A. Thawing, expansion and freezing of Adipose-derived stem/stromal cells (ADSC)
1. Refer to the user guide for StemProÔ Human Adipose-Derived Stem Cell Kit (ThermoFisher, R7788110), from here on referred to as the “StemProÔ user guide”.
2. Prepare the complete MesenPRO RS medium, by adding 10 ml of RS growth supplement and 5 ml of glutamine solution according to the manual. In addition, add 5 ml of antibiotic-antimycotic solution and filter sterilize through a 500 ml Stericup filtration flask.
3. Refer to StemProÔ user guide and follow the procedure for thawing ADSC.
4. Change media on ADSC every 3 days using MesenPRO RS complete medium.
5. Passage and expand ADSC once the cells have become ~90% confluent using TrypLE Express, according to the StemProÔ user guide. Do not allow ADSC to become 100% confluent when expanding cells.
6. Continue to expand cells until there are ~10 100mm dishes, which typically yields 1-1.5 million cells per plate.
7. Prepare freezing solution according to StemProÔ user guide, which consists of 20% fetal bovine serum, 10% DMSO in MesenPRO RS complete medium.
8. Freeze ~10 vials of ADSCs with 1 million cells per vial, according to the StemProÔ user guide.
B. Generation of Beiging-8 (B-8) medium
1. The first step of generating the B-8 medium is developing the defined medium (DM). Into a 500 ml Stericup add the following to generate the DM:
· 50 ml of Albumin (20%, vol/vol in DMEM/F-12)
· 5 ml of antibiotic-antimycotic solution (100X)
· 5 ml of glutaGRO (100X)
· 5 ml of minimal essential amino acids (100X)
· 0.5 ml of Trace Elements A, B and C
· sodium ascorbate (50 mg/ml, final concentration)
· transferrin (10 mg/ml, final concentration)
· 432.75 ml of DMEM/F-12
· Filter-sterilized the DM.
2. Aliquot and heat 5 ml of DM in a 15 ml conical tuble, along with a 500 ml aliquot of IBMX (500 mM), to 65°C for 15 minutes.
3. To the remaining DM add the following to generate B-8:
· IGF-1 (200 ng/ml, final concentration; stock: 1 mg/ml)
· FGF2 (8 ng/ml, final concentration; stock 25 mg/ml)
· BMP7 (100 ng/ml, final concentration; stock 100 ug/ml)
· Y27632 (10 mM, final concentration; stock 10 mM)
· Rosiglitazone (2 mM, final concentration; stock 20 mM)
· Dexamethasone (1 mM, final concentration; stock 10 mM)
· Triiodo-L-thyronine (1 nM, final concentration; stock 20 mM)
· Once the IBMX (500 mM, final concentration; stock 500 mM) is heated and fully re-suspended, rapidly add it to the 5 ml of heated DM. This may then be added to the remaining DM—which will generate the complete B-8 medium.
· Medium may be stored at 4°C for up to 2 weeks.
C. Differentiation of ADSC to beige adipocytes.
1. Thaw an aliquot of ADSC as described in the StemProÔ user guide, and expand the cells as needed for planned downstream applications. Cells may be passaged for ~10 times without major cell senescence (a 1:4 or 1:5 split may be used for routine passaging).
2. Plate ADSC into each well of a 6-well plate (5000 cells/cm2) using MesenPRO RS complete medium and change medium every 3 days.
Optional: a 2nd plate of ADSC can be generated to serve as an ADSC control plate or to generate white adipocytes using the Stempro adipogenesis differentiation kit (A1007001).
3. Grow ADSC until they reach 100% confluency prior to differentiation induction. This is a critical step for efficient differentiation.
4. Pre-warm an aliquot of B-8 media in a 37°C water bath.
5. Aspirate media from 6-well plate and wash cells 2X’s in DPBS to remove any residual media.
6. Add 2 ml of pre-warmed B-8 media to each well of the 6-well plate containing confluent ADSCs. This is the starting point (day 0) for the differentiation to beige adipocytes.
The ADSC from the control plate may be imaged and collected for RNA/protein analysis or used to generate white adipocytes beginning at this time point.
7. The media on the beige adipocyte plate should be changed every 48 hours for three weeks.
8. On day 20-21, Forskolin (20 mM, final concentration) may be added to the beige cells for 24 hours for protein or 6 hours for RNA.
9. Beige adipocytes can then be collected in triplicate for RNA/protein analysis or other downstream applications using conventional approaches.
1. Initial differentiation of ADSC to beige cells may have the appearance of apoptotic cells or undergoing cell death. However, by days 5-6, small lipid-filled vesicles should become apparent in the cells.
2. By day 21, cell culture should be strikingly different from ADSC with uniform multiloculular lipid droplets. Stopping cultures early (~day 15) results in significantly reduced levels of UCP1.
3. Transcript markers most useful for evaluation include UCP1, DIO2, ADIPOQ and PPARGC1. These markers are not detectable in ADSC but will be significantly elevated upon differentiation to beige adipocytes. Forskolin will result in further a 2-3 fold induction of UCP1, and a >5-fold induction in DIO2.
4. Immunostaining of the cultures will reveal that >90% are positive for UCP1.