This research was supported in part by Program for Creating Start-ups from Advanced Research and Technology (START Program) from the Japan Society for the Promotion of Science (ST261006IP, TM) and by Program for the Strategic Research Foundation at Private Universities (2014-2019) (S1411018, TM) from the Ministry of Education, Sports, Science and Technology.

Samples of human subcutaneous fat were obtained from patients undergoing surgery in the Departments of Plastic Surgery, Urology, Pediatric Surgery and Orthopedic Surgery of Nihon University Itabashi Hospital (Tokyo, Japan). The patients gave written informed consent, and the Ethics Committee of Nihon University School of Medicine approved the study.
1. Tissue Preparation
<ol>
	<li>Bring the tissue sample from the operating room to the laboratory.</li>
	<li>Wash 1-2 g of adipose tissue with 5 ml of PBS(-) at room temperature (RT) once, in a 50 ml tube and then place the sample in a 10 cm plastic dish.</li>
</ol>
2. Collagenase Digestion
<ol>
	<li>Remove the sample from the PBS(-), then add 10 ml of sterile 0.1% (w/v) collagenase solution (Collagenase type II) to the dish containing the tissue sample.</li>
	<li>Mince the tissue with surgical scissors for approximately 15 min, until it reaches a pureed consistency.</li>
	<li>Subsequently, digest the minced tissue in the 0.1% (w/v) collagenase solution at 37 &#176;C for 30 min (60 rpm) in a 50 ml tube with gentle agitation on a shaker.</li>
	<li>Filter the digested sample through a 100 &#181;m filter into a 50 ml tube. Next, pass 10 ml of DCM (see Table of Materials) supplemented with 2% FBS through the filter.</li>
</ol>
3. Cell Isolation
<ol>
	<li>Centrifuge the filtered cells at 100 x g for 1 min at RT.</li>
	<li>At this point, prepare 5 ml of DCM supplemented with 2% FBS in a 50 ml tube.</li>
	<li>Next, draw up the floating fat cells using a 1,000 &#181;l pipette and add the cells to a 50 ml tube containing medium.</li>
	<li>Subsequently, shake the 50 ml tube gently to mix the DCM medium with the cells.</li>
	<li>Then, centrifuge the tube at 100 x g for 1 min at RT.</li>
	<li>Discard the medium at the bottom of the tube using an 18 G needle and a 20 ml syringe.</li>
	<li>Next, add 10 ml of DCM supplemented with 2% FBS to the collected cells.</li>
	<li>Mix the DCM with the cells by shaking the 50 ml tube gently.</li>
	<li>Centrifuge the tube at 100 x g for 1 min at RT.</li>
</ol>
4. Plating Cells
<ol>
	<li>Add 41 ml of DCM or DMEM supplemented with 20% FBS to a 12.5 cm flask.</li>
	<li>Next, using a 200 &#181;l pipette add 40 &#181;l of fat cells to the flask.</li>
	<li>Then, fill up the flask with DCM or DMEM supplemented with 20% FBS. CRITICAL STEP: At this point, ensure that the cells are spread throughout the medium. Visually check the detached cells and afterwards keep the flask on the lid of a round petri dish to keep it flat.</li>
	<li>Finally, place the flask inside the incubator and leave it undisturbed in a 5% CO2 incubator at 37 &#176;C for 7 days.</li>
	<li>Following a week of incubation, invert the flask and check for DFAT cells. Next, assess DFAT cell generation proficiency as described10 since at this point fibroblast-like cells (DFAT cells) are located in regions distinct from fat cells.</li>
	<li>Add 5 ml of DCM or DMEM supplemented with 20% FBS to the flask following removal of the previous cell culture medium. Allow the DFAT cells to grow for another week after changing the medium.</li>
	<li>Using a cell counter, count the number of DFAT cells generated under various cell culture conditions (DCM vs. DMEM).</li>
</ol>

<ol><li>Lanzoni, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concise+review%3A+clinical+programs+of+stem+cell+therapies+for+liver+and+pancreas%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Concise review: clinical programs of stem cell therapies for liver and pancreas.</a> Stem Cells. 31 (10), 2047-2060 (2013).</li>
<li>de Girolamo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem%2Fstromal+cells%3A+a+new+%22cells+as+drugs%22+paradigm.+Efficacy+and+critical+aspects+in+cell+therapy%5BTitle%5D)+AND+%22Curr.+Pharm.+Des%22%5BJournal%5D)">Mesenchymal stem/stromal cells: a new "cells as drugs" paradigm. Efficacy and critical aspects in cell therapy.</a> Curr. Pharm. Des. 19 (13), 2459-2473 (2013).</li>
<li>Lindroos, B., Suuronen, R., Miettinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+potential+of+adipose+stem+cells+in+regenerative+medicine%5BTitle%5D)+AND+%22Stem+Cell.+Rev%22%5BJournal%5D)">The potential of adipose stem cells in regenerative medicine.</a> Stem Cell. Rev. 7 (2), 269-291 (2011).</li>
<li>Yan, J., Tie, G., Xu, T. Y., Cecchini, K., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+as+a+treatment+for+peripheral+arterial+disease%3A+current+status+and+potential+impact+of+type+II+diabetes+on+their+therapeutic+efficacy%5BTitle%5D)+AND+%22Stem+Cell.+Rev%22%5BJournal%5D)">Mesenchymal stem cells as a treatment for peripheral arterial disease: current status and potential impact of type II diabetes on their therapeutic efficacy.</a> Stem Cell. Rev. 9 (3), 360-372 (2013).</li>
<li>Ringden, O., Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stromal+cells+as+treatment+for+chronic+GVHD%5BTitle%5D)+AND+%22Bone+Marrow+Transplant%22%5BJournal%5D)">Mesenchymal stromal cells as treatment for chronic GVHD.</a> Bone Marrow Transplant. 46 (2), 163-164 (2011).</li>
<li>Matsumoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mature+adipocyte-derived+dedifferentiated+fat+cells+exhibit+multilineage+potential%5BTitle%5D)+AND+%22J.+Cell.+Physiol%22%5BJournal%5D)">Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.</a> J. Cell. Physiol. 215 (1), 210-222 (2008).</li>
<li>Lessard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+human+adipose+stem+cells+through+dedifferentiation+of+mature+adipocytes+in+ceiling+cultures%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.</a> J. Vis. Exp. (97), (2015).</li>
<li>Lessard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+dedifferentiating+human+mature+adipocytes+from+the+visceral+and+subcutaneous+fat+compartments%3A+fibroblast-activation+protein+alpha+and+dipeptidyl+peptidase+4+as+major+components+of+matrix+remodeling%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.</a> PLoS One. 10 (3), 0122065(2015).</li>
<li>Peng, X., et al. Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPeng%2C+X+%22Biomed.+Res.+Int%22">Biomed. Res. Int</a>. 2015, 673651(2015).</li>
<li>Kono, S., Kazama, T., Kano, K., Harada, K., Uechi, M., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenotypic+and+functional+properties+of+feline+dedifferentiated+fat+cells+and+adipose-derived+stem+cells%5BTitle%5D)+AND+%22Vet.+J%22%5BJournal%5D)">Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells.</a> Vet. J. 199 (1), 88-96 (2014).</li>
<li>Bellin, M., Marchetto, M. C., Gage, F. H., Mummery, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cells%3A+the+new+patient%5BTitle%5D)+AND+%22Nat.+Rev.+Mol.+Cell+Biol%22%5BJournal%5D)">Induced pluripotent stem cells: the new patient.</a> Nat. Rev. Mol. Cell Biol. 13 (11), 713-726 (2012).</li>
</ol>

The authors have nothing to disclose.

Mature adipocytes that undergo in vitro dedifferentiation, a process known as ceiling culture, may revert to a more primitive phenotype and gain proliferative abilities. These cells are referred to as dedifferentiated fat (DFAT) cells. The multilineage differentiation potential of DFAT cells was evaluated. Flow cytometry analysis and gene expression analysis revealed that DFAT cells were highly homogeneous in comparison to ASCs6. In fact, the cell-surface antigen profile of DFAT cells is very similar ...

Cell therapy and tissue engineering are hot topics in the field of regenerative medicine1-5. While these therapeutic modalities hold great promise, several technical issues currently hamper their clinical use. In this regard, as in the generation of iPS cells, all tissue engineering therapies must produce cells free of external gene transductions in order to maintain patient safety. Accordingly, we were the first group to successfully produce human DFAT cells6. Several other research groups have since adopted our method to generate DFAT cells of mammalian origin7-9, further highlighting the usefulness of our model.
Over the course of several studies, we have found that the quality of the cell culture environment may be modified by adjusting the content of the cell medium. This finding has led to an increase in the success rate of DFAT cell production and improved cell quality; both critical factors in efficiently generating cells for future clinical trials. In this regard, an improved DFAT culture medium (DCM, a medium similar to mesenchymal stem cell medium, which contains recombinant human insulin, serum albumin, L-glutamic acid, several fatty acids, and cholesterol) and a method for DFAT cell generation and proliferation was developed (more information about the contents of DCM is available upon request). Using this method high quality DFAT cells were generated with the ability to differentiate into several cell types including adipogenic, osteogenic, and chondrogenic cells. Altogether, this validated cell culture protocol enhances the quality of DFAT cells and may be quite useful for enhancing clinical applications of cell therapy and tissue engineering.

<table><tbody><tr><td>CSTI303-MSC medium&amp;nbsp;</td><td>CSTI</td><td>87-671</td><td>This medium is defined as DCM in the text</td></tr><tr><td>PBS(-)</td><td>Wako</td><td>166-23555</td><td>It does not contain Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>DMEM medium</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>172012</td><td></td></tr><tr><td>Collagenase type II</td><td>Sigma</td><td>C-6885</td><td></td></tr><tr><td>Scissors</td><td>Takasago Medical Industry Co., Ltd</td><td>TKZ-F2194-1</td><td></td></tr><tr><td>Shaker</td><td>TAITEC</td><td>Bioshaker V.BR-36</td><td></td></tr><tr><td>Falcon Cell Strainer 100 &amp;mu;m Yellow</td><td>CORNING LIFE SCIENCES&amp;nbsp;</td><td>DL 352360</td><td></td></tr><tr><td>Falcon 12.5 cm&amp;sup2; Rectangular Canted Neck Cell Culture Flask with Blue Vented Screw Cap</td><td>CORNING LIFE SCIENCES&amp;nbsp;</td><td>353107</td><td></td></tr><tr><td>18 G needle</td><td>NIPRO</td><td>02-002</td><td></td></tr><tr><td>20 ml Syringe&amp;nbsp;</td><td>NIPRO</td><td>08-753</td><td></td></tr><tr><td>Z Series Coulter Counter</td><td>BECKMAN COULTER</td><td>383550</td><td></td></tr></tbody></table>

an efficient method to obtain dedifferentiated fat cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

In this study, the method and toolkit for DFAT cell generation was improved (Figure 1). Our method allows us to generate DFAT cells using both DCM and DMEM medium containing 20% FBS (Figure 2A). As such, we compared the efficiency of DCM and DMEM in generating DFAT cells. In this regard, DCM enhanced DFAT cell proliferation by three times when compared to DMEM, regardless of the number of adipocytes (Figure 2A and B). Usi...

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An Efficient Method to Obtain Dedifferentiated Fat Cells

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

an-efficient-method-to-obtain-dedifferentiated-fat-cells

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Developmental Biology

7.9K Views.  Nihon University. We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Video: An Efficient Method to Obtain Dedifferentiated Fat Cells

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

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 ...

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

Behavior

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 ...

Mouse Heart and Skeletal Muscle Digestion for Fibro-Adipogenic Progenitor Culture

Fibro-Adipogenic Progenitor Isolation, Expansion, and Differentiation from the Spiny Mouse Model

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are tissue-resident cells that are able to differentiate into adipocytes, fibroblasts, and chondrocytes. Fibro-adipogenic progenitors are fundamental for orchestrating tissue regeneration as they are responsible for extracellular matrix remodeling after injury. This study focuses on investigating the specific role of fibro-adipogenic progenitors in spiny mouse cardiac repair and skeletal muscle regeneration. To this end, a protocol has been optimized for the purification of spiny mouse fibro-adipogenic progenitors by flow cytometry from enzymatically dissociated skeletal and cardiac muscle. The population obtained from this protocol is capable of expanding in vitro, and can be differentiated to myofibroblasts and adipocytes. This protocol offers a valuable tool for researchers to examine the distinctive properties of spiny mouse, and to compare them to the Mus musculus. This will provide insights that could advance the understanding of regenerative mechanisms in this intriguing model.

Author Spotlight: Streamlining Cell Analysis with an Innovative Protocol for Studying Spiny Mouse Models

This protocol outlines the isolation of skeletal and cardiac muscle fibro-adipogenic progenitors (FAPs) from spiny mouse (Acomys) via enzymatic dissociation and fluorescence-activated cell sorting. The FAPs obtained from this protocol can be effectively expanded and differentiated to myofibroblasts and adipocytes.

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are ...

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.

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 ...

Immunology and Infection

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Bioengineering

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

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

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