The Dental Biology Unit is directed by Dr. Songtao Shi and includes Drs. Masako Miura, Byoung-Moo Seo, Wataru Sonoyama, Yasuo Miura and Carolyn Coppe. Our research is focused on understanding how dental/craniofacial associated mesenchymal stem cells may involve in human diseases and more importantly how to utilize mesenchymal stem cells for potential therapies in clinics. Mesenchymal stem cells are multipotent stem cells, capable of differentiating into a variety of cell types including, but not limited to, osteoblasts/odontoblasts, adipocytes, neural cells,chondrocytes, and muscle cells. A balanced function between osteoblasts (bone-forming cells) and osteoclasts (bone resorption cells) is required for maintaining normal bone density. Functional deficiency of osteoblasts may lead to an osteoporosis phenotype. Although caspase-3 (Casp3) activity was found to involve in apoptosis of osteoblasts, the exact role of Casp3 in bone development and skeletal diseases is largely unknown. In Casp3 deficient mice, we found delayed ossification and decreased bone density, implying that normal Casp3 activity may be critical for maintaining the integrity of bone. Thus, we next examined osteoclast function of Casp3 deficient mice and found a significantly decreased osteoclast activity in Casp3 deficient mice, indicating that osteoclast-mediated bone resorption was not a major cause of observed bone defects for Casp3 deficient mice. Therefore, we focused on investigating cell proliferation and differentiation of osteoblast progenitor, bone marrow-derived mesenchymal stem cell (MSC), of Casp3 deficient mice. The number of colonies generated from 3-week-old Casp3 deficient mice was significantly lower than the age-matched wild type (WT) mice, suggesting a decreased number of osteoprogenitors in Casp3 deficient mice. The number of proliferating MSC in Casp3 deficient mice was significantly decreased compared to WT MSC assesed by the bromodeoxyuridine (BrdU) incorporation assay. In the mean time, when cultured under the osteogenic inductive condition, Casp3 deficient MSC accumulated less calcium compared to WT MSC. Additionally, the bone-forming capacity of MSC was examined by in vivo transplantion into immunocompromised mice. Casp3 deficient MSC generated significantly less bone at 8 weeks post-transplantation compared to WT MSC. Next, we observed that TGFbeta could induce an accelerated replicative senescence in Casp3 deficient MSC. In contrast, BMP-2 treatment did not induce an increased senescence in Casp3 deficient MSC, indicating there was a TGFbeta specificity in the process of senscense. To elucidate the mechanism of how accelerated senescence occurs in Casp3 deficient MSC, we examined the involvement of the TGFbeta mediated signaling proteins and cell cycle-related proteins. Casp3 deficient MSC showed elevated expressions of TGFbeta receptor type I (TGFRI) and Smad2 under the regular culture condition. Following TGFbeta treatment, the expressions of Smad2 and phospho-Smad2 (p-Smad2) were up-regulated compared to WT MSC, indicating that TGF beta/Smad2 pathway was over-activated in Casp3 deficient MSC. In contrast, TGFbeta receptor type II (TGFRII) and Smad3 were not up-regulated in Casp3 deficient MSC. Moreover, the expressions of Cdk2 and Cdc2 were down-regulated in Casp3 deficient MSC along with the up-regulated expressions of their counterparts, p21 and p53, which may be one of the mechanisms leading to cell cycle arrest and eventual replicative senescence. These data implied that Casp3 deficiency causes over-activated TGFbeta signaling and attenuated the cell cycle in MSC. A Casp3 inhibitor, Z-DEVD-FMK, was administered to mice to examine whether inhibition of Casp3 activity could achieve similar results as observed in Casp3 deficient mice. We found that bone density was significantly decreased in ovariectomized (OVX) mice after treatment with Z-DEVD-FMK compared to vehicle (DMSO)-treated OVX mice. Similar to the Casp3 deficient mice, there was no increase in bone resorption in Z-DEVD-FMK-treated OVX mice compared to DMSO-treated OVX mice, suggesting that osteoclast function is not a major factor for the decreased bone density observed in Casp3 inhibitor-treated OVX mice. This study showed that Casp3 activity is critical for osteogenic differentiation of MSC and demonstrated that the mechanism involves replicative senescence of MSC. These data point to the conclusion that any in vivo application of Casp3 inhibitor must consider the influence on the bone density, especially in postmenopausal women. Human postnatal stem cells possess a great potential to be utilized in stem-cell-mediated clinical therapies. Determining how to effectively preserve postnatal stem cells has therefore become a critical issue for postnatal stem cell research. We found that functional postnatal stem cells can be retrieved from six-month cryopreserved human periodontal ligament (PDL), indicating that frozen human tissue contains retrievable postnatal stem cells. These cryopreserved PDL stem cells (C-PDLSCs) maintained normal PDL stem cell characteristics, including expression of the mesenchymal stem cell surface molecule STRO-1, single colony strain generation, multipotential differentiation, cementum/PDL-like tissue regeneration and a normal diploid karyotype. Collectively, these studies provide valuable evidence demonstrating a practical approach to preserve solid human tissues for subsequently postnatal stem cell isolation and tissue regeneration. Efficient haematopoiesis occurs mainly in the bone marrow (BM) micro-environment and depends on cooperation between bone marrow derived MSC and the hematopoietic stem cells (HSCs). However, the mechanisms that coordinate the activity of these two distinct stem cells are not fully understood. We and our collaborator Dr. Li Zhang (University of Maryland) show that integrin beta2, a surface protein present on HSCs, is also expressed on MSC, and can be used efficiently as a surface marker to enrich MSC from bone marrow aspirates by cell sorting. Genetic inactivation of integrin beta2 impaired osteogenic differentiation of MSC through inhibition of osteogenic master protein Cbfa1, leading to defective bone formation in vivo, and decreased bone mineral density in the deficient animal, whereas it had no significant effect on the in vivo osteoclastic activity. The defective osteogenesis of the integrin beta2-deficient MSC could be rescued by expression of full length but not a cytoplasmic domain-truncated beta2. Altogether, this study demonstrates that integrin beta2 is critical to the functions of the MSC, thus implicating a potential predisposition of bone defects in the integrin beta2-deficient patients.
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