Symphytum officinale augments osteogenesis in human bone marrow-derived mesenchymal stem cells in vitro as they differentiate into osteoblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Mesenchymal stem cells (MSCs) are multipotent stem cells possessing regenerative potential. Symphytum officinale (SO) is a medicinal plant and in homoeopathic literature, believed to accelerate bone healing. AIM OF THE STUDY: This study aimed to determine if homoeopathic doses of SO could augment osteogenesis in MSCs as they differentiate into osteoblasts in vitro. MATERIALS AND METHODS: Bone marrow samples were obtained from patients who underwent bone grafting procedures (n = 15). MSCs were isolated, expanded and characterized by flow cytometry (CD90, CD105). Cytotoxicity of SO was evaluated by MTT assay. Osteogenic differentiation was induced in MSCs with ß-glycerophosphate, ascorbic acid and dexamethasone over 2 weeks. Different homoeopathic doses of SO (MT, 3C, 6C, 12C and 30C) were added to the basic differentiation medium (BDM) and efficiency of MSCs differentiating into osteoblasts were measured by evaluating expression of Osteocalcin using flow cytometry, and alkaline phosphatase activity using ELISA. Gene expression analyses for osteoblast markers (Runx-2, Osteopontin and Osteocalcin) were evaluated in differentiated osteoblasts using qPCR. RESULTS: Flow cytometry (CD90, CD105) detected MSCs isolated from bone marrow (93-98%). MTT assay showed that the selected doses of SO did not induce any cytotoxicity in MSCs (24 hours). The efficiency of osteogenic differentiation (2 weeks) for different doses of Symphytum officinale was determined by flow cytometry (n = 10) for osteoblast marker, Osteocalcin, and most doses of Symphytum officinale enhanced osteogenesis. Interestingly, gene expression analysis for Runx-2 (n = 10), Osteopontin (n = 10), Osteocalcin (n = 10) and alkaline phosphatase activity (n = 8) also showed increased osteogenesis with the addition of Symphytum officinale to BDM, specially mother tincture. CONCLUSIONS: Our findings suggest that homoeopathic dose (specially mother tincture) of Symphytum officinale has the potential to enhance osteogenesis.

Characterization of cancer stem cells from different grades of human colorectal cancer.

Colorectal cancer (CRC) is one of the most common solid tumors worldwide. Recent evidence suggests that a population of cancer cells, called cancer stem cells (CSCs), is responsible for tumor heterogeneity, invasion, metastasis, therapeutic resistance, and recurrence of CRC. The isolation and characterization of CSCs using cell surface markers have been reported previously with varying results. In this study, we investigated a panel of four putative CSC markers, CD44, CD24, CD166, and EpCAM, to define CRC-CSC. Paraffin embedded tissue samples from different grades of primary, untreated CRC were analyzed for the expression of four CSC markers CD44, CD326, CD24, and CD166, using immunohistochemistry. Flow cytometric analysis of CRC-CSC from HT29 (low grade) and HCT116 (high grade) human colorectal cancer cell lines was done. Marker-based isolation of CSC and non-CSC-bulk-tumor cells from HT29 was done using FACS, and tumor sphere assay was performed. There was a statistically significant difference (p < 0.05) in the expression of CD44, CD326, and CD166 between cases and controls. A novel cutoff distribution of CD44 and CD166 was suggested to help for better immunohistochemical analysis of CRC. Higher prevalence of CSC was seen in high-grade CRC as compared to low-grade CRC. Sorted and cultured CD44 + CD166+ cells formed tumor spheres, suggesting that these cells, having properties of self renewal and anchorage independent proliferation, were in fact CSC. Hence, CD44 and CD166 may serve as good CRC-CSC markers when used together with novel cutoff immunohistochemistry (IHC) expression levels.