We acknowledge Dr. Rawda Samir Mohamed, MSc, Veterinarian Specialist, Faculty of Pharmacy, The British University of Egypt (BUE) for helping with the dissection of the rats.
We also would like to acknowledge and appreciate the efforts of the Faculty of Mass Communication, The British University in Egypt (BUE) for the production and editing of the video of this manuscript.
We would like to thank Miss Fatma Masoud, MSc, Assistant Lecturer of English, The British University in Egypt (BUE) for the revision and English language proofreading of the manuscript.
This work was partially funded by the Center for Drug Research and Development (CDRD), Faculty of Pharmacy, The British University in Egypt (BUE), Cairo, Egypt.

<ol>
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All the co-authors declare no conflict of interests associated with this work.

In this protocol, we managed to present a detailed protocol for the isolation of Ad-MSCs from rat epididymal fat and the differentiation of these Ad-MSCs into IPCs. In fact, rat epidydimal fat is an easily attainable source of adipose tissue for obtaining Ad-MSCs and does not require any special equipment, neither for collection nor for processing15,26,27. The isolated Ad-MSCs showed excellent culture expansion and exhibited all...

Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are among the most widely used cell types for regenerative medicine1,2. They are classified as adult stem cells and characterized by multilineage differentiation potential and self-renewal capacity3. MSCs can be isolated and obtained from various sources, including adipose tissue, bone marrow, peripheral blood, umbilical cord tissue and blood, hair follicles, and teeth4,5.
The isolation of stem cells from adipose tissue is seen as both appealing and promising due to their easy access, rapid expansion in vitro, and high yield6. Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be isolated from different species such as humans, bovines, mice, rats, and, more recently, goats7. It has been proven that Ad-MSCs are now potential candidates for tissue engineering and gene/cell therapy that can even be used to develop an autologous alternative for the long-term repair of soft tissue injury or defects7,8.
The International Society for Cell and Gene Therapy (ISCT) has defined three minimum criteria that must be exhibited by MSCs for full characterization9. First, they must be plastic adherent. Second, MSCs should express mesenchymal stem cell surface markers such as CD73, CD90, and CD105 and lack expression of the hematopoietic markers CD45, CD34, CD14 or CD11b, CD79&#945; or CD19, and HLA-DR. Finally, MSCs should exhibit the ability to differentiate into the three mesenchymal lineages: adipocytes, osteocytes, and chondrocytes. Interestingly, MSCs can also differentiate into other lineages such as neuronal cells, cardiomyocytes, hepatocytes, and epithelial cells10,11.
In fact, MSCs possess unique properties that enable them to be applied as potential therapeutic agents in regenerative therapy for different diseases. MSCs can secrete soluble factors to induce an immunomodulatory environment that provides therapeutic benefits12. In addition, MSCs can migrate toward sites of injury and tumor microenvironments to deliver targeted therapy; however, the mechanisms are not fully elucidated13. In addition, MSCs have the ability to secrete exosomes, extracellular vesicles in the nanoscale that carry a cargo of non-coding RNAs, protein, and soluble factors, which lately emerged as a novel mechanism of the MSCs&#39; therapeutic potential in various diseases14.
More importantly, MSCs have generated marked attention for their potential to differentiate into insulin-producing cells (IPCs), either by genetic modification15,16 or through utilizing various extrinsic-inducing factors within the culture media in vitro17. The IPC induction period varies greatly, as it depends on the used induction protocol and the utilized extrinsic factors. The process of differentiation can last from days to months, and it requires a combination of exogenous-inducing factors that must be added and/or withdrawn in different stages. Many of these factors that have been used for endocrine pancreatic differentiation are biologically active compounds that have been shown to promote the proliferation or differentiation/neogenesis of insulin-secreting &#946;-cells and/or increase the insulin content of IPCs18,19,20,21. It is noteworthy here that MSCs have also been reported to have therapeutic effects in diabetes and its complications via several mechanisms, including their secretome, as well as a wide array of immuno-modulatory actions22,23,24.
In this protocol, we present a detailed stepwise protocol for the isolation and characterization of Ad-MSCs from rat epididymal fat, followed by a simple, relatively short protocol for the generation of IPCs from Ad-MSCs.

<table><tbody><tr><td>Albumin, bovine serum Fraction V</td><td>MP Biomedicals</td><td></td><td></td></tr><tr><td>Alcian Blue 8GX</td><td>Sigma-Aldrich, USA</td><td>A3157</td><td></td></tr><tr><td>Alizarin Red S</td><td>Sigma-Aldrich, USA</td><td>A5533</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>Antibody for Rat CD90, FITC</td><td>Stem Cell Technologies</td><td>60024FI</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A3912</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>CD105 Monoclonal Antibody, FITC</td><td>Thermo Fisher Scientific, Invitrogen, USA</td><td>MA1-19594</td><td></td></tr><tr><td>CD34 Polyclonal Antibody</td><td>Thermo Fisher Scientific, Invitrogen, USA</td><td>PA5-85917</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific, USA</td><td></td><td></td></tr><tr><td>Collagenase type I, powder</td><td>Gibco, Thermo Fisher, USA</td><td>17018029</td><td></td></tr><tr><td>D-Glucose anhydrous, extra pure</td><td>Fisher Scientific, Germany</td><td>G/0450/53</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific, Germany</td><td>BP231-100</td><td></td></tr><tr><td>Dithizone staining</td><td>Sigma-Aldrich, USA</td><td>D5130</td><td></td></tr><tr><td>DMEM - High Glucose 4.5 g/L</td><td>Lonza, Switzerland</td><td>12-604F</td><td></td></tr><tr><td>DMEM - Low Glucose 1 g/L</td><td>Lonza, Switzerland</td><td>12-707F</td><td></td></tr><tr><td>DMEM/F12 medium</td><td>Lonza, Switzerland</td><td>BE12-719F</td><td></td></tr><tr><td>DNAse/RNAse free water</td><td>Gibco Thermo Fisher, USA</td><td>10977-035</td><td></td></tr><tr><td>Ethanol absolute, Molecular biology grade</td><td>Sigma-Aldrich, Germany</td><td>24103</td><td></td></tr><tr><td>Exendin-4</td><td>Sigma-Aldrich, Germany</td><td>E7144</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco Thermo Fisher, Brazil</td><td>10270-106</td><td></td></tr><tr><td>Formaldehyde 37%</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>Isopropanol, Molecular biology grade</td><td>Fisher Scientific, USA</td><td>BP2618500</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco Thermo Fisher, USA</td><td>25030-024</td><td></td></tr><tr><td>Magnesium Chloride (Anhydrous)</td><td>Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>Mesenchymal Stem Cell Functional identification kit</td><td>R&amp;amp;D systems Inc., MN, USA</td><td>SC006</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich, Germany</td><td>N0636</td><td></td></tr><tr><td>Oil Red Stain</td><td>Sigma-Aldrich, USA</td><td>O0625</td><td></td></tr><tr><td>Penicillin-Streptomycin-Amphotericin</td><td>Gibco Thermo Fisher, USA</td><td>15240062</td><td></td></tr><tr><td>Phosphate buffered saline, 1X, without Ca/Mg</td><td>Lonza, Switzerland</td><td>BE17-516F</td><td></td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>Rat Insulin ELISA Kit</td><td>Cloud-Clone Corp., USA</td><td>CEA682Ra</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>Sodium Phosphate Dibasic (Anhydrous)</td><td>Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>Sodium Phosphate Monobasic (Anhydrous)</td><td>Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>SYBR Green Maxima</td><td>Thermo Scientific, USA</td><td>K0221</td><td></td></tr><tr><td>Syringe filter, 0.2 micron</td><td>Corning, USA</td><td>431224</td><td></td></tr><tr><td>TRIzol</td><td>Thermo Scientific, USA</td><td>15596026</td><td></td></tr><tr><td>Trypan blue</td><td>Gibco Thermo Fisher, USA</td><td>15250061</td><td></td></tr><tr><td>Trypsin-Versene-EDTA, 1X</td><td>Lonza, Switzerland</td><td>CC-5012</td><td></td></tr><tr><td>Verso cDNA synthesis kit</td><td>Thermo Scientific, USA</td><td>AB-1453/A</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich, Germany</td><td>M3148</td><td></td></tr></tbody></table>

isolation of rat adipose tissue mesenchymal stem cells for differentiation into insulin-producing cells

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

Isolation and characterization of Ad-MSCs 
As shown in Figure 2, the isolated cells from adipose tissue showed a heterogeneous population of rounded and fibroblast-like cells starting from the next day of isolation (Figure 2A). 4 days post isolation, the fibroblast cells started to increase in number and size and grow as a homogenous population by passage 1 (Figure 2B,C). These cells continued ...

Watch this Scientific Journal Video about Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells at JoVE.com

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

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

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

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Research

JoVE Journal

Developmental Biology

7.2K Views.  Ain Shams University. Het algemene doel van dit protocol is om rood vetweefsel, gedroogde mesenchymale stamcellen te isoleren en te karakteriseren en te differentiëren naar insulineproducerende cellen met behulp van eenvoudige methoden. Mesenchymale stamcellen hebben de wegen gevonden op het gebied van genetica. Ze kunnen worden geïsoleerd uit verschillende bronnen zoals beenmerg, vetweefsel en perinataal weefsel.Ze accepteren uitstekende culturele kenmerken. Ze zijn multi-potent en kunnen mogelijk versch...

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

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

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

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

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

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

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

Biology

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 ...

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

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

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

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

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

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 ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

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

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