Int J Stem Cells.  2020 Nov;13(3):377-385. 10.15283/ijsc20024.

Isolation and Characterization of Human Suture Mesenchymal Stem Cells In Vitro

• Kong L
• Wang Y
• Ji Y
• Chen J
• Cui J
• Shen W

• KMID: 2508911
• DOI: http://doi.org/10.15283/ijsc20024

Abstract

Background and Objectives
Cranial sutures play a critical role in adjustment of skull development and brain growth. Premature fusion of cranial sutures leads to craniosynostosis. The aim of the current study was to culture and characterize human cranial suture mesenchymal cells in vitro.
Methods
The residual skull tissues, containing synostosed or contralateral suture from three boys with right coronal suture synostosis, were used to isolate the suture mesenchymal cells. Then, flow cytometry and multilineage differentiation were performed to identify the typical mesenchymal stem cell (MSC) properties. Finally, we used quantitative real-time polymerase chain reaction (RT-PCR) to detect the mRNA expression of osteogenesis and stemness related genes.
Results
After 3 to 5 days in culture, the cells migrated from the tissue explants and proliferated parallelly or spirally. These cells expressed typical MSC markers, CD73, CD90, CD105, and could give rises to osteocytes, adipocytes and chondrocytes. RT-PCR showed relatively higher levels of Runx2, osteocalcin and FGF2 in the fused suture MSCs than in the normal cells. However, BMP3, the only protein of BMP family that inhibits osteogenesis, reduced in synostosed suture derived cells. The expression of effector genes remaining cell stemness, including Bmi1, Gli1 and Axin2, decreased in the cells migrated from the affected cranial sutures.
Conclusions
The MSCs from prematurely occlusive sutures overexpressed osteogenic related genes and down-regulated stemness-related genes, which may further accelerate the osteogenic differentiation and suppress the self-renewal of stem cells leading to craniosynostosis.

Keyword

Cranial suture; Craniosynostosis; Human; Mesenchyme stem cell

Figure

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  Fig. 1 Isolation and culture of suture mesenchymal stem cells. (A) Craniofacial computed tomographic scan with three-dimensional reconstruction of three patients before operation showed typically right coronal suture fusion. Representative H&E staining of contralateral patent suture (B) and prematurely fused suture (C). (D) The tissues, containing fused or contralateral cranial suture, in the culture dishes. (E) The cells were migrating from the cranial suture tissue. (F, G) Inverted microscopic view of suture mesenchymal cells exhibiting almost homogeneous spindle morphology and stained with 0.25% crystal violet blue.

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  Fig. 2 Identification of suture mesenchymal stem cells. (A) FACS analysis of mesenchymal stem cells migrated from the explanted sutures. Alizarin red (B), Oil red (C) and Alcian blue (D) staining indicates that the suture mesenchymal cells can undergo osteogenic, adipogenic and chondrogenic differentiation.

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  Fig. 3 Expression of osteogenic phenotypic genes and FGFR family genes in suture cells. (A-C) Osteogenic differentiation in the mesenchymal stem cells, from normal patent sutures (Normal group) or prematurely synostosed sutures (Synostosed group), was tested. Con, control group without osteogenic induction; Ost, osteogenic differentiation group with osteogenic induction. (A) Representative pictures of Alizarin Red S staining. Both Cells were cultured with osteogenic induction media for six days. (B, C) mRNA levels of Runx2 and OCN by RT-PCR analysis. (D∼G) RT-PCR analysis of BMP3, FGF2, FGFR2 and FGFR3 in normal and synostosed sutures-derived mesenchymal cells. Results are mean±SD. *p<0.05, **p<0.01, and ***p<0.001 versus normal cells. OCN, osteocalcin.

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  Fig. 4 Expression of stemness-related genes in suture cells. (A, B) Colony formation of two mesenchymal cell types and the number of colonies per well was calculated. (C∼G) mRNA levels of Lef1, Bmi1, Nanog, Gli1 and Axin2 in normal and synostosed sutures-derived mesenchymal cells using quantitative PCR. Results are mean±SD. *p<0.05, **p<0.01, and ***p<0.001 versus normal cells. Normal, normal patent sutures derived cells and Synostosed, prematurely synostosed sutures derived cells.

Reference

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