A single day of TGF-β1 exposure activates chondrogenic and hypertrophic differentiation pathways in bone marrow-derived stromal cells (BMSC)

We profile the change in gene expression of BMSC and ACh in chondrogenic differentiation cultures when exposed to TGF-β1 for variable durations (0, 1, 3, 7, or 21 days) over a 21 day micro-pellet culture. For BMSC differentiation cultures, as little as 1-day of TGF-β1 exposure resultsedin similar gene expression on day 21 as in BMSC cultures that were exposed to TGF-β1 for 3, 7, or 21 days of the total culture period. In contrast, ACh chondrogenic cultures do not respond to TGF-β1 in a similar fashion, and gene expression differences are observed with the early withdrawl of TGF-β1. When comparing the global gene expression of BMSC and ACh cultures, the gene expression of these cell types diverged, rather then converged over time, despite being cultured under the same conditions. Purpose: To determine the duration of TGF-β1 exposure required to initiate chondrogenic differentiation of bone marrow-derived stromal cells (BMSC), also known as "mesenchymal stem cells (MSC)". Method: Micro-pellet cultures were established in custom-made microwell plates (Microwell-mesh plates), such that each pellet was derived from 5,000 cells each. Three BMSC and two ACh donors were analysed. We analyzed cultures before induction (day 0) and harvested on day 1, 3, 7, and 21 from chonrogenic cultures continuously exposed to TGF-β1. Additionally, for one BMSC and one ACh donor, we analyzed micro-pellets cultured until day 21, with exposure to TGF-β1 for only the first 1, 3, or 7 days of the total 21-day culture. Results: For BMSC chondrogenic differentiation cultures, regardless of the TGF-β1 exposure time (1, 3, 7, or 21 days), all the cultures converged on a similar gene expression profile by day 21. Despite the phenotypic appearance of cartilage-like tissue in BMSC differentiation cultures, based on gene expression, these cells diverged from that of ACh cultures over the same culture conditions and durations. Conclusion: We found that a single day of TGF-β1 exposure was sufficient to trigger BMSC chondrogenic differentiation and tissue formation, similar to 21 days of TGF-β1 exposure. Despite cessation of exogenous TGF-β1 exposure following 24 hours, intrinsic programming mediated further chondrogenic and hypertrophic BMSC differentiation.