Figure 1. MesenCult™-ACF Chondrogenic Differentiation Medium Induces Robust Chondrogenic Differentiation of Human MSCs

Human BM-derived MSCs were cultured in MesenCult™-ACF Medium then differentiated to the chondrogenic lineage using MesenCult™-ACF Chondrogenic Differentiation Medium. Robust chondrogenic differentiation was observed (A) starting with as few as 3 x 10⁵ MSCs, or (B) when differentiating for just 14 days starting with 5 x 10⁵ MSCs.

Figure 2. Chondrogenic Differentiation of Human MSCs Is More Robust With Fewer Hypertrophic Chondrocytes Using MesenCult™-ACF Chondrogenic Differentiation Medium Compared to Competitor Media

Human BM-derived MSCs culture-expanded for up to two passages in MesenCult™-ACF Medium, serum-based medium, or one of two commercially available media (Competitor 1 (Ex1) and Competitor 2 (Ex2)), were then differentiated to the chondrogenic lineage starting with 5 x 10⁵ MSCs and either using MesenCult™-ACF Chondrogenic Differentiation Medium or one of several commercially available chondrogenic differentiation media (Ch1, Ch2 or Ch3) for 21 days. More robust and uniform chondrogenic differentiation was observed when the MSCs were differentiated in MesenCult™-ACF Chondrogenic Differentiation Medium compared to the other commercially available chondrogenic differentiation media (Ch1, Ch2 and Ch3), irrespective of the expansion medium used to culture the MSCs prior to differentiation. Cultures differentiated using MesenCult™-ACF Chondrogenic Differentiation Medium displayed an abundance of isogenous groups (yellow arrows), suggesting there is proliferation of differentiating chondrocyte progenitors. Few hypertrophic chondrocytes (black arrows) are seen in cultures differentiated with MesenCult™-ACF Chondrogenic Differentiation Medium, suggesting the maintenance of chondrogenic activity throughout the culturing period.

Figure 3. MesenCult™-ACF Chondrogenic Differentiation Medium Induces Stronger and More Sustained Chondrogenic Transcript Expression Compared to Competitor Media

Human BM-derived MSCs expanded in (A) MesenCult™-ACF Medium, (B) a serum-based medium or (C) Competitor 2 (Ex2) medium, were differentiated for 21 days with MesenCult™-ACF Chondrogenic Differentiation Medium and Competitor 2 (Ch2) chondrogenic differentiation medium. Regardless of the expansion medium initially used to culture the MSCs, differentiation using MesenCult™-ACF Chondrogenic Differentiation Medium led to a substantial up-regulation of the chondrogenic transcripts compared to Ch2. In addition, expression of the terminally-differentiated hypertrophic transcript Mmp13 was higher for Ch2 differentiated cultures compared to cultures differentiated with MesenCult™-ACF Chondrogenic Differentiation Medium.

Figure 4. Mouse MSCs Cultured in MesenCult™-ACF Chondrogenic Differentiation Medium Differentiate to Chondrocytes

Mouse compact bone-derived MSCs were cultured using the MesenCult™ Proliferation Kit with MesenPure™ (Mouse, Catalog #05512) for 2 passages then differentiated by pellet culture with MesenCult™-ACF Chondrogenic Differentiation Medium for 21 days under normoxic (20% O²) conditions. Strong chondrogenic differentiation is indicated by dark-blue staining of the cartilage extracellular matrix and an abundance of isogenous chondrocyte groups.

Figure 5. Human MSC Chondrogenic Differentiation With AggreWell™800 Plates

Using centrifugation, 1 x 10^6 human MSCs were distributed evenly among 800 µm microwells in one well of an AggreWell™800 plate. Small aggregates of only ~3,300 cells per pellet were then differentiated to chondrocytes using MesenCult™-ACF Chondrogenic Differentiation Medium for 21 days under normoxic (20% O2) conditions.

Figure 6. Procedure Overview: Human MSC Chondrogenic Differentiation Using MesenCult™-ACF Chondrogenic Differentiation Medium

Aggregate culture is a useful method for inducing chondrogenic differentiation of human BM- and adipose-derived MSCs in a three-dimensional in vitro culture environment. MSCs are efficiently differentiated to the chondrogenic lineage using MesenCult™-ACF Chondrogenic Differentiation Medium in 14 - 21 days with 3 - 5 x 10⁵ cells.