li>Incubate the filtered samples for 5 min at room temperature. 
	Note: This step is important to allow for the adipocytes to float at the top of the tube and some of the stromal vascular cells to settle on the bottom of the tube.</li>
	<li>Utilize a 20 mL syringe and a 20 G x 6 in pipetting needle to remove the stromal vascular fraction layer and transfer it to a clean 50 mL conical tube.</li>
	<li>Add 10 mL of KRBBS and allow the samples to sit on the bench for 5 min, at room temperature.</li>
	<li>Repeat steps 2.12&#8211;2.14 twice. 
	Note: These steps will ensure that there is no contamination of the stromal vascular cells with the floating adipocytes.</li>
	<li>Keep the stromal vascular fractions from both depots on ice for further processing, as described in the steps below. 
	Note: Do not leave the cells on ice for more than 1 h, as this may have an impact on the cell viability. The adipocytes can be flash-frozen in liquid nitrogen for long-term storage or used fresh for experiments.</li>
</ol>
3. Isolation of Adipose Tissue Endothelial Cells
<ol>
	<li>Spin the stromal vascular fractions (SVF) at 500 x g for 5 min, at 4 &#176;C.</li>
	<li>Remove the samples from the centrifuge and carefully pour off the supernatant; keep the pellet containing the SVF cells.</li>
	<li>Gently resuspend the cell pellets in 5 mL of PBS. 
	NOTE: If more than 5 g of an AT depot was processed into multiple aliquots (see step 2.4), pool the cell pellets together.</li>
	<li>Spin the samples at 500 x g for 5 min, at 4 &#176;C.</li>
	<li>Remove the supernatant, resuspend the cells in 1 mL of PBS, and count the cells. 
	NOTE: Here, an automatic cell counter was used for cell counting. The cell viability was obtained by mixing 12 &#181;L of cell suspension with 12 &#160;&#181;L of an acridine orange/propidium iodide (AO/PI) solution (see Table of Materials). The average number of cells per gram of AT for each depot is: (a) OM Depot: 1.69 x 105 &#177; 4.51 x 104; (b) SC Depot: 1.24 x 105 &#177; 6.45 x 104. The viability of the cells from both depots was &#62;90%.</li>
	<li>Pellet the samples at 500 x g for 5 min, at 4 &#176;C. 
	NOTE: A CD34 immunomagnetic selection protocol (see Table of Materials) was adapted for steps 3.7&#8211;3.15.</li>
	<li>Resuspend the pellet in 100 &#181;L of CD34 isolation buffer. 
	NOTE: The total cell counts require the following re-suspension volumes: (a) &#60;2 x 107 cells: resuspend the pellet in 100 &#181;L; (b) 2 x 108&#8211;5 x 108 cells: resuspend the pellet in 1 mL. In steps 3.9&#8211;3.16, the volumes of the reagents used were scaled down for &#60;2 x 107 cells.</li>
	<li>Add 10 &#181;L of CD34 cocktail and incubate the sample for 15 min, at room temperature.</li>
	<li>Add 5 &#181;L of magnetic beads and incubate the sample for 10 min, at room temperature.</li>
	<li>Add 2.5 mL of CD34 isolation buffer to each sample and place the samples into the magnet, for 5 min, at room temperature.</li>
	<li>Invert the magnet for 5 s to pour off the isolation buffer.</li>
	<li>Remove the samples from the magnet and repeat steps 3.10 and 3.11 4x.</li>
	<li>Remove the tube from the magnet and add 2 mL of CD34 isolation buffer.</li>
	<li>Pellet the cells at 500 x g for 5 min, at 4 &#176;C.</li>
	<li>Resuspend the cell pellets in 1 mL of CD34 isolation buffer and count the cells. 
	NOTE: Here, the average number of cells per gram of AT is: (a) OM Depot: 4.75 x 104 &#177; 3.36 x 104; (b) SC Depot: 1.06 x 104 &#177; 9.53 x 103. A CD31 plastic immunobead protocol was adapted for steps 3.16&#8211;3.34 (see Table of Materials).</li>
	<li>Pellet cells at 500 x g for 5 min and resuspend the pellet in 250 &#181;L of Buffer B and 250 &#181;L of wash buffer.</li>
	<li>Thoroughly resuspend the CD31 beads by vortexing the tube.</li>
	<li>Add 20 &#181;L of CD31 beads. 
	NOTE: Due to total cell counts &#62;1 x 106, the volume was scaled down according to the manufacturer&#8217;s input.</li>
	<li>Incubate the sample with rocking for 30 min, at room temperature.</li>
	<li>Attach a strainer to a sterile 50 mL conical tube. 
	NOTE: The larger opening of the strainer must be on top.</li>
	<li>Prior to the cell separation, add 1 mL of wash buffer to equilibrate the strainer. Apply the mixture generated under step 3.16 to the strainer.</li>
	<li>Wash the strainer with 5 mL of wash buffer in a circular motion for a total volume of 20 mL. Attach the connector to a sterile 50 mL conical tube and close the luer-lock.</li>
	<li>Attach the strainer to the connector. Add 1 mL of wash buffer along the wall of the strainer. Add 1 mL of activated buffer D along the wall of the strainer. Swirl the sample gently and incubate it for 10 min, at room temperature.</li>
	<li>Add 1 mL of wash buffer. Separate the cells from the beads by pipetting up and down 10x. 
	NOTE: Avoid generating air bubbles.</li>
	<li>Open the luer-lock and allow the detached cells to flow into the 50 mL conical tube. Wash the strainer 10x with 1 mL of wash buffer each time.</li>
	<li>Discard the connector and strainer and centrifuge the samples at 300 x g for 10 min, at 4 &#176;C. Resuspend the cell pellet in 2 mL of complete EGM-2 Media for culture. 
	Note: The average number of cells per gram of AT at this point is: (a) OM Depot: 5.92 x 103 &#177; 4.27 x 103; (b) SC Depot: 4.12 x 103 &#177; 2.1 x 103. The viability of cells from both depots was over 90%.</li>
</ol>
4. Induction of Adipogenesis in Isolated Endothelial Cells
<ol>
	<li>Grow the cells in 6-well tissue culture-treated plates with complete EGM-2 media in a 37 &#176;C, 5% CO2 incubator. Replace the media every 3&#8211;4 days until the cells reach 80&#8211;90% confluence. 
	NOTE: The population doubling is between 2&#8211;3 days.</li>
	<li>Expand the cells by plating them on 100 mm Petri dishes and split the cells 1:3 once. At this stage, the cells are on passage 2.</li>
	<li>Count the cells using an automatic cell counter (see Table of Materials).</li>
	<li>Seed approximately 100&#8211;200,000 cells into 6-well plates ensuring duplicate wells for each depot and culture them in complete EGM-2 media for 2 days.</li>
	<li>Aspirate off any growth media and replace it with 2 mL of DMEM/F12 with 5% FBS and 50 &#181;g/mL of penicillin/streptomycin for the control cells and replace it with 2 mL of Adipogenesis media (see step 1.4) for the treatment cells.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified 5% CO2 incubator for 13 days, replacing the respective media every 3 to 4 days. 
	NOTE: Cells will begin to accumulate lipids after 4 days of treatment.</li>
	<li>Conduct ORO and Nile red staining according to published methods15-17.</li>
	<li>To isolate cells for an RNA extraction, remove the media from the 6-well induction plates and wash it with 1 mL of sterile PBS.</li>
	<li>Add 350 &#181;L of a guanidium thiocyanate-phenol-chloroform solution (see Table of Materials) to each well and incubate it at room temperature for 5&#8211;10 min.</li>
	<li>Scrape the cells off the plate using a cell scraper and transfer the cells to a 1.5 mL microcentrifuge tube. 
	Note: The samples can be stored in the -80 &#176;C freezer for RNA extraction and further processing at a later date.</li>
	<li>Extract mRNA from the samples using an adapted RNA extraction protocol (see Table of Materials).</li>
	<li>Perform a real-time polymerase chain reaction (RT-PCR) to assess the gene expression using the following probes: adiponectin, UCP-1, and CIDEA (see Table of Materials). 
	NOTE: Here, RT-PCR procedures were carried out using the following standard protocol: denaturation (95 &#176;C; 15 s), annealing, and extension (60 &#176;C; 1 min) for 40 cycles. The samples that expressed genes with CT values &#62;35 were considered undetectable.</li>
</ol>

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<li>Ong, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+specific+cell-surface+markers+of+adipose-derived+stem+cells+from+subcutaneous+and+visceral+fat+depots%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">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>
<li>Ong, W. K., Sugii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose-derived+stem+cells%3A+fatty+potentials+for+therapy%5BTitle%5D)+AND+%22The+International+Journal+of+Biochemistry+%26+Cell+Biology%22%5BJournal%5D)">Adipose-derived stem cells: fatty potentials for therapy.</a> The International Journal of Biochemistry & Cell Biology. 45 (6), 1083-1086 (2013).</li>
<li>Tchkonia, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abundance+of+two+human+preadipocyte+subtypes+with+distinct+capacities+for+replication%2C+adipogenesis%2C+and+apoptosis+varies+among+fat+depots%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Endocrinoloy+and+Metabolism%22%5BJournal%5D)">Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots.</a> American Journal of Physiology-Endocrinoloy and Metabolism. 288 (1), E267-E277 (2005).</li>
<li>van de Vyver, M., Andrag, E., Cockburn, I. L., Ferris, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thiazolidinedione-induced+lipid+droplet+formation+during+osteogenic+differentiation%5BTitle%5D)+AND+%22Journal+of+Endocrinology%22%5BJournal%5D)">Thiazolidinedione-induced lipid droplet formation during osteogenic differentiation.</a> Journal of Endocrinology. 223 (2), 119-132 (2014).</li>
</ol>

The authors have nothing to disclose.

The focus of this paper is to provide a methodology for the isolation, expansion and adipogenic induction of CD34+CD31+ endothelial cells from visceral and subcutaneous depots of human adipose tissue.
Methodologies have been reported for the isolation of endothelial cells from various vascular beds of rodents or humans that involve primarily techniques using CD31 antibodies either fluorescently labeled or coupled to magnetic beads18,19...

Recent evidence shows that both in mice and in humans, a subset of cells residing in the adipose tissue vasculature can be differentiated into either white or beige/brown adipocytes1,2,3. The phenotype of such cells is a subject of controversy, with evidence supporting endothelial cells, smooth muscle/pericyte, or a spectrum of intermediate phenotypes4,5,6,7. The scope of developing this methodology was to test the adipogenic potential of CD34+CD31+ endothelial cells isolated from different fat depots from obese humans. Other studies in the literature are focusing on the adipogenic potential of the total stromal vascular fraction or of the known adipocyte progenitors2,8,9. Since currently existing technologies can target specifically adipose tissue endothelial cells for drug delivery10, understanding the potential of such cells to undergo adipogenic induction towards white or beige adipocytes is important for future targeted therapies.
Different groups reported the combination of CD31 and CD34 markers as surrogates to isolate endothelial cells from human adipose tissue11,12,13. Typically, the isolation is performed using two sequential steps and a positive selection using magnetic beads. In this report, immunoseparation using CD34+ magnetic beads combined with CD31 plastic beads was utilized. We found this technique superior to the sequential magnetic immunoseparation with respect to the preservation of typical cobblestone endothelial morphology. Also, we were able to generate enough cells required for the expansion and adipogenic induction starting from as little as 1&#8211;2 g of fat. A small sample biopsy of subcutaneous fat is enough to produce the required quantity of cells for downstream applications. This aspect is potentially important, particularly if this method will be utilized for screening for a responsiveness to adipogenic induction in human subjects.
Unlike other systems reported in the literature, this method utilizes only two ingredients for the adipogenic induction of the CD34+CD31+ cells: a PPAR&#947; agonist&#8212;rosiglitazone&#8212;and human insulin. Importantly, the amount of insulin used falls within the normal/high range of circulating post-absorptive insulin in humans14. The degree of responsiveness to insulin of the cells in vitro, measured by Akt phosphorylation, does not correlate with their ability to respond to the induction cocktail. Interestingly, using this induction cocktail and experimental conditions, a mix of white and beige/brown cells were obtained as determined by the size and numbers of intracellular lipid droplets and the expression of molecular markers. This straightforward and cost-effective induction protocol along with the quantitative evaluation of the phenotype of the responder cells (white vs. beige) allows for a screening of agents that can potentially alter the balance of differentiated beige:white adipocytes.
This method also provides a translational approach for understanding the underlying mechanisms of adipogenesis of vascular endothelial progenitors in human adipose tissue. Using this specific isolation/differentiation technique, investigators can interrogate various pathways responsible for adipogenesis in a subset of vascular endothelial cells from various fat depots in lean and obese humans.

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</td><td> &lt;p&gt;Kit&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;cDNA Synthesis&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1708841&lt;/p&gt; </td><td> &lt;p&gt;Supermix&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;JumpStart PCR Polymerase&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;D9307-250UN&lt;/p&gt; </td><td> &lt;p&gt;Hot start, with PCR Buffer N&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Magnesium Chloride Solution&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;M8787-5ML&lt;/p&gt; </td><td> &lt;p&gt;3 mM final in PCR reaction&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;dNTPs&lt;/p&gt; </td><td> &lt;p&gt;Promega&lt;/p&gt; </td><td> &lt;p&gt;U1515&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TaqMan AdipoQ&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;Hs00605917_m1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TaqMan CIDEA&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;Hs00154455_m1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TaqMan RPL27&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;Hs03044961_g1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TaqMan UCP1&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;Hs00222453_m1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;BCA Assay&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;QPBCA-1KT&lt;/p&gt; </td><td> &lt;p&gt;Kit&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Bis-acrylamide&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1610146&lt;/p&gt; </td><td> &lt;p&gt;40% stock solution&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Ammonium Persulfate&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1610700&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TEMED&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1610800&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Tris&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;T1503&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Glycine&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1610718&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Sodium Dodecal Sulfate&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;L3771&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;EDTA&lt;/p&gt; </td><td> &lt;p&gt;Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;S311-100&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Bromophenol Blue&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;B8026&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Blot Membrane&lt;/p&gt; </td><td> &lt;p&gt;EMD Millipore&lt;/p&gt; </td><td> &lt;p&gt;IPFL00010&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Methanol&lt;/p&gt; </td><td> &lt;p&gt;Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;A452-SK4&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Odyssey Blocking Buffer, Tris&lt;/p&gt; </td><td> &lt;p&gt;LI-COR&lt;/p&gt; </td><td> &lt;p&gt;927-50000&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Anti-AKT antibody&lt;/p&gt; </td><td> &lt;p&gt;Cell Signaling Technology&lt;/p&gt; </td><td> &lt;p&gt;2920S&lt;/p&gt; </td><td> &lt;p&gt;Mouse monoclonal&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-pAKT antibody&lt;/p&gt; </td><td> &lt;p&gt;Cell Signaling Technology&lt;/p&gt; </td><td> &lt;p&gt;9271S&lt;/p&gt; </td><td> &lt;p&gt;Rabbit polyclonal&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-UCP1 antibody&lt;/p&gt; </td><td> &lt;p&gt;Abcam&lt;/p&gt; </td><td> &lt;p&gt;ab10983&lt;/p&gt; </td><td> &lt;p&gt;Rabbit polyclonal&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-Mouse IgG antibody&lt;/p&gt; </td><td> &lt;p&gt;LI-COR&lt;/p&gt; </td><td> &lt;p&gt;926-68070&lt;/p&gt; </td><td> &lt;p&gt;Goat Polyclonal, IRDye 680RD&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-Rabbit IgG antibody&lt;/p&gt; </td><td> &lt;p&gt;LI-COR&lt;/p&gt; </td><td> &lt;p&gt;926-32211&lt;/p&gt; </td><td> &lt;p&gt;Goat Polyclonal, IRDye 800CW&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-Rabbit IgG antibody&lt;/p&gt; </td><td> &lt;p&gt;Jackson ImmunoResearch&lt;/p&gt; </td><td> &lt;p&gt;111-025-003&lt;/p&gt; </td><td> &lt;p&gt;Goat Polyclonal, TRITC&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Phosphate Buffer Saline&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;10010049&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;37% Formaldehyde Solution&lt;/p&gt; </td><td> &lt;p&gt;Electron Microscopy Sciences&lt;/p&gt; </td><td> &lt;p&gt;15686&lt;/p&gt; </td><td> &lt;p&gt;4% solution for cell fixation&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Normal Goat Serum&lt;/p&gt; </td><td> &lt;p&gt;Vector Laboratories&lt;/p&gt; </td><td> &lt;p&gt;S-1000&lt;/p&gt; </td><td> &lt;p&gt;10% blocking solution&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Triton X-100&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;X-100&lt;/p&gt; </td><td> &lt;p&gt;0.1% permeabilization solution&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;DAPI&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;D1306&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Calcein AM&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;65-0853-39&lt;/p&gt; </td><td> &lt;p&gt;Cell fluorescent visualization&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Matrigel Basement Membrane Matrix&lt;/p&gt; </td><td> &lt;p&gt;Corning Life Sciences&lt;/p&gt; </td><td> &lt;p&gt;356231&lt;/p&gt; </td><td> &lt;p&gt;Growth factor reduced&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;DiI labeled Acetylated LDL&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;L3484&lt;/p&gt; </td><td></td></tr></tbody></table>

isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

Our protocol aims to provide an in vitro approach to determine the adipogenic potential of CD34+CD31+ vascular cells from different depots of human adipose tissue. A simplified flowchart diagram is shown in Figure 1A. The first step using a positive selection of CD34 expressing cells results in &#62; 95% CD34+ cells in the population of the freshly isolated cells (Figure 1A). Importantly, this marker is lost after the ce...

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Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

isolation-expansion-adipogenic-induction-cd34-cd31-endothelial-cells

Research

JoVE Journal

Immunology and Infection

11.8K Views.  Eastern Virginia Medical School. The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Video: Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

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

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

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 ...

Biology

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

Medicine

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

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 ...

Culturing Preadipocytes Isolated from Murine Perivascular Adipose Tissue

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Author Spotlight: The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown ...

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 ...

Culturing Murine Adipocytes and Explants for Functional and Imaging Studies

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and mobilization, thermoregulation, and the secretion of endocrine factors. The expansion of white adipose tissue due to caloric imbalance results in both the enlargement of existing adipocytes and the generation of additional adipocytes from adipocyte progenitor cells. Obesity-driven changes to white adipose tissue, including those affecting adipocytes, are associated with numerous comorbidities, such as type 2 diabetes and 13 types of cancer. A significant barrier to studying how adipocytes contribute to disease is the inability to readily isolate and culture mature adipocytes. This article describes a protocol to isolate murine lean and obese adipocytes from the subcutaneous and visceral fat depots of male and female C57BL/6 mice. The protocol details how isolated primary adipocytes can be cultured in a membrane adipocyte aggregate system for up to 2 weeks, facilitating their functional analysis in co-culture experiments, lipolysis assays, or through the collection of conditioned media containing adipocyte-secreted factors. Additionally, the protocol outlines methods for culturing adipose tissue explants in basement membrane matrix domes and imaging primary isolated adipocytes. Importantly, this approach can be integrated with existing protocols for the isolation of adipose tissue-resident adipocyte&#160;progenitor cells using fluorescence-activated cell sorting (FACS). Together, these protocols provide researchers with tools to functionally study adipocytes, adipocyte progenitor cells, and whole adipose tissue from lean and obese, male and female mice.

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and ...