METHOD: DPSC from murine incisors were isolated through either the outgrowth (DPSC-OG) or the enzymatic digestion (DPSC-ED) method. Cells at passage 4 were used in this study. The cells were characterized through morphology and expression of cell surface markers. The cells' doubling time when cultured using different seeding densities was calculated and analyzed using one-way ANOVA and Tukey's multiple comparison post-test. The ability of cells to differentiate to chondrocyte and osteoblast was evaluated through staining and analysis on the matrices secreted.
RESULTS: Gene expression analysis showed that DPSC-OG and DPSC-ED expressed dental pulp mesenchymal stem cell markers, but not hematopoietic stem cell markers. The least number of cells that could have been used to culture DPSC-OG and DPSC-ED with the shortest doubling time was 5 × 10(2) cells/cm(2) (11.49 ± 2.16 h) and 1 × 10(2) cells/cm(2) (10.55 h ± 0.50), respectively. Chondrocytes differentiated from DPSC-ED produced 2 times more proteoglycan and at a faster rate than DPSC-OG. FTIR revealed that DPSC-ED differentiated into osteoblast also secreted matrix, which more resembled a calvaria.
DISCUSSION: Isolation approaches might have influenced the cell populations obtained. This, in turn, resulted in cells with different proliferation and differentiation capability. While both DPSC-OG and DPSC-ED expressed mesenchymal stem cell markers, the percentage of cells carrying each marker might have differed between the two methods. Regardless, enzymatic digestion clearly yielded cells with better characteristics than outgrowth.
Methods: Human ALDH1+ BCSCs were grown in serum-free Dulbecco's Modified Eagle Medium (DMEM)/F12, while MCF-7 and MDA-MB-231 were cultured in DMEM supplemented with 10% foetal bovine serum under standard conditions. Total RNA was extracted using the Tripure Isolation Reagent. The relative mRNA expressions of OCT4, ALDH1A1 and CD44 associated with stemness as well as TGF-β1, TβR1, ERα1 and MnSOD associated with aggressiveness in BCSCs and MCF-7 cells were determined using the quantitative real-time PCR (qRT-PCR).
Results: The mRNA expressions of OCT4 (5.19-fold ± 0.338; P = 0.001), ALDH1A1 (3.67-fold ± 0.523; P = 0.006), CD44 (2.65-fold ± 0.307; P = 0.006), TGF-β1 (22.89-fold ± 6.840; P = 0.015), TβR1 (3.74-fold ± 1.446; P = 0.045) and MnSOD (4.6-fold ± 1.096; P = 0.014) were higher in BCSCs than in MCF-7 but were almost similar to MDA-MB-231 cells. In contrast, the ERα1 expression of BCSCs (0.97-fold ± 0.080; P = 0.392) was similar to MCF-7 cells, indicating that BSCSs are oestrogen-dependent breast cancer cells.
Conclusion: The oestrogen-dependent BCSCs express stemness and aggressiveness genes at a higher level compared to oestrogen-dependent MCF-7 but are almost similar to oestrogen-independent MDA-MB-231 cells.
OBJECTIVE: The present study aims to investigate the effects of a standardized raw extract of C. asiatica (RECA) on hydrogen peroxide (H2O2)-induced oxidative stress and apoptotic death in neural-like cells derived from mouse embryonic stem (ES) cell line.
METHODS: A transgenic mouse ES cell (46C) was differentiated into neural-like cells using 4-/4+ protocol with addition of all-trans retinoic acid. These cells were then exposed to H2O2 for 24 h. The effects of RECA on H2O2-induced neural-like cells were assessed through cell viability, apoptosis, and reactive oxygen species (ROS) assays, as well as neurite length measurement. The gene expression levels of neuronal-specific and antioxidant markers were assessed by RT-qPCR analysis.
RESULTS: Pre-treatment with H2O2 for 24 hours, in a dose-dependent manner, damaged neural-like cells as marked by a decrease in cell viability, substantial increase in intracellular ROS accumulation, and increase in apoptotic rate compared to untreated cells. These cells were used to treat with RECA. Treatment with RECA for 48 h remarkably restored cell survival and promoted neurite outgrowth in the H2O2- damaged neurons by increasing cell viability and decreasing ROS activity. RT-qPCR analysis revealed that RECA upregulated the level of antioxidant genes such as thioredoxin-1 (Trx-1) and heme oxygenase-1 (HO-1) of treated cells, as well as the expression level of neuronal-specific markers such as Tuj1 and MAP2 genes, suggesting their contribution in neuritogenic effect.
CONCLUSION: Our findings indicate that RECA promotes neuroregenerative effects and exhibits antioxidant properties, suggesting a valuable synergistic activity of its phytochemical constituents, thus, making the extract a promising candidate in preventing or treating oxidative stress-associated Alzheimer's disease.
METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5 mls single doses of 2 × 107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography.
RESULTS: Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; p = .001. Immunocytochemistry was positive for these chondrogenic markers. 12 months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; P = .001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance.
CONCLUSIONS: Both ADSCs and BMSCs had increased chondrogenic gene expressions using TGF-β3 and BMP-6. The treated knees had improved cartilage scores; PKH26 can provide elongated tracking, while EMG results revealed improved joint recoveries. These could be suitable therapies for osteoarthritis.
Methods: BMuc were subjected to 10 d of induction factors to investigate the potential of cells to differentiate into corneal lineages.
Results: Corneal stem cell markers β1-integrin, C/EBPδ, ABCG2, p63, and CK3 were upregulated in the gene expression analysis in induced BMuc, whereas CK3 and p63 showed significant protein expression in induced BMuc compared to the uninduced cells. BMuc were then left to reach 80% confluency after differential trypsinization. The cells were harvested and cultivated on a commercially available untreated air-dried amniotic membrane (AM) in a Transwell system in induction medium. The corneal constructs were fabricated and then implanted into damaged rat corneas for up to 8 weeks. A significant improvement was detected in the treatment group at 8 weeks post-implantation, as revealed by slit lamp biomicroscopy analysis. The structure and thickness of the corneal layer were also analyzed using histological staining and time-domain optical coherence tomography scans and were found to resemble a native corneal layer. The protein expression for CK3 and p63 were continuously detected throughout the corneal epithelial layer in the corneal construct.
Conclusions: In conclusion, human BMuc can be induced to express a corneal epithelial-like phenotype. The addition of BMuc improves corneal clarity, prevents vascularization, increases corneal thickness and stromal alignment, and appears to have no adverse effect on the host after implantation.