Stempro adipogenesis differentiation medium

hADSCs (cat#R7788-115, Invitrogen, CA, USA) were cultured in MesenPro RS™ Medium (Invitrogen, CA, USA) supplemented with 100 units/mL of penicillin and 100 μg/mL of streptomycin (Cellgro, VA, USA), in a humidified incubator at 37°C and 5% CO₂. In order to differentiate into adipocytes, cells were grown for 2 days of post-confluence, after which the medium was replaced with StemPro® Adipogenesis Differentiation Medium (Life technologies, CA, USA). Media was changed every 3 days during the specific periods of cultivation. The differentiation scheme is depicted in Figure 1(a).10.1080/21623945.2019.1590929-F0001Figure 1.

Measurement of the effect of KD025 on adipogenesis in hADSCs.

hADSCs were differentiated by culturing in differentiation media (DM) with or without KD025 at the indicated concentrations. (a) Experimental scheme of differentiation. (b) Cells were stained with ORO at day 15, and microscopic images were taken after the start of differentiation (indicated as day 0). Cells were exposed to 0.3, 0.5, 1, and 3 μM of KD025. (c) Lipid accumulation of (b) was assessed by measuring absorbance at 520 nm. (d) Cells were differentiated with or without 3 μM KD025 until day 24. Microscopic pictures of cells are presented. (e) Lipid accumulation of (d) was assessed by measuring absorbance at 520 nm. **p < 0.01; ***p < 0.001 vs. control.

Subconfluent (80% of confluency) MSCs of different lines (n = 4) were seeded at 2 × 10⁴ MSCs/cm² into 24-well plates with 1 ml of DMEM + 10% FBS. After 24 hrs, DMEM-medium was replaced by STEMPro Osteogenesis Differentiation medium and STEMPro Adipogenesis Differentiation medium (both from Life Technologies) for differentiation into osteoblasts and adipocytes, respectively. Cells were cultured at 37°C and 5% CO₂ with media exchange every 2–3 days. Osteoblastic differentiation of MSCs was determined by Alizarin Red (Sigma-Aldrich) staining after 22 days, and adipocytic differentiation by Oil Red O (Sigma-Aldrich) staining after 18 days of culture.

Proliferation of DiD-labeled or unlabeled cells was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies, USA). Differentiation of DiD-haMSCs was performed as previously reported [2 (link)]. Briefly, for adipogenic and osteogenic differentiation, cells were seeded and grown in StemPro® adipogenesis differentiation medium and StemPro® osteogenesis differentiation medium (Gibco, USA) for 3 weeks, respectively. For chondrogenic differentiation, micromass culture was used and cells were cultured in StemPro® chondrogenesis differentiation medium (Gibco, USA) for 4 weeks. The differentiation media were re-fed every 3 days. Subsequently, Oil Red O staining, Alizarin Red S staining, and Alcian Blue staining were performed to visualize the lipid droplet, calcium deposition, and cartilage, respectively. Cells were harvested at the end point of tri-lineage differentiation for real time polymerase chain reaction (PCR) to assess the specific gene expression of ADIPONECTIN (5′-CGTGATGGCAGAGATGGCACT-3′ for forward and 5′-GCGAATGGGTACATTGGGAACAG-3′ for reverse), OSTEOCALCIN (5′-TCCAAGCAGGAGGGCAATAAG-3′ for forward and 5′-GCGTTTGTAGGCGGTCTTCAAG-3′ for reverse), and COLLAGEN TYPE 2 ALPHA 1 (COL2α1) (5′-TCGCACTTGCCAAGACCTGAA-3′ for forward and 5′-GGTCTCTCCAAACCAGATGTG-3′ for reverse) associated with adipogenesis, osteogenesis, and chondrogenesis, respectively.