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

Lorena Di Pietro,Sabrina Ceccariglia,Marta Barba,Chiara Prampolini,Paolo Frassanito,Ornella Parolini,Alessia Vita,Wanda Lattanzi,Gianpiero Tamburrini,Enrico Guadagni,Luca Massimi,Davide Bonvissuto

doi:10.3390/ijms21124356

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.

Highlights

Craniofacial bones are flat bones formed through intramembranous ossification, differently from most of the other bones of the human skeleton that develop through endochondral ossification [1].Intramembranous bone ossification is a direct process in which osteoblasts differentiate directly from mesenchymal cells, without the formation of cartilage precursors.In the craniofacial region, intramembranous bone development starts with the aggregation of mesenchymal stromal cells (MSC) into condensation centres, where they grow and proliferate, forming clusters [2]
The expression pattern of AXIN2 and GLI1 in calvarial bone specimens was analysed by immunofluorescence in unfused- and fused-suture tissue samples derived from sagittal nonsyndromic forms of CS (NCS) patients
The two markers were tested in a double staining with either the Ki-67 proliferation marker or the THY1 MSC surface antigen, with the aim to assess the presence of a proliferating MSC subpopulation positive for AXIN2 and GLI1

Summary

Introduction

Craniofacial bones are flat bones formed through intramembranous ossification, differently from most of the other bones of the human skeleton that develop through endochondral ossification [1].Intramembranous bone ossification is a direct process in which osteoblasts differentiate directly from mesenchymal cells, without the formation of cartilage precursors.In the craniofacial region, intramembranous bone development starts with the aggregation of mesenchymal stromal cells (MSC) into condensation centres, where they grow and proliferate, forming clusters [2]. Craniofacial bones are flat bones formed through intramembranous ossification, differently from most of the other bones of the human skeleton that develop through endochondral ossification [1]. Intramembranous bone ossification is a direct process in which osteoblasts differentiate directly from mesenchymal cells, without the formation of cartilage precursors. Intramembranous bone development starts with the aggregation of mesenchymal stromal cells (MSC) into condensation centres, where they grow and proliferate, forming clusters [2]. Once the cluster reaches a critical size, MSC in the centre start to differentiate into osteoblasts. The growth of immature bone occurs at the osteogenic fronts, where cells actively proliferate. When an osteogenic front joins its neighbour, the two fronts either merge to create a single bone or give rise to a suture [3]

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