Name: isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue
Uploaded: 2018-07-17T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

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% antib...

<ol>
	<li>Tang, W., 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=White+fat+progenitor+cells+reside+in+the+adipose+vasculature.">White fat progenitor cells reside in the adipose vasculature.</a> Science. 322 (5901), 583-586 (2008).</li><li>Lee, Y. H., Petkova, A. P., Granneman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+adipogenic+niche+for+adipose+tissue+remodeling+and+restoration.">Identification of an adipogenic niche for adipose tissue remodeling and restoration.</a> Cell Metabolism. 18 (3), 355-367 (2013).</li><li>Min, S. Y., 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+'brite/beige'+adipocytes+develop+from+capillary+networks,+and+their+implantation+improves+metabolic+homeostasis+in+mice.">Human 'brite/beige' adipocytes develop from capillary networks, and their implantation improves metabolic homeostasis in mice.</a> Nature Medicine. 22 (3), 312-318 (2016).</li><li>Gupta, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zfp423+expression+identifies+committed+preadipocytes+and+localizes+to+adipose+endothelial+and+perivascular+cells.">Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells.</a> Cell Metabolism. 15 (2), 230-239 (2012).</li><li>Tran, K. V., 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=The+vascular+endothelium+of+the+adipose+tissue+gives+rise+to+both+white+and+brown+fat+cells.">The vascular endothelium of the adipose tissue gives rise to both white and brown fat cells.</a> Cell Metabolism. 15 (2), 222-229 (2012).</li><li>Rodeheffer, M. S., Birsoy, K., Friedman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+white+adipocyte+progenitor+cells+in+vivo.">Identification of white adipocyte progenitor cells in vivo.</a> Cell. 135 (2), 240-249 (2008).</li><li>Scott, M. A., Nguyen, V. T., Levi, B., James, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+methods+of+adipogenic+differentiation+of+mesenchymal+stem+cells.">Current methods of adipogenic differentiation of mesenchymal stem cells.</a> Stem Cells and Development. 20 (10), 1793-1804 (2011).</li><li>Macotela, Y., 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=Intrinsic+differences+in+adipocyte+precursor+cells+from+different+white+fat+depots.">Intrinsic differences in adipocyte precursor cells from different white fat depots.</a> Diabetes. 61 (7), 1691-1699 (2012).</li><li>Lee, Y. H., Petkova, A. P., Mottillo, E. P., Granneman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+of+bipotential+adipocyte+progenitors+recruited+by+beta3-adrenoceptor+activation+and+high-fat+feeding.">In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding.</a> Cell Metabolism. 15 (4), 480-491 (2012).</li><li>Xue, Y., Xu, X., Zhang, X. Q., Farokhzad, O. C., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+diet-induced+obesity+in+mice+by+adipose+tissue+transformation+and+angiogenesis+using+targeted+nanoparticles.">Preventing diet-induced obesity in mice by adipose tissue transformation and angiogenesis using targeted nanoparticles.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (20), 5552-5557 (2016).</li><li>Villaret, 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=Adipose+tissue+endothelial+cells+from+obese+human+subjects:+differences+among+depots+in+angiogenic,+metabolic,+and+inflammatory+gene+expression+and+cellular+senescence.">Adipose tissue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence.</a> Diabetes. 59 (11), 2755-2763 (2010).</li><li>Miranville, 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=Improvement+of+postnatal+neovascularization+by+human+adipose+tissue-derived+stem+cells.">Improvement of postnatal neovascularization by human adipose tissue-derived stem cells.</a> Circulation. 110 (3), 349-355 (2004).</li><li>Sengenes, C., Lolmede, K., Zakaroff-Girard, A., Busse, R., Bouloumie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preadipocytes+in+the+human+subcutaneous+adipose+tissue+display+distinct+features+from+the+adult+mesenchymal+and+hematopoietic+stem+cells.">Preadipocytes in the human subcutaneous adipose tissue display distinct features from the adult mesenchymal and hematopoietic stem cells.</a> Journal of Cellular Physiology. 205 (1), 114-122 (2005).</li><li>Moller, D. E., Flier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance--mechanisms,+syndromes,+and+implications.">Insulin resistance--mechanisms, syndromes, and implications.</a> The New England Journal of Medicine. 325 (13), 938-948 (1991).</li><li>Pittenger, M. F., 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=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284 (5411), 143-147 (1999).</li><li>Novikoff, A. B., Novikoff, P. M., Rosen, O. M., Rubin, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+relationships+in+cultured+3T3-L1+preadipocytes.">Organelle relationships in cultured 3T3-L1 preadipocytes.</a> The Journal of Cell Biology. 87 (1), 180-196 (1980).</li><li>Satish, L., 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=Expression+analysis+of+human+adipose-derived+stem+cells+during+in+vitro+differentiation+to+an+adipocyte+lineage.">Expression analysis of human adipose-derived stem cells during in vitro differentiation to an adipocyte lineage.</a> BMC Medical Genomics. 8 (41), (2015).</li><li>Gimbrone, M. A., Cotran, R. S., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+vascular+endothelial+cells+in+culture.+Growth+and+DNA+synthesis.">Human vascular endothelial cells in culture. Growth and DNA synthesis.</a> The Journal of Cell Biology. 60 (3), 673-684 (1974).</li><li>Burridge, K. A., Friedman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment+and+vascular+bed+origin+influence+differences+in+endothelial+transcriptional+profiles+of+coronary+and+iliac+arteries.">Environment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries.</a> American Journal of Physiology-Heart and Circulatory Physiology. 299 (3), H837-H846 (2010).</li><li>Paruchuri, S., 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+pulmonary+valve+progenitor+cells+exhibit+endothelial/mesenchymal+plasticity+in+response+to+vascular+endothelial+growth+factor-A+and+transforming+growth+factor-beta2.">Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2.</a> Circulation Research. 99 (8), 861-869 (2006).</li><li>Hewett, P. W., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microvessel+endothelial+cells:+isolation,+culture+and+characterization.">Human microvessel endothelial cells: isolation, culture and characterization.</a> In Vitro Cellular &amp; Development Biology. 29 (11), 823-830 (1993).</li><li>Hewett, P. W., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lung+microvessel+endothelial+cells:+isolation,+culture,+and+characterization.">Human lung microvessel endothelial cells: isolation, culture, and characterization.</a> Microvascular Research. 46 (1), 89-102 (1993).</li><li>Hewett, P. W., Murray, J. C., Price, E. A., Watts, M. E., Woodcock, M. <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+characterization+of+microvessel+endothelial+cells+from+human+mammary+adipose+tissue.">Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue.</a> In Vitro Cellular &amp; Development Biology. 29 (4), 325-331 (1993).</li><li>Xiao, L., McCann, J. V., Dudley, A. C. <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+expansion+of+tumor-specific+endothelial+cells.">Isolation and culture expansion of tumor-specific endothelial cells.</a> Journal of Visualized Experiments. (105), e53072 (2015).</li><li>Berry, R., Rodeheffer, M. S., Rosen, C. J., Horowitz, M. 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+tissue+residing+progenitors+(adipocyte+lineage+progenitors+and+adipose+derived+stem+cells+(ADSC).">Adipose tissue residing progenitors (adipocyte lineage progenitors and adipose derived stem cells (ADSC).</a> Current Molecular Biology Reports. 1 (3), 101-109 (2015).</li><li>Zhou, L., 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=In+vitro+evaluation+of+endothelial+progenitor+cells+from+adipose+tissue+as+potential+angiogenic+cell+sources+for+bladder+angiogenesis.">In vitro evaluation of endothelial progenitor cells from adipose tissue as potential angiogenic cell sources for bladder angiogenesis.</a> PLoS One. 10 (2), e0117644 (2015).</li><li>Curat, C. 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=From+blood+monocytes+to+adipose+tissue-resident+macrophages:+induction+of+diapedesis+by+human+mature+adipocytes.">From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes.</a> Diabetes. 53 (5), 1285-1292 (2004).</li><li>Sidney, L. E., Branch, M. J., Dunphy, S. E., Dua, H. S., Hopkinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+evidence+for+CD34+as+a+common+marker+for+diverse+progenitors.">Concise review: evidence for CD34 as a common marker for diverse progenitors.</a> Stem Cells. 32 (6), 1380-1389 (2014).</li><li>Traktuev, D. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+of+multipotent+CD34-positive+adipose+stromal+cells+share+pericyte+and+mesenchymal+surface+markers,+reside+in+a+periendothelial+location,+and+stabilize+endothelial+networks.">A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks.</a> Circulation Research. 102 (1), 77-85 (2008).</li><li>Frontini, A., Giordano, A., Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+of+adipose+tissues:+a+niche+of+adipogenesis.">Endothelial cells of adipose tissues: a niche of adipogenesis.</a> Cell Cycle. 11 (15), 2765-2766 (2012).</li><li>Sengenes, C., Miranville, A., Lolmede, K., Curat, C. A., Bouloumie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endothelial+cells+in+inflamed+adipose+tissue.">The role of endothelial cells in inflamed adipose tissue.</a> Journal of Internal Medicine. 262 (4), 415-421 (2007).</li><li>Ong, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+specific+cell-surface+markers+of+adipose-derived+stem+cells+from+subcutaneous+and+visceral+fat+depots.">Identification of specific cell-surface markers of adipose-derived stem cells from subcutaneous and visceral fat depots.</a> Stem Cell Reports. 2 (2), 171-179 (2014).</li><li>Ong, W. K., Sugii, S. <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:+fatty+potentials+for+therapy.">Adipose-derived stem cells: fatty potentials for therapy.</a> The International Journal of Biochemistry &amp; Cell Biology. 45 (6), 1083-1086 (2013).</li><li>Tchkonia, T., 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=Abundance+of+two+human+preadipocyte+subtypes+with+distinct+capacities+for+replication,+adipogenesis,+and+apoptosis+varies+among+fat+depots.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazolidinedione-induced+lipid+droplet+formation+during+osteogenic+differentiation.">Thiazolidinedione-induced lipid droplet formation during osteogenic differentiation.</a> Journal of Endocrinology. 223 (2), 119-132 (2014).</li></ol>

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.

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...

Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

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 ...

This method can help answer key questions in the obesity field, such as identifying the key factors and mechanisms that prevent a robust adipogenic response during adipose tissue remodeling. The main advantage of this technique is that it allows isolation of CD34+CD31+endothelial cells from human adipose tissue starting with small amounts of tissue. We first had the idea for this method when we wanted to identify cells in human adipose vasculature that respond to adipogenic stimulation.Visual demonstration of this method is critical, as the cell isolation steps are difficult to practice due to the oftentimes limited access to fresh human adipose tissue. Collect human omental and subcutaneous adipose tissue from human subjects undergoing bariatric surgery. Immediately after tissue extraction, keep the human omental and subcutaneous adipose tissue in separate vials containing Hank's buffered salt solution with 50 micrograms per milliliter of penicillin streptomycin at room temperature.Once the sample arrives at the lab after the tissue extraction, carefully clean and remove fibrotic and cauterized sections of the tissue by using 70%ethanol-swabbed scissors and tweezers. Weigh the adipose tissue and partition it into five gram aliquots for the collagenase digestion. Use two pairs of scissors to finely mince five grams of the adipose tissue in five milliliters of a collagenase solution in a scintillation vial at room temperature.Add an additional 10 milliliters of collagenase solution to the minced tissue for 15 milliliters total. Digest the samples for one hour at 37 degrees Celsius in a water bath with continuous shaking. Following digestion, pour the samples into the 20 milliliter blunt end syringe.Filter the sample through 250 micron nylon mesh into the 50 milliliter conical tube to separate adipocytes and stromal vascular cells from undigested tissue. Wash out the scintillation vial with 10 milliliters of KRBBS, and pour the wash through the filter to collect residual cells before incubating the filtered samples for five minutes at room temperature. Then, utilize a 20 milliliter syringe and a 20 gauge by six inch pipetting needle to remove stromal vascular fraction layer and transfer it to a clean 50 milliliter conical tube.Add 10 milliliters of KRBBS and allow the samples to sit on the bench for five minutes at room temperature. Keep the stromal vascular fractions from both depots on ice for further processing. Spin the stromal vascular fractions at 500 times G for five minutes at 4 degrees Celsius.Remove the samples from the centrifuge and carefully pour off the supernatant, keeping the pellet containing the stromal vascular fraction cells. Gently re-suspend the cell pellets in five milliliters of PBS. After spinning the samples again, remove the supernatant, re-suspend the cells in one milliliter of PBS, and count the cells.Pellet the samples for a third time, and re-suspend the pellet in 100 microliters of CD34 isolation buffer. Add 10 microliters of CD34 cocktail and incubate the sample for 15 minutes at room temperature. Then add five microliters of magnetic beads and incubate the sample for 10 minutes at room temperature.Next, add 2.5 milliliters of CD34 isolation buffer to each sample, and place the samples into the magnet for five minutes at room temperature. Invert the magnet for five seconds to pour off the isolation buffer. After repeating this process four times, remove the tube from the magnet and add two milliliters of CD34 isolation buffer.Following another centrifugation, re-suspend the CD34+cell pellets in one milliliter of CD34 isolation buffer and count the cells. Pellet the cells again, and re-suspend the pellet in 250 microliters of Buffer B and 250 microliters of wash buffer. Next, thoroughly re-suspend the CD31 beads by vortexing the tube, then add 20 microliters of CD31 beads to the re-suspended cell pellet and incubate the sample with rocking for 30 minutes at room temperature.Prior to the cell separation, add one milliliter of wash buffer to equilabrate the strainer. Then, apply the prepared mixture of re-suspended cells in CD31 beads to the strainer. Wash the strainer with five milliliters of wash buffer in a circular motion for a total volume of 20 milliliters.Attach the connector to a sterile 50 milliliter conical tube and close the leur lock. After attaching the strainer to the connector, add one milliliter of wash buffer along the wall of the strainer, then add one milliliter of activated Buffer D along the wall of the strainer. Swirl the sample gently, and incubate it for 10 minutes at room temperature.Next, add one milliliter of wash buffer and separate the cells from the beads by pipetting up and down 10 times. Open the leur lock and allow the detached cells to flow into the 50 milliliter conical tube. Wash the strainer 10 times with one milliliter of wash buffer each time.Discard the connector and strainer, and centrifuge the samples at 300 times G for 10 minutes at 4 degrees Celsius. Finally, re-suspend the cell pellet in two milliliters of complete EGM2 media for culture. Grow the cells in six-well tissue culture treated plates with complete EGM2 media in a 37 degrees Celsius, 5%carbon dioxide incubator.Replace the media every three to four days until the cells reach 80 to 90%confluence. Then, seed approximately 100 to 200 thousand cells into six-well plates, ensuring duplicate wells for each depot, and culture them in complete EGM2 media for two days. After two days, aspirate off any growth media.Replace the media with two milliliters of DMEM/F-12 media with 5%FBS and 50 micrograms per milliliter of penicillin streptomycin for the control cells. For the treatment cells, replace the media with two milliliters of adipogenesis media. Incubate the cells at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for 13 days replacing the respective media every three to four days.Representative results showing the typical endothelial cobblestone-like morphology of the CD34+CD31+cells after two passages in culture is shown. Also demonstrated is the ability of cells to form tubules when cultured in Matrigel as expected. Uniform uptake of fluorescently labeled acetylated LDL shows the presence of a homogenous cell population with endothelial identity.Shown here is Oil Red O staining of neutral lipid droplets in CD34+CD31+cells following 13 days of adipogenic induction. Representative responses of cells from omental and subcutaneous depots of three different human subjects are shown to illustrate the large variability in response to adipogenic induction between fat depots of the same donor and between donors. This image displays fluorescent intracellular lipid staining in CD34+C31+cells after adipogenic induction for 13 days.The image shows cells with unilocular lipids, multilocular lipids, and no lipid accumulation. Real time PCR shows the expression of the pan adipocyte marker adiponectin and the brown beige markers CIDEA and UCP-1. These results confirm that cells following adipogenic induction express molecular markers consistent with a mixed white beige adipocyte phenotype.Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it is important to remember to use fresh human adipose tissue that was collected and not stored longer than two hours. Following this procedure, other methods like flow cytometry, immunohistochemistry, and RT-PCR can be performed to identify specific subsets of responding cells, and expression of mature adipocyte markers that can be utilized to identify a beige brown adipocyte phenotype.After it's development, this technique paved the way for researchers in the filed of obesity to explore roles of adipose tissue, vascular cells, and adipogenesis in humans. After watching this video, you should have a good understanding of how to isolate, culture, and expand CD34+31+cells. Don't forget that working with human tissues can be extremely hazardous, and precautions, such as bio-safety regulations, should always be taken while performing this procedure.The implications of this technique extend toward therapy of obesity associated comorbidities, such as insulin resistance, because it identifies adipogenic response of cells to a potential therapeutic intervention.

Research

JoVE Journal

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...