BackgroundBetter understanding of the signaling pathways that regulate human bone marrow stromal stem cell (hBMSC) differentiation into bone-forming osteoblasts is crucial for their clinical use in regenerative medicine. Chemical biology approaches using small molecules targeting specific signaling pathways are increasingly employed to manipulate stem cell differentiation fate.MethodsWe employed alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin R staining to assess mineralized matrix formation of cultured hBMSCs. Changes in gene expression were assessed using an Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. For in vivo ectopic bone formation experiments, hMSCs were mixed with hydroxyapatite–tricalcium phosphate granules and implanted subcutaneously into the dorsal surface of 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius Red staining were used to detect bone formation in vivo.ResultsWe identified several compounds which inhibited osteoblastic differentiation of hMSCs. In particular, we identified ruxolitinib (INCB018424) (3 μM), an inhibitor of JAK-STAT signaling that inhibited osteoblastic differentiation and matrix mineralization of hMSCs in vitro and reduced ectopic bone formation in vivo. Global gene expression profiling of ruxolitinib-treated cells identified 847 upregulated and 822 downregulated mRNA transcripts, compared to vehicle-treated control cells. Bioinformatic analysis revealed differential regulation of multiple genetic pathways, including TGFβ and insulin signaling, endochondral ossification, and focal adhesion.ConclusionsWe identified ruxolitinib as an important regulator of osteoblast differentiation of hMSCs. It is plausible that inhibition of osteoblast differentiation by ruxolitinib may represent a novel therapeutic strategy for the treatment of pathological conditions caused by accelerated osteoblast differentiation and mineralization.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-1068-x) contains supplementary material, which is available to authorized users.

Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional heterogeneity of hBMSC cultures. In the present study, we characterized in detail two hTERT-BMSC clonal cell populations termed here CL1 and CL2 that represent an opposing phenotype with respect to morphology, markers expression: alkaline phosphatase (ALP) and CD146, and ex vivo differentiation potential. CL1 differentiated readily to osteoblasts, adipocytes, and chondrocytes as shown by expression of lineage specific genes and proteins. Whole genome transcriptome profiling of CL1 versus CL2 revealed enrichment in CL1 of bone-, mineralization-, and skeletal muscle-related genes, for example, ALP, POSTN, IGFBP5 BMP4, and CXCL12. On the other hand, CL2 transcriptome was enriched in immune modulatory genes, for example, CD14, CD99, NOTCH3, CXCL6, CFB, and CFI. Furthermore, gene expression microarray analysis of osteoblast differentiated CL1 versus CL2 showed significant upregulation in CL1 of bone development and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures.

BackgroundNanoparticles are small-scale substances (<100 nm) with unique properties. Therefore, nanoparticles pose complex health risk implications. The objective of this study was to detect whether treatment with quercetin (Qur) and/or arginine (Arg) ameliorated nephrotoxicity induced by two different doses of nano zinc oxide (n-ZnO) particles.MethodZnO nanoparticles were administered orally in two doses (either 600 mg or 1 g/Kg body weight/day for 5 conscutive days) to Wister albino rats. In order to detect the protective effects of the studied antioxidants against n-ZnO induced nepherotoxicity, different biochemical parameters were investigated. Moreover, histopathological examination of kidney tissue was performed.ResultsNano zinc oxide-induced nephrotoxicity was confirmed by the elevation in serum inflammatory markers including: tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6); and C-reactive protein (CRP). Moreover, immunoglobulin (IGg), vascular endothelium growth factor (VEGF), and nitric oxide (NO) were significantly increased in rat serum. Serum urea and creatinine levels were also significantly increased in rats intoxicated with n-ZnO particles compared with the control group. Additionally, a significant decrease in the non-enzymatic antioxidant reduced glutathione (GSH) was shown in kidney tissues and serum glucose levels were increased. These biochemical findings were supported by a histopathological examination of kidney tissues, which showed that in the animals that received a high dose of n-ZnO, numerous kidney glomeruli underwent atrophy and fragmentation. Moreover, the renal tubules showed epithelial desquamation, degeneration and necrosis. Some renal tubules showed casts in their lumina. Severe congestion was also observed in renal interstitium. These effects were dose dependent. Cotreatment of rats with Qur and/or Arg along with n-ZnO significantly improved most of the deviated tested parameters.ConclusionsThe data show that Qur has a beneficial effect against n-ZnO oxidative stress and related vascular complications. Also, its combination with Arg proved to be even more effective in ameliorating nano zinc oxide nephrotoxicity.