Lineage Tracing of Resident Tendon Progenitor Cells during Growth and Natural Healing

Abstract

Unlike during embryogenesis, the identity of tissue resident progenitor cells that contribute to postnatal tendon growth and natural healing is poorly characterized. Therefore, we utilized 1) an inducible Cre driven by alpha smooth muscle actin (SMACreERT2), that identifies mesenchymal progenitors, 2) a constitutively active Cre driven by growth and differentiation factor 5 (GDF5Cre), a critical regulator of joint condensation, in combination with 3) an Ai9 Cre reporter to permanently label SMA9 and GDF5-9 populations and their progeny. In growing mice, SMA9+ cells were found in peritendinous structures and scleraxis-positive (ScxGFP+) cells within the tendon midsubstance and myotendinous junction. The progenitors within the tendon midsubstance were transiently labeled as they displayed a 4-fold expansion from day 2 to day 21 but reduced to baseline levels by day 70. SMA9+ cells were not found within tendon entheses or ligaments in the knee, suggesting a different origin. In contrast to the SMA9 population, GDF5-9+ cells extended from the bone through the enthesis and into a portion of the tendon midsubstance. GDF5-9+ cells were also found throughout the length of the ligaments, indicating a significant variation in the progenitors that contribute to tendons and ligaments. Following tendon injury, SMA9+ paratenon cells were the main contributors to the healing response. SMA9+ cells extended over the defect space at 1 week and differentiated into ScxGFP+ cells at 2 weeks, which coincided with increased collagen signal in the paratenon bridge. Thus, SMA9-labeled cells represent a unique progenitor source that contributes to the tendon midsubstance, paratenon, and myotendinous junction during growth and natural healing, while GDF5 progenitors contribute to tendon enthesis and ligament development. Understanding the mechanisms that regulate the expansion and differentiation of these progenitors may prove crucial to improving future repair strategies.

Gdf5 progenitors give rise to fibrocartilage cells that mineralize via hedgehog signaling to form the zonal enthesis

“…(1) GDF5Cre-Confetti. In order to measure the clonal expansion of enthesis progenitors, constitutive GDF5Cre mice generously provided by Dr. David Kingsley (Rountree, et al 2004; Dyment, et al 2014) were crossed with R26R-Confetti mice (Gt(ROSA)26Sor tm1(CAG-Brainbow2.1)Cle /J, Jackson Labs). The R26R-Confetti mice harbor a CAG promoter followed by a floxed STOP cassette and downstream brainbow 2.1 sequence all within the Rosa26 locus.…”

“…These tissues possess a range of mechanical properties, structural organization, and embryological origins (Charvet, et al 2012; Lu and Thomopoulos. 2013; Dyment, et al 2014; Zelzer, et al 2014). The interfaces that connect muscle to tendon (i.e., myotendinous junction) and tendon to bone (i.e., enthesis) are crucial for efficient load transfer and subsequent joint motion.…”

“…At the interphase of these two populations, a unique enthesis progenitor pool has been identified that co-express Scx and Sox9 (Soeda, et al 2010; Blitz, et al 2013; Sugimoto, et al 2013). Enthesis cells during this timeframe also express growth and differentiation factor (Gdf5), unlike cells in the underlying primary cartilage (Rountree, et al 2004; Dyment, et al 2014). At later embryonic stages, enthesis cells express Gli1 and cells within unmineralized regions of the enthesis maintain this expression during later stages of postnatal growth (Schwartz, et al 2015).…”

Tendon injury and repair – A perspective on the basic mechanisms of tendon disease and future clinical therapy

“…The tendon core consists of densely packed Type-I collagen matrix and the fibroblastic cells that maintain it [50]. Although increasingly realized to have distinct functions in the context of tendon disease and repair, the physiological roles of many of the cells within both compartments and possible communication between the compartments is still poorly understood [50][51][52][53][54]. We proceed by outlining what is known about the intrinsic compartment that Fig.…”

“…Strikingly little is known regarding the actual mechanisms by which originally healthy tendon accumulates damage, and then how the intrinsic and extrinsic compartments activate and coordinate tissue remodeling [18,[21][22][23]. Only slightly more is known about this process after acute injury, however studies using animal models of acute injury and repair are beginning to shed some light [53,54].…”