Adipogenic Differentiation of Human/Mouse Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are functionally defined by their capacity to self renew and their ability to differentiate into multiple cell types including adipocytes, chondrocytes, and osteocytes. This protocol describes the adipogenic differentiation of human and mouse MSCs using the StemXVivo™ Human/Mouse Osteogenic/Adipogenic Base Media (Catalog # CCM007) and StemXVivo Adipogenic Supplement (Catalog # CCM011).

Please read the protocol in its entirety before starting.

Supplies Required

Reagents

• Bone marrow derived MSCs
• StemXVivo Human/Mouse Osteogenic/Adipogenic Base Media (R&D Systems, Catalog # CCM007)
• StemXVivo Adipogenic Supplement (R&D Systems, Catalog # CCM011)
• Penicillin-Streptomycin (100x)

Materials

• 10 cm tissue culture plates
• 15 mL centrifuge tubes
• Serological pipettes
• Pipettes and pipette tips

Equipment

• 37° C, 5% CO₂ humidified incubator
• Centrifuge
• Hemocytometer
• Microscope
• Water bath

Reagent & Media Preparation

Note: Sterile technique is required when handling the reagents.

• StemXVivo Osteogenic/Adipogenic Base Media - Thaw the StemXVivo Osteogenic/Adipogenic Base Media at 2 - 8° C or room temperature. Aliquot any unused thawed media and store at -20° C in a manual defrost freezer. Thawed media may be stored in the dark at 2 - 8° C for up to 1 month.
• Completed StemXVivo Osteogenic/Adipogenic Base Media - Add Penicillin-Streptomycin to the StemXVivo Osteogenic/Adipogenic Base Media at a 1:100 dilution.
  Note: If Penicillin-Streptomycin is not needed for the experiment, it can be omitted.
• Completed StemXVivo Adipogenic Differentiation Media - If a precipitate forms, warm the Adipogenic Supplement vial in a 37° C water bath for 5 minutes. Vortex until the precipitate dissolves. Add StemXVivo Adipogenic Supplement to the completed StemXVivo Osteogenic/Adipogenic Base Media at a 1:100 dilution.

Procedure

Figure 1

Note: When handling biohazard materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.

1. Pre-warm the Completed StemXVivo Osteogenic/Adipogenic Base Media in a 37° C water bath. This procedure uses 10 mL for each 10 cm tissue culture plate used.
2. Resuspend 1 x 10⁶ MSCs into 10 mL of the pre-warmed Completed StemXVivo Osteogenic Base Media.
   Note: If using another size tissue culture vessel, seed cells at approximately 2.1 x 10⁴ cells/cm²/0.2 - 0.3 mL.
3. Add this cell suspension to a 10 cm tissue culture plate and incubate overnight in a 37° C, 5% CO₂ incubator. Cells should be 100% confluent after overnight incubation. If they are not confluent, replace media every 2 - 3 days with Osteogenic/Adipogenic Base Media until 100% confluency is reached.
4. At 100% confluency, replace the media with 10 mL of pre-warmed completed StemXVivo Adipogenic Differentiation Media to induce adipogenesis.
5. Every 3 - 4 days remove and discard the spent media and replace with 10 mL of freshly prepared, pre-warmed Completed StemXVivo Adipogenic Differentiation Media.
   Note: Differentiation is complete after 7 - 21 days, at which time adipogenic induced cells will have morphological changes and lipid vacuoles (Figure 2) and express markers of the adipocyte lineage (Figure 3).

   Figure 2. Adipogenic Differentiation of Human MSCs. Human mesenchymal stem cells differentiated in vitro for 21 days using the StemXVivo Human/Mouse Osteogenic/Adipogenic Base Media (Catalog # CCM007) and StemXVivo Human/Mouse Adipogenic Supplement (Catalog # CCM011) were stained for the adipogenic lineage-specific marker, FABP-4 using a Goat Anti-Human FABP-4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3150) and a NorthernLights 557-conjugated Anti-Goat Secondary Antibody (Catalog # NL001; red) and counterstained with DAPI (blue).

   Figure 3. Adipogenic Differentiation of Mouse MSCs. The Mouse Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC010) was used to differentiate mouse MSCs into the adipogenic lineage. Mature differentiated cells were labeled using the adipogenic lineage-specific antibody to FABP4 supplied with the kit (red). The nuclei were counterstained with DAPI (blue). Similar results may be obtained using the StemXVivo Human/Mouse Osteogenic/Adipogenic Base Media (Catalog # CCM007) and StemXVivo Human/Mouse Adipogenic Supplement (Catalog # CCM011).