Osteogenic 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 a technique to promote the osteogenic differentiation of human and mouse MSCs.

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 Human Osteogenic Supplement (R&D Systems, Catalog # CCM008) or StemXVivo Mouse Osteogenic Supplement (R&D Systems, Catalog # CCM009)
• Penicillin-Streptomycin (100x)

Materials

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

Equipment

• 37º 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 Osteogenic Differentiation Media - Add StemXVivo Osteogenic Supplement to the completed StemXVivo Osteogenic/Adipogenic Base Media at a 1:20 dilution. Note: With the exception of Step 5, the reagent and material preparation for mouse and human completed StemXVivo Osteogenic/Adipogenic Base Media and completed StemXVivo Osteogenic Differentiation Media are the same using either the mouse or the human supplement.

Figure 1

Procedure

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 2.3 - 2.5 x 10⁵ MSCs in 10 mL of the pre-warmed Completed StemXVivo Osteogenic/Adipogenic Base Media.
   Note: If using another size tissue culture vessel, seed cells at approximately 4.2 x 10³ cells/cm²/0.2 - 0.3 mL media.
3. Add this cell suspension to a 10 cm tissue culture plate. The cells should be 50 - 70% confluent in 1 - 2 days.
4. At 50 - 70% confluency, replace the media with 10 mL of pre-warmed completed StemXVivo Osteogenic Differentiation Media to induce osteogenesis.
5. Every 3 - 4 days (for mouse every 2 - 3 days) remove and discard spent media and replace with 10 mL of pre-warmed completed StemXVivo Ostegenic Differentiation Media.
   Note: Dispense media down the side of the plate so as not to disrupt cells.
6. After 2 - 3 weeks induced cells will exhibit phenotypic changes consistent with osteogenic differentiation (Figures 2 and 3).

   Figure 2. Osteocyte Differentiation of Human MSCs. Human mesenchymal stem cells differentiated in vitro for 21 days using StemXVivo Human/Mouse Osteogenic/Adipogenic Base Media (Catalog # CCM007) and Human StemXVivo Osteogenic Supplement (Catalog # CCM008) were stained for the osteogenic lineage marker, Osteocalcin using a Mouse Anti-Human/Rat Osteocalcin Monoclonal Antibody (Catalog # MAB1419) and NorthernLights 557-conjugated Anti-mouse Secondary Antibody (Catalog # NL007; red). Cells were counterstained with DAPI (blue).

   Figure 3. Osteocyte Differentiation of Mouse MSCs. The Mouse Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC010) was used to differentiate MSCs into the osteogenic lineage. Mature differentiated cells were stained for the osteogenic lineage marker Osteopontin 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 Mouse StemXVivo Osteogenic Supplement (Catalog # CCM009).