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-b1 (TGF-b1). 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-b1 exposure was sufficient to trigger chondrogenic differentiation cascades resulting in outcomes similar to micro-pellets exposed to TGF-b1 for 21 days. RNA-sequencing analysis demonstrated that one day of TGF-b1 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-b1 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-b1 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-b1.

Intermittent parathyroid hormone (1–34) supplementation of bone marrow stromal cell cultures may inhibit hypertrophy, but at the expense of chondrogenesis

“…The second was the duration of chondrogenic culture (6 weeks in the previous study [23] and 2 weeks in our study). In previous work, we demonstrated that pellets formed from 2 × 10 5 cells each suffer profound gradients and yield heterogenous tissues [25,26]. In large-diameter pellets, differentiation appears to occur first at the peripheral edge of the pellet, with differentiation in the core being delayed, likely due to metabolite limitation in large-diameter tissues.…”

“…We posit that common large-diameter cartilage tissue models, which suffer large diffusion gradients and tissue heterogeneity, confound efforts to identify optimal BMSC chondrogenic induction protocols. In previous work, we demonstrated that it was possible to obtain more definitive insights into BMSC chondrogenesis, in vitro, if a more homogenous micro-pellet model system was used [24][25][26]36]. In previous studies, it was reported that intermittent and constant PTH treatment of cells could result in profoundly alternate differentiation Fig.…”

“…c The nylon mesh retains individual micro-pellets in discrete microwells over the culture period. Image C was slightly modified from an image originally provided by abpLearning (medical-animations.com, Australia and [26]) using SoftImage (Autodesk, Montreal, Canada) and gifted to the Doran Laboratory at 2% O 2 and 5% CO 2 in a 37°C incubator and media exchanged every other day (or 48 h). Our group previously showed that BMSC chondrogenesis was improved at 2% O 2 [24,25].…”

“…In previous work, we developed the Microwell-mesh [24], a high-throughput microwell platform that enables the manufacture of hundreds of homogeneous cartilage micro-pellets (sometimes referred to as microtissues) and maintains these micro-pellets in individual microwells over extended culture periods. As a result of their smaller size (0.5-1 mm diameter), relative to traditional pellet cultures, micro-pellets are more sensitive to differentiation signalling factors [26].…”

Transforming growth factor-beta stimulates human bone marrow-derived mesenchymal stem/stromal cell chondrogenesis more so than kartogenin

“…The authors concluded that KGN preconditioning likely improved chondrogenic differentiation of umbilical cord blood-derived mesenchymal stromal cells by committing them to a pre-cartilaginous stage with enhanced JNK phosphorylation and suppressed β-catenin 21 . Our team recently reported that exposing human BMSC to a single day of TGF-β1 yielded differentiation outcomes similar to BMSC exposed to TGF-β1 for 21 days 22 . This observation is specifically relevant to this paper for two reasons: (1) in the human BMSC micropellet model, a single day of TGF-β1 is sufficient to trigger chondrogenic induction, while 14 days of continuous KGN had minimal impact on human BMSC chondrogenesis; and (2) the mechanism by which a single day of TGF-β1 exposure drives BMSC differentiation appears to be fundamentally different than the mechanism by which KGN pre-conditioning of human umbilical cord-derived mesenchymal stromal cells promoted subsequent chondrogenesis in response to TGF-β3 21 .…”