GLI1 and AXIN2 Are Distinctive Markers of Human Calvarial Mesenchymal Stromal Cells in Nonsyndromic Craniosynostosis

Abstract

All skeletal bones house osteogenic stem cell niches, in which mesenchymal stromal cells (MSC) provide progenitors for tissue growth and regeneration. They have been widely studied in long bones formed through endochondral ossification. Limited information is available on the composition of the osteogenic niche in flat bones (i.e., skull vault bones) that develop through direct membranous ossification. Craniosynostosis (CS) is a congenital craniofacial defect due to the excessive and premature ossification of skull vault sutures. This study aimed at analysing the expression of GLI1, AXIN2 and THY1 in the context of the human skull vault, using nonsyndromic forms of CS (NCS) as a model to test their functional implication in the aberrant osteogenic process. The expression of selected markers was studied in NCS patients’ calvarial bone specimens, to assess the in vivo location of cells, and in MSC isolated thereof. The marker expression profile was analysed during in vitro osteogenic differentiation to validate the functional implication. Our results show that GLI1 and AXIN2 are expressed in periosteal and endosteal locations within the osteogenic niche of human calvarial bones. Their expression is higher in MSC isolated from calvarial bones than in those isolated from long bones and tends to decrease upon osteogenic commitment and differentiation. In particular, AXIN2 expression was lower in cells isolated from prematurely fused sutures than in those derived from patent sutures of NCS patients. This suggests that AXIN2 could reasonably represent a marker for the stem cell population that undergoes depletion during the premature ossification process occurring in CS.

Personalized Bone Reconstruction and Regeneration in the Treatment of Craniosynostosis

“…They also showed that the implantation of MSC-graft leads to suture regeneration by restoring Gli1 + cells subpopulation within cranial suture both through exogenously implanted Gli1+ MSCs and endogenous MSCs derived from the dura mater [108]. Our group has indeed recently demonstrated that GLI1 represents a specific marker for MSC in the human calvarial niche [21].…”

“…The suture mesenchyme represents a unique skeletogenic stem cell niche in the developing skull, serving as a transient reservoir of mesenchymal stromal cells (MSC) and osteoprogenitors (see Figure 1) [21,22]. Regardless of the initial trigger underlying CS etiology, it is believed that in these disorders the calvarial suture stem cell niche undergoes an accelerated exhaustion, which ultimately drives the premature ossification of the suture mesenchyme [20,23].…”

“…In particular, our group studied the molecular mechanisms implicated in the overactive osteogenic cascade of mesenchymal stromal cells isolated from calvarial tissues of nonsyndromic CS patients, with idiopathic etiology. By studying this cellular model, we identified functional biomarkers (LMP3, BBS9, RUNX2, AXIN2, and GLI1), to be exploited also in the design of molecular targeted therapeutic approaches aimed at regulating the osteogenic commitment of stem cells in the suture niche [10,12,15,21]. Furthermore, Barreto and coworkers developed a 2D culture system based on cells isolated from nonsyndromic CS patients' sutures, to demonstrate that fused-vs-patent suture cells display differential gene expression profiles underlying different stiffness-mediated responses [188].…”

The Intertwined Evolution and Development of Sutures and Cranial Morphology

“…Once closed, sutures appear to lose their MSC population. The Gli1+ population is lost at the site of the fused interfrontal suture in mice ( Zhao et al, 2015 ), whilst there is a reduction in the Axin2 population at prematurely fused sutures in craniosynostosis patients ( Di Pietro et al, 2020 ). Thus, suggesting that an open suture status is necessary for the suture to contain a reservoir of MSCs enabling it to function as a site of interstitial bone growth and support normal craniofacial development.…”

“…Marsupials exhibit a lower level of closure than placentals, and sutures are reported to remain open throughout life ( Rager et al, 2014 ). It is thus possible that variation in suture closure between placentals and marsupials may be indicative of differing postnatal functional roles, especially given the significance of suture fusion status in supporting postcranial bone growth ( Zhao et al, 2015 ; Di Pietro et al, 2020 ).…”