A FoxA2+ long-term stem cell population is necessary for growth plate cartilage regeneration after injury.

Longitudinal bone growth, achieved through endochondral ossification, is accomplished by a cartilaginous structure, the physis or growth plate, comprised of morphologically distinct zones related to chondrocyte function: resting, proliferating and hypertrophic zones. The resting zone is a stem cell-rich region that gives rise to the growth plate, and exhibits regenerative capabilities in response to injury. We discovered a FoxA2+group of long-term skeletal stem cells, situated at the top of resting zone, adjacent the secondary ossification center, distinct from the previously characterized PTHrP+ stem cells. Compared to PTHrP+ cells, FoxA2+ cells exhibit higher clonogenicity and longevity. FoxA2+ cells exhibit dual osteo-chondro-progenitor activity during early postnatal development (P0-P28) and chondrogenic potential beyond P28. When the growth plate is injured, FoxA2+ cells expand in response to trauma, and produce physeal cartilage for growth plate tissue regeneration.

Overexpression of transcription factor FoxA2 in the developing skeleton causes an enlargement of the cartilage hypertrophic zone, but it does not trigger ectopic differentiation in immature chondrocytes.

We previously found that FoxA factors are necessary for chondrocyte differentiation. To investigate whether FoxA factors alone are sufficient to drive chondrocyte hypertrophy, we build a FoxA2 transgenic mouse in which FoxA2 cDNA is driven by a reiterated Tetracycline Response Element (TRE) and a minimal CMV promoter. This transgenic line was crossed with a col2CRE;Rosa26rtTA/+ mouse line to generate col2CRE;Rosa26rtTA/+;TgFoxA2+/- mice for inducible expression of FoxA2 in cartilage using doxycycline treatment. Ectopic expression of FoxA2 in the developing skeleton reveals skeletal defects and shorter skeletal elements in E17.5 mice. The chondro-osseous border was frequently mis-shaped in mutant mice, with small islands of col.10+ hypertrophic cells extending in the metaphyseal bone. Even though overexpression of FoxA2 causes an accumulation of hypertrophic chondrocytes, it did not trigger ectopic hypertrophy in the immature chondrocytes. This suggests that FoxA2 may need transcriptional co-factors (such as Runx2), whose expression is restricted to the hypertrophic zone, and absent in the immature chondrocytes. To investigate a potential FoxA2/Runx2 interaction in immature chondrocytes versus hypertrophic cells, we separated these two subpopulations by FACS to obtain CD24+CD200+ hypertrophic chondrocytes and CD24+CD200- immature chondrocytes and we ectopically expressed FoxA2 alone or in combination with Runx2 via lentiviral gene delivery. In CD24+CD200+ hypertrophic chondrocytes, FoxA2 enhanced the expression of chondrocyte hypertrophic markers collagen 10, MMP13, and alkaline phosphatase. In contrast, in the CD24+CD200- immature chondrocytes, neither FoxA2 nor Runx2 overexpression could induce ectopic expression of hypertrophic markers MMP13, alkaline phosphatase, or PTH/PTHrP receptor. Overall these findings mirror our in vivo data, and suggest that induction of chondrocyte hypertrophy by FoxA2 may require other factors in addition to Runx2 (i.e., Hif2α, MEF2C, or perhaps unknown factors), whose expression/activity is rate-limiting in immature chondrocytes.