Micro-pellet culture reveals that bone marrow mesenchymal stromal cell (BMSC) chondrogenic induction is triggered by a single day of TGF-β1 exposure

Abstract

Despite immense promise, engineering of stable cartilage tissue from bone marrow-derived stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") remains elusive. Relative cartilage-like matrix deposition is commonly used to guide BMSC chondrogenic optimisation efforts. However, matrix deposition is heterogeneous in most models, and notably, it lags behind cell fate decisions. We reason that the lag time between cell fate decision and matrix accumulation, coupled with matrix heterogeneity, has obscured basic BMSC biological characteristics, such as differentiation kinetics. Here, we utilize a customized microwell platform to assemble hundreds of small-diameter BMSC micro-pellets and characterized chondrogenic differentiation kinetics in response to the canonical signaling molecule, transforming growth factor-{beta}1 (TGF-{beta}1). Micro-pellets provide a homogeneous readout, and our experimental design accounts for the significant time delay between growth factor signal and deposition of cartilage-like matrix. While 14-to-21-day induction protocols are routine, BMSC micro-pellet cultures reveal that a single day of TGF-{beta}1 exposure was sufficient to trigger chondrogenic differentiation cascades resulting in outcomes similar to micro-pellets exposed to TGF-{beta}1 for 21 days. RNA-sequencing analysis demonstrated that one day of TGF-{beta}1 exposure was also sufficient to induce hypertrophic cascades in BMSC, not observed in articular chondrocytes. Refocusing chondrogenic induction optimisation efforts from weeks to the first hours or days of culture, using homogeneous model systems, may benefit efforts to build stable cartilage formed by BMSCs. SignificanceThe macro-pellet model, and assumptions generated using it, have permeated BMSC-based cartilage tissue engineering strategies since the 1990s. Using a micro-pellet model, we show that BMSC chondrogenic kinetics are significantly more rapid than historical macro-pellets data suggests, and that BMSC chondrogenic and hypertrophic commitment is instructed by a single day of TGF-{beta}1 exposure. This highly relevant study demonstrates that: (1) macro-pellets, which are large heterogeneous tissue models confound the differentiation kinetics visible in micro-pellet models; (2) induction strategies should focus on the first hours or days of culture; (3) even a single day of TGF-{beta}1 exposure drives BMSC to form hypertrophic tissue in vivo, requiring early intervention to prevent hypertrophy; and (4) articular chondrocytes and BMSCs respond distinctly to TGF-{beta}1.