The Research On Biological Behaviors Of BMMSCs And DPSCs Derived From A Patient With Cleidocranial Dysplasia

Cleidocranial dysplasia (CCD) is an autosomal-dominant, heritable skeletal disease that is characterized by absent or hypoplastic clavicles, persistently open or delayed closure of sutures, wormian bones, delayed eruption of permanent dentition, supernumerary teeth, short stature, and other skeletal changes. Mutations in the runt-related transcription factor 2 gene (RUNX2, also known as CBFA1, PEBP2aA, and AML3) located on chromosome 6p21 have been identified as the cause of CCD. RUNX2 is a key transcriptional modulator in the process of mesenchymal stem cell (MSCs) differentiation into osteoblasts; it plays a fundamental role in osteoblast maturation and homeostasis and is essential for normal bone development. So far, there is few investigations about Chinese cases with CCD. The effects of RUNX2 mutations on stem cells in CCD patients are unknown. Here, a patient with the clinical diagnosis of CCD (RUNX2^+/m) and his family were investigated. To explore the effect of the defective RUNX2 protein on the biological function of MSCs derived from the patient, RUNX2^+/m BMMSCs (bone marrow mesenchymal stem cells, BMMSCs) and DPSCs (dental pulp stem cells, DPSCs) were isolated and cultured from the CCD patient, and RUNX2^+/m BMMSCs and DPSCs differentiation and physiological function were examined by comparison with normal MSCs function. The study was divided into three parts described as follows.1. Clinical examination and analysis, and identification of mutations of a patient with cleidocranial dysplasiaIn present study, we managed carefully the clinical examinations for the patient with opened fontanelle and examined dentin specimens from the patient using scanning electron microscopy (SEM) analysis. Clinical characteristics of the patient in our study demonstrated a thin build, short stature, narrow thorax and shrugged shoulders that were easily apposable, brachydactyly, hypoplastic clavicles, opened anterior and posterior fontanelle. Panoramic radiograph shows dental anomalies, including retained deciduous teeth, supernumerary teeth in the molar region and impacted permanent teeth with delayed development and abnormal roots. So the patient was diagnosed as cleidocranial dysplasia. To identify mutations in the RUNX2 gene in the CCD patient, we analyzed genomic DNA of the patient, his healthy parents, and unrelated normal controls. A novel insertion/frameshift mutation (c. 1116–1119insC) was identified within exon 7 in the patient, whereas his healthy parents and normal controls did not carry the mutation. This results further indicated RUNX2 mutation results in CCD and the novel insertion/frameshift mutation in the RUNX2 gene (c. 1116–1119insC) caused the severe CCD phenotype. SEM analysis of a tooth from the CCD patient and a corresponding normal individual indicated that peritubular and intertubular dentin in the CCD patient displayed loose collagen bundles and inadequate mineralization compared with normal teeth. The result indicated that RUNX2 gene may be involved in dental development.2. The research on biological behaviors of BMMSCs and DPSCs derived from a patient with cleidocranial dysplasiaBone extracellular matrix deposition or bone formation by differentiated osteoblasts begins late during development and lasts throughout life. Human mesenchymal stem cells from bone marrow or dental pulp can respectively differentiate into osteoblasts and odontoblasts in vitro and generate bone/dentin-like mineralized tissue when transplanted subcutaneously into immunocompromised mice. However, the relationship between MSCs and bone/tooth development is still unclear. In this study, we investigated MSCs derived from a patient with cleidocranial dysplasia. We found that the RUNX2 mutation caused decreased proliferative ability and osteogenic potential of MSCs isolated from the bone marrow and dental pulp of the CCD patient (RUNX2^+/m) compared to control individuals (RUNX2^+/+). Furthermore, Surprisingly, our results showed that there was higher expression of osteoblast-specific genes (runt-related transcription factor 2 gene RUNX2, alkaline phosphatase ALP) in RUNX2^+/m BMMSCs compared to RUNX2^+/+ BMMSCs, but expression of OCN (osteocalcin, OCN) was not significantly different, perhaps due to negative feedback by the RUNX2 mutation. In summary, by studying a rare human genetic disease, we identified a novel insertion/frameshift mutation in the RUNX2 gene (c. 1116–1119insC) that caused a severe CCD phenotype and altered the biological function of RUNX2^+/m MSCs. The reduced ability of RUNX2^+/m MSCs to differentiate into osteoblasts might contribute to the defects related to bones and teeth in the CCD patient and therefore provide a molecular explanation for these defects.3. The study of Wnt/β-catenin signaling pathway of BMMSCs derived from a patient with cleidocranial dysplasiaGrowing evidence indicates that the Wnt signaling pathway plays a critical role in stem/progenitor cell self-renewal and regulates the differentiation potential of adult human MSCs in adult tissues. To investigate whether the decreased differentiation potential of RUNX2^+/m BMMSCs could be rescued, we cultured RUNX2^+/m and RUNX2^+/+ BMMSCs in the presence of exogenous recombinant human Wnt3a or DKK-1 to activate or inactivate the Wnt/β-catenin signaling pathway. Our results showed that expression of bone-related genes, including OCN, RUNX2 and ALP, in RUNX2^+/+ BMMSCs was upregulated in the presence of exogenous DKK-1 and downregulated in the presence of exogenous Wnt3a. Expression of OCN, RUNX2 and ALP in RUNX2^+/m BMMSCs was also downregulated in the presence of exogenous Wnt3a, but it was not changed in the presence of exogenous DKK-1. Western blot analysis of RUNX2 demonstrated similar results as the real time RT-PCR data. Our results indicated that the decreased differentiation potential could not be rescued by manipulation of the Wnt/β-catenin signaling pathway.