Plastic Surgery Research Council

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Osteogenic Differentiation Of Msc As A Model Study Of The Postnatal Progressive Crouzon Syndrome
Joanna K. Ledwon, PhD1, Yekaterina Galat, BS2, Jolanta M. Topczewska, PhD1, Arun K. Gosain, MD1.
1Northwestern University Feinberg School of Medicine, Department of Surgery Plastic Division, Stanley Manne Children's Research Institute, Ann and Robert H. Lurie Children's Hospital of Chicago., Chicago, IL, USA, 2Department of Pediatrics, Developmental Biology Program, Stanley Manne Children's Research Institute, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA.

PURPOSE
A patient with Crouzon syndrome, presenting as postnatal progressive pansynostosis, was identified as heterozygous for a mutation in Fibroblast Growth Factor Receptor 2 (FGFR2) 1025G>A resulting in C342Y substitution. This mutation is usually associated with the coronal suture fusion not with pansynostosis. Here, we aim to create an in vitro model using mesenchymal stem cells (MSC) isolated from the patient's periosteum to identify the cause for the unusual presentation.
METHODS
The MSC isolated from the periosteum of Crouzon patient and healthy control were expanded and sorted by FACS to obtain a homogeneous CD73+CD90+CD105+ triple positive cell population. After sorting, cells were grown in MSC NutriStem XF medium until 80% confluency was reached. The osteogenic differentiation was induced with MSCgo Osteogenic XF medium and continued for 21 days. RNA expression profiles for the typical markers of osteogenic differentiation: BGLAP, SPP1, ALPL, RUNX2, COL1A1, NOG, BMP4 and other genes such as SFRP2 and ENPP1 were monitored every 7 days of differentiation by qRT-PCR. Results were normalized to the housekeeping gene GAPDH. Statistical analyses were performed with the Student's t-test, and p-values ≤ 0.05 were considered significant. Bone matrix deposition was detected by alizarin red staining.
RESULTS
The FGFR2C342Y/+ cell culture was characterized by increased proliferation, as the 80% confluence was reached one day faster than the control (n=3). The qRT-PCR data (Fig. 1) collected for each time point of cell differentiation revealed an increased expression of RUNX2, FGF signaling target, consistent with the dominant character of the mutation. We also observed a continual increase in FGFR2 transcripts suggesting transcriptional stimulation of the pathway. Initially lower COL1A1 expression was gradually increasing with C342Y cell differentiation. In contrast, initially high expression of ALPL and SPP1 genes in C342Y cells declined with time. The pattern of expression of BGLAP, the marker of mature osteoblasts, was shifted in time, initially lower than control it reached its maximum expression at 14 days when the control expression was already declining. BMP4 expression was initially undetectable in the C342Y cells but increased later. Similar shift was observed for SFRP2 transcription. The alizarin red staining for the bone matrix showed heterogeneity of C342Y culture and inefficient mineralization (Fig. 2). Decreased mineralization ability of the patient's cells was supported by lower ENPP1 expression.

CONCLUSION
Our studies demonstrated that MSC isolated from FGFR2C342Y/+ patient differentiated similar to the control. However, C342Y culture was characterized by increased proliferation and elevated markers of osteoblasts precursors. The transcriptional shift toward delayed maturation of the osteoblasts and inefficient bone mineralization suggests that our MSC model recapitulates the delayed progression of suture ossification. In the future, we will investigate how insufficient level of BMP and excess of WNT signaling contribute to the observed phenotype.


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