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.

Keywords: AXIN2; GLI1; bone development; cranial suture; mesenchymal stromal cells; nonsyndromic craniosynostosis; osteogenesis; personalised medicine; regenerative medicine; stem cell niche.

Figure 1

Visual description of the architecture of calvarial flat bone. Two sheets (outer and inner tables) of compact bone enclose a layer of spongy bone (diploë). Green arrows highlight the endosteal, periosteal and inter-suture locations of MSC niches (figure modified from https://smart.servier.com/).

Figure 2

AXIN2 and THY1 expression in calvarial bone. (a) The haematoxylin/eosin staining displays the histological structure of a cross-section of the flat calvarial bone, showing the periosteum and endosteum location. Localisation of AXIN2/Ki-67 and AXIN2/THY1 expression in (b,c) fused and (d,e) unfused suture tissue sections, assessed by immunofluorescence. Green arrows highlight AXIN2+ cells, red arrows point out (b,d) Ki-67+ cells and (c,e) THY1+ cells. Yellow arrows indicate AXIN2+/THY1+ and AXIN2+/Ki-67+ cells. Pictures are representative of 90 microscopic fields from nine slides (technical replicates) at 20× magnification of three patients (biological replicates) (see Methods for details).

Figure 3

GLI1 and THY1 expression in calvarial bone. (a) The haematoxylin/eosin staining displays the histological structure of a cross-section of the flat calvarial bone, showing the periosteum and endosteum location. Localisation of GLI1/Ki-67 and GLI1/THY1 in (b,c) fused and (d,e) unfused suture tissue sections, assessed by immunofluorescence. Green arrows highlight cells GLI1+, red arrows point out (b,d) Ki-67+ cells and (c,e) THY1+ cells. Yellow arrows indicate GLI1+/THY1+ and GLI1+/Ki-67+ cells. The haematoxylin/eosin staining displays the histological structure of the flat calvarial bone. Pictures are representative of 90 microscopic fields from nine slides (technical replicates) at 20× magnification of three patients (biological replicates) (see Methods for details).

Figure 4

Comparative expression profiling of marker genes in iliac crest BM-MSC and suture cells CMSC. Transcript levels of: (a) THY1; (b) ITGAV; (c) TEK; (d) ENPEP; (e) GLI1; and (f) AXIN2, evaluated by qPCR in N- and P-CMSC, compared with BM-MSC. RQ, Relative Quantity (see Methods for details). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 5

Expression profiles of osteo-specific genes. Transcript levels of: (a,f) RUNX2; (b,g) ALP; (c,h) ON; (d,i) OPN; and (e,j) OCN, evaluated by qPCR in N-CMSC and P-CMSC, respectively, cultured with osteogenic medium (OM) for one (1 wk) and three weeks (3 wk). Cells cultured in standard growth medium (GM) were tested as controls at each time point. RQ, Relative Quantity (see Methods for details). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 6

Expression profiles of putative niche marker genes. Transcript levels of: (a,d) THY1; (b,e) GLI1; and (c,f) AXIN2, evaluated by qPCR in N-CMSC and P-CMSC, respectively, cultured with osteogenic medium (OM) for one (1 wk) and three weeks (3 wk). Cells cultured in standard growth medium (GM) were tested as controls at each time point. RQ, Relative Quantity (see Methods for details). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 7

Expression of GLI1 and THY1 in CMSC during osteogenic induction. Markers’ expression analysed by immunofluorescence in (a–c) N-CMSC and (e–g) P-CMSC, cultured with osteogenic medium (OM) for one (1 wk) and three weeks (3 wk), compared to cells cultured in growth medium (GM). Nuclei are stained in blue with DAPI. The intensity of fluorescence was quantified by the ImageJ software for both (d) N-CMSC and (h) P-CMSC. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Pictures are representative of 30 microscopic fields of three different cell culture samples (see Methods for details).

Figure 8

Expression of AXIN2 and THY1 in CMSC during osteogenic induction. Markers’ expression analysed by immunofluorescence in (a–c) N-CMSC and (e–g) P-CMSC, cultured with osteogenic medium (OM) for one (1 wk) and three weeks (3 wk), compared to cells cultured in growth medium (GM). Nuclei are stained in blue with DAPI. The intensity of fluorescence has been quantified by the ImageJ software for both (d) N-CMSC and (h) P-CMSC. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Pictures are representative of 30 microscopic fields of three different cell culture samples (see Methods for details).

Figure 9

Comparative expression profiling of AXIN2 gene and protein in N-versus-P-CMSC. (a) Transcript and (b) protein levels of AXIN2 compared in P-CMSC versus N-CMSC, in standard growth conditions (GM) and following one (1 wk) and three weeks (3 wk) of osteogenic commitment (OM). RQ, Relative Quantity (see Methods for details). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 10

AXIN2 knockdown results. Expression analysis of: (a) AXIN2; (b) RUNX2; (c) ALP; (d) ON; (e) OPN; and (f) OCN, evaluated by qPCR upon AXIN2 silencing in N-CMSC (si-N-CMSC) cultured with osteogenic medium for one week (OM 1wk). Untreated N-CMSC were used as controls for both si-N-CMSC and P-CMSC. The graphs show the average values derived from three different experiments on three different patient’s derived cells. RQ, Relative Quantity (see Methods for details). * p < 0.05; ** p < 0.01; *** p < 0.001.