A Protocol to Induce Brown and Beige Adipocyte Differentiation From Murine and Human Adipose-Derived SVF
诱导小鼠与人源脂肪来源 SVF 分化为棕色与米色脂肪细胞的操作流程
(*contributed equally to this work) 发布: 2025年12月05日第15卷第23期 DOI: 10.21769/BioProtoc.5522 浏览次数: 830
评审: Philipp WörsdörferAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2020
Abstract
Adipose cells vary functionally, with white adipocytes storing energy and brown/beige adipocytes generating heat. Mouse and human subcutaneous white adipose tissue (WAT)-derived stromal vascular fraction (SVF) provides mesenchymal stem cells (MSCs) that can be differentiated into thermogenic adipocytes using pharmacological cocktails. After six days of browning induction, these cells exhibited significant upregulation of thermogenic markers (UCP1, Cidea, Dio2, PRDM16) along with adipogenic genes (PPARγ, aP2), showing enhanced thermogenic potential. This in vitro system offers a practical platform to study adipogenesis and thermogenic regulation.
Key features
• The protocol aims to differentiate murine and human SVF from subcutaneous fat into brown/beige adipocytes for assays.
• This approach employs a straightforward methodology that effectively differentiates SVF cells using optimal chemicals, their concentrations, and durations.
Keywords: Adipocyte differentiation (脂肪细胞分化)
Thermogenesis (产热)
SVF (SVF)
Beige adipocytes (米色脂肪细胞)
Brown adipocytes (棕色脂肪细胞)
MSCs (间充质干细胞)
Graphical overview
Background
Obesity, as defined by WHO, is a chronic complex condition characterized by excessive adipose tissue that can adversely affect health [1]. Adipose tissue, often known as "fat," serves as a passive energy reservoir and is involved in various physiological functions, including the regulation of food consumption, energy balance, insulin production, immune response, and the maintenance of body temperature [2,3]. Mammals possess three different types of adipocytes—white, brown, and beige adipocytes—each located in specific anatomical regions and characterized by distinct gene expression profiles [4]. White adipose tissue (WAT) is extensively found in subcutaneous areas and surrounding internal organs. Its cellular structure is characterized by a prominent single lipid droplet, minimal mitochondria, and limited cytoplasm, serving primarily as an energy reservoir. Additionally, WAT functions as an endocrine organ, releasing adipokines like leptin, lipotropin, and TNFα, which play crucial roles in regulating energy metabolism and sustaining physiological balance [5]. Brown adipose tissue (BAT), on the other hand, has many mitochondria and multilocular lipid droplets and is heavily innervated and vascularized. Its major function is thermoregulation, which involves oxidizing fatty acids (FAs) to maintain body temperature [6]. Another form of adipose tissue, known as beige adipose, is found in WAT depots but exhibits brown- like characteristics including multilocular lipid droplets and high mitochondrial density [7,8]. Brown and beige adipocytes are often referred to as “thermogenic adipocytes.” This process is facilitated by uncoupling protein 1 (UCP1) and is referred to as non-shivering thermogenesis [9,10].
Although beige adipocytes exhibit functional similarities to brown adipocytes, these two thermogenic cells differ in their developmental origins. In fact, beige adipocytes originate from the same progenitor cells as white adipocytes [11]. The process by which beige adipocytes are formed is commonly referred to as browning, typically occurring in subcutaneous fat deposits. Collectively, these findings indicate that both extrinsic and intrinsic cellular factors play a role in determining the fate of adipocyte differentiation. In this protocol, we used mouse and human stromal vascular fraction (SVF) isolated from fat depots to identify and evaluate methods for inducing differentiation into brown and beige adipocytes. Slight modifications were introduced in the earlier protocol [12] in our laboratory, where they have been replicated and refined to implement fundamental procedures with reduced toxicity and time interval. It includes the addition of optimized novel chemicals (1 nM T3 and 125 μM Indomethacine), decreased concentration of the earlier used compounds (20 nM insulin, 1 μM dexamethasone, and 0.5 μM IBMX), and shorter time duration for adipocyte differentiation.
Materials and reagents
Biological materials
1. Subcutaneous fat depots (scWAT) of mice
2. Subcutaneous fat depots (scWAT) of human
Reagents
1. Dulbecco’s modified Eagle’s medium (DMEM), high glucose (Corning, catalog number: 10-013-CV), store at 4 °C
2. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: A5256801), store at -20 °C
3. Penicillin-streptomycin (Pen/Strep) (Thermo Fisher Scientific, catalog number: 15070063), store at -20 °C
4. Phosphate-buffered saline (PBS), 1× (Corning, catalog number: 21-040-CMX12)
5. Collagenase/dispase (Roche, catalog number: 10269638001)
6. TRIzol reagent (Fisher Scientific, catalog number: 15596018)
7. Chloroform (Sigma-Aldrich, catalog number: 472476)
8. Ultrapure DNase/RNase-free distilled water (nuclease-free water) (Fisher Scientific, catalog number: 10-977-023)
9. iScriptTM cDNA Synthesis kit (Bio-Rad, catalog number: 1708890)
10. iQTM SYBR Green Supermix (Bio-Rad, catalog number: 1708880)
11. Insulin (20 nM final concentration) (Sigma-Aldrich, catalog number: I1507), powder storage temperature -20 °C, store at 4 °C after dilution
12. Triiodothyronine (T3) (1 nM final concentration) (Sigma-Aldrich, catalog number: T2877), store at -20 °C
13. Indomethacin (125 μM final concentration) (Sigma-Aldrich, catalog number: I7378), store at -20 °C
14. Dexamethasone (1 μM final concentration) (Sigma-Aldrich, catalog number: D4902), store at -20 °C
15. Rosiglitazone (1 μM final concentration) (Sigma-Aldrich, catalog number: 557366-M), store at -20 °C
16. 3-Isobutyl-1-methylxanthine (IBMX) (0.5 μM final concentration) (Sigma-Aldrich, catalog number: I5879), store at -20 °C
17. Calcium chloride (Sigma-Aldrich, catalog number: C5670)
18. Isopropanol (Sigma-Aldrich, catalog number: 190764)
Solutions
1. Stock solutions: 0.5 mg/mL (87.2 μM) insulin, 1 mM T3, 100 mM Indomethacin, 10 mM Dexamethasone, 0.25 M IBMX, 10 mM Rosiglitazone, and 10 mM CaCl2 (see Recipes)
2. Growth medium (see Recipes)
3. Induction medium (see Recipes)
4. Maintenance medium (see Recipes)
5. Reverse transcription mix (see Recipes)
6. qPCR mix (see Recipes)
7. Washing medium (see Recipes)
Recipes
1. Stock solutions
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Insulin | 0.5 mg/mL (87.2 μM) | 1 mg of insulin dissolved in 2 mL of distilled water (pH = 2, add 0.5 μL HCl 16N). Filter the stock solution through a 0.22 μm sterile filter. |
| T3 | 1 mM | 1.3 mg of T3 dissolved in 2 mL of ethanol. Note: T3 requires amber storage due to light sensitivity. |
| Indomethacin | 100 mM | 71.558 mg of Indomethacin in 2 mL of ethanol. To help with solubilization, it may be heated to 75 °C for 1 min using a heating block. |
| Dexamethasone | 10 mM | 7.85 mg of Dexamethasone in 2 mL of ethanol. Note: Dexamethasone requires amber storage due to light sensitivity. |
| IBMX | 0.25 M | 55.56 mg of IBMX in 1 mL of ethanol. To help with solubilization, it may be heated to 75 °C for 1 min using a heating block. |
| Rosiglitazone | 10 mM | 7.15 mg of Rosiglitazone in 2 mL of ethanol. Note: Rosiglitazone requires amber storage due to light sensitivity. |
| CaCl2 | 10 mM | 1.11 mg of CaCl2 (anhydrous) in 1 mL of sterile distilled water. Filter sterilize with 0.22 μm and store at 4 °C. |
Note: Stocks are aliquoted and stored for 2–3 months at -20 °C, except for insulin (working vial), which needs to be stored at 4 °C. Avoid multiple freeze-thaw cycles to maintain the stability of the chemicals.
2. Growth medium
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| DMEM, high glucose | 90% | 450 mL |
| FBS | 10% | 50 mL |
| Pen/Strep | 1% | 5 mL |
3. Induction medium
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Growth medium | 1 mL/well of 12-well plate | |
| Insulin | 20 nM | Diluted from stock in growth medium |
| T3 | 1 nM | Diluted from stock in growth medium |
| Indomethacin | 125 μM | Diluted from stock in growth medium |
| Dexamethasone | 1 μM | Diluted from stock in growth medium |
| IBMX | 0.5 μM | Diluted from stock in growth medium |
| Rosiglitazone | 1 μM | Diluted from stock in growth medium |
| Total | n/a | 1 mL/well of 12-well plate |
4. Maintenance medium
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Growth Medium | 1 mL/well of 12-well plate | |
| Insulin | 20 nM | Diluted from stock in growth medium |
| T3 | 1 nM | Diluted from stock in growth medium |
| Total | n/a | 1 mL/well of 12-well plate |
5. Reverse transcription mix (per 1 sample)
| Reagent | Volume |
|---|---|
| Nuclease-free water | Variable |
| iScript reaction mix (5×) | 4 μL |
| iScript RT | 1 μL |
| RNA (1 μg) | Variable |
| Total | 20 μL |
6. qPCR mix (per 1 sample)
| Reagent | Volume |
|---|---|
| iQTM SYBR Green Supermix | 5 μL |
| Forward primer (final concentration 300–500 nM) | 0.5 μL |
| Reverse primer (final concentration 300–500 nM) | 0.5 μL |
| Nuclease-free water | 2 μL |
| cDNA (100–500 ng) | 2 μL |
| Total | 10 μL |
7. Washing medium
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| PBS | 1× | 9.99 mL |
| Calcium (Ca2+) | 10 μM | 10 μL of 10 mM CaCl2 stock solution |
| Total | 10 mL |
Laboratory supplies
1. Sterile forceps and scissors
2. 70 μm cell strainer (BD Falcon, catalog number: 352350)
3. 12-well cell culture plate (Corning, catalog number: 12-565-321)
4. 0.22 μm sterile filter (NalgeneTM, catalog number: 7262520)
Equipment
1. Heat block (Fisher Scientific, model: 88870001)
2. Tissue culture incubator (37 °C, 5% CO2) (Eppendorf, New Brunswick, model: Galaxy 170R)
3. Water bath (MRC, model: SWBR27-2)
4. Centrifuge (Eppendorf, model: 5430R/refrigerated)
5. Tissue culture hood (Heal force: HF Safe-1200 LC Biosafety Cabinet, Class II, Type II)
6. Inverted microscope (Thermo Fisher Scientific, model: EVOS M3000)
7. Automated cell counter (Thermo Fisher Scientific, model: Countess 3)
8. PCR machine (Thermo Fisher Scientific, model: VeritiTM Thermal Cycler 96 well)
9. NanoDrop Lite Plus (Thermo Fisher Scientific, model: NDLPLUSGL)
10. qPCR machine (Thermo Fisher Scientific, model: QuantStudio 3)
11. Shaking incubator (Helix)
Software and datasets
1. Graphpad Prism 9® software
Procedure
文章信息
稿件历史记录
提交日期: Aug 20, 2025
接收日期: Oct 19, 2025
在线发布日期: Nov 4, 2025
出版日期: Dec 5, 2025
版权信息
© 2025 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Yadav, R. R., Mishra, N. and Verma, N. (2025). A Protocol to Induce Brown and Beige Adipocyte Differentiation From Murine and Human Adipose-Derived SVF. Bio-protocol 15(23): e5522. DOI: 10.21769/BioProtoc.5522.
- Verma, N., Perie, L. and Mueller, E. (2020). The mRNA levels of heat shock factor 1 are regulated by thermogenic signals via the cAMP-dependent transcription factor ATF3. J Biol Chem. 295(18): 5984–5994. https://doi.org/10.1074/jbc.ra119.012072
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分类
干细胞 > 成体干细胞 > 间充质干细胞
发育生物学 > 细胞生长和命运决定
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