Although previous studies have reported the effects of extensive subculturing on proliferation rates and osteogenic potential of human mesenchymal stem cells (hMSCs), the results remain controversial. The aim of our study was to characterize the proliferation and osteogenic potential of hMSCs during serial subculture, and also to identify proteins that are differentially regulated in hMSCs during serial subculture and osteogenic differentiation using proteome analysis. Here we show that the proliferation and osteogenic capacity of hMSCs decrease during serial subculturing. Several proteins were shown to be differentially regulated during serial subculture; among these the expression of T-complex protein 1 a subunit (TCP-1a), a protein known to be associated with cell proliferation, cell cycle, morphological changes, and apoptosis, gradually decreased during serial subculture. Among proteins that were differentially regulated during osteogenic differentiation, chloride intracellular channel 1 (CLIC1) was downregulated only during the early passages eukaryotic translation elongation factor, and acidic ribosomal phosphoprotein P0 was downregulated during the middle passages, while annexin V, LIM, and SH3 domain protein 1 (LASP-1), and 14-3-3 protein gamma (YWHAG) were upregulated during the later passage. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of osteogenic differentiation potential under serial subculturing. ß
Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density (BMD) and increased risk of fracture. We studied the effects of transplantation of mesenchymal stem cells (MSCs) overexpressing receptor activator of nuclear factor-kappaB (RANK)-Fc and CXC chemokine receptor-4 (CXCR4) using retrovirus on ovariectomy (OVX)-induced bone loss in mice. Ten-week-old adult female C57BL/6 mice were divided into six groups as follows: Sham-operated mice treated with phosphate-buffered saline (PBS) (Sham-op + PBS); OVX mice intravenously transplanted with syngeneic MSCs overexpressing RANK-Fc-DsRED and CXCR4-GFP (RANK-Fc + CXCR4); RANK-Fc-DsRED and GFP (RANK-Fc + GFP); CXCR4-GFP and DsRED (CXCR4 + RED); DsRED and GFP (RED + GFP); or treated with PBS only (OVX + PBS). Measurement of BMD showed that introduction of RANK-Fc resulted in significant protection against OVX-induced bone loss compared to treatment with PBS (-0.1% versus -6.2%, P < 0.05) at 8 weeks after cell infusion. CXCR4 + RED group also significantly prevented bone loss compared to OVX + PBS group (2.7% versus -6.2%, P < 0.05). Notably, the effect of RANK-Fc + CXCR4 was greater than that of RANK-Fc + GFP (4.4% versus -0.1%, P < 0.05) while it was not significantly different from that in CXCR4 + RFP group (4.4% versus 2.7%, P = 0.055) at 8 weeks. Transplantation of MSCs with control virus (RED + GFP group) also resulted in amelioration of bone loss compared to OVX + PBS group (-1.7% versus -6.2%, P < 0.05). Fluorescence-activated cell sorting (FACS) and real-time quantitative PCR (qPCR) analysis for GFP from bone tissue revealed enhanced cell trafficking to bone by co-overexpression of CXCR4. In conclusion, we have demonstrated that intravenous transplantation of syngeneic MSCs overexpressing CXCR4 could promote increased in vivo cell trafficking to bone in OVX mice, which could in itself protect against bone loss but also enhance the therapeutic effects of RANK-Fc.
Although various chemokines have pro-tumorigenic actions in cancers, the effects of CXCL16 remain controversial. The aim of this study was to investigate the molecular characteristics of CXCL16-expressing papillary thyroid cancers (PTCs). CXCL16 expressions were significantly higher in PTCs than benign or normal thyroid tissues. In the TCGA dataset for PTCs, a higher CXCL16 expression was associated with M2 macrophage- and angiogenesis-related genes and poor prognostic factors including a higher TNM staging and the BRAFV600E mutation. PTCs with a higher expression of 3-gene panel including CXCL16, AHNAK2, and THBS2 showed poor recurrence-free survivals than that of the lower expression group. Next, shCXCL16 was introduced into BHP10-3SCp cells to deplete the endogenous CXCL16, and then, the cells were subcutaneously injected to athymic mice. Tumors from the BHP10-3SCpshCXCL16 exhibited a delayed tumor growth with decreased numbers of ERG+ endothelial cells and F4/80+ macrophages than those from the BHP10-3SCpcontrol. CXCL16-related genes including AHNAK2 and THBS2 were downregulated in the tumors from the BHP10-3SCpshCXCL16 compared with that from the BHP10-3SCpcontrol. In conclusion, a higher CXCL16 expression was associated with macrophage- and angiogenesis-related genes and aggressive phenotypes in PTC. Targeting CXCL16 may be a good therapeutic strategy for advanced thyroid cancer.
PPARg has critical role in the differentiation of mesenchymal stem cells into adipocytes while suppressing osteoblastic differentiation. We generated transgenic mice that overexpress PPARg specifically in osteoblasts under the control of a 2.3-kb procollagen type 1 promoter (Col.1-PPARg). Bone mineral density (BMD) of 6-to 14-week-old Col.1 À PPARg male mice was 8% to 10% lower than that of their wildtype littermates, whereas no difference was noticed in Col.1-PPARg female mice. Col.1-PPARg male mice exhibited decreased bone volume (45%), trabecular thickness (23%), and trabecular number (27%), with a reciprocal increase in trabecular spacing (51%). Dynamic histomorphometric analysis also revealed that bone-formation rate (42%) and mineral apposition rate (32%) were suppressed significantly in Col.1-PPARg male mice compared with their wild-type littermates. Interestingly, osteoclast number and surface also were decreased by 40% and 58%, respectively, in Col.1-PPARg male mice. In vitro whole-marrow culture for osteoclastogenesis also showed a significant decrease in osteoclast formation (approximately 35%) with the cells from Col.1-PPARg male mice, and OPG/RANKL ratio was reduced in stromal cells from Col.1-PPARg male mice. Although there was no significant difference in BMD in Col.1-PPARg female mice up to 30 weeks, bone loss was accelerated after ovariectomy compared with wild-type female mice (À3.9% versus À6.8% at 12 weeks after ovariectomy, p < .01), indicating that the effects of PPARg overexpression becomes more evident in an estrogen-deprived state in female mice. In conclusion, in vivo osteoblast-specific overexpression of PPARg negatively regulates bone mass in male mice and accelerates estrogen-deficiency-related bone loss in female mice. ß
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