In the field of regenerative medicine, numerous potential applications of mesenchymal stem cells (MSCs) can be envisaged, due to their ability to differentiate into a range of tissues on the basis of the substrate on which they grow. With the advances in nanotechnology, carbon nanotubes (CNTs) have been widely explored for use as cell culture substrate in tissue engineering applications. In this study, canine bone marrow-derived MSCs were considered as the cellular model for an in vitro study to elucidate the collective cellular processes, using three different varieties of thin films of functionalized carbon nanotubes (COOH-single-walled CNTs [SWCNTs], COOH-multiwalled CNTs [MWCNTs] and polyethylene glycol [PEG]-SWCNTs), which were spray dried onto preheated cover slips. Cells spread out better on the CNT films, resulting in higher cell surface area and occurrence of filopodia, with parallel orientation of stress fiber bundles. Canine MSCs proliferated at a slower rate on all types of CNT substrates compared to the control, but no decline in cell number was noticed during the study period. Expression of apoptosis-associated genes decreased on the CNT substrates as time progressed. On flow cytometry after AnnexinV-fluorescein isothiocyanate/propidium iodide (PI) staining, total number of apoptotic and necrotic cells remained lower in COOH-functionalized films compared to PEG-functionalized ones. Collectively, these results indicate that COOH-MWCNT substrate provided an environment of low cytotoxicity. Canine MSCs were further induced to differentiate along osteogenic, chondrogenic, and neuronal lineages by culturing under specific differentiation conditions. The cytochemical and immunocytochemical staining results, as well as the expression of the bone marker genes, led us to hypothesize that the COOH-MWCNT substrate acted as a better cue, accelerating the osteogenic differentiation process. However, while chondrogenesis was promoted by COOH-SWCNT, neuronal differentiation was promoted by both COOH-SWNCT and COOH-MWCNT. Taken together, these findings suggest that COOH-functionalized CNTs represent a promising scaffold component for future utilization in the selective differentiation of canine MSCs in regenerative medicine.

Sustained and controlled release of neurotrophic factors in target tissue through nanomaterial based delivery system could be a better strategy for nerve tissue regeneration. The present study aims to prepare the nerve growth factor (NGF) encapsulated chitosan nanoparticles (NGF-CNPs) and its evaluation on neuronal differentiation potentiality of canine bone marrow derived mesenchymal stem cells (cBM-MSCs). The NGF-CNPs were prepared by ionotropic gelation method with tripolyphosphate (TPP) as an ionic cross-linking agent. Observations on physiochemical properties displayed the size of nanoparticles as 80-90 nm with positive zeta potential as well as an ionic interaction between NGF and nanoparticle. NGF loading efficiency was found to be 61% while its sustained release was observed by an in vitro release kinetics study. These nanoparticles were found to be cytocompatible to cBM-MSCs when supplemented at a concentration upto 4 mg/ml in culture media. The NGF-CNP supplemented culture media was able to transdifferentiate the preinduced cBM-MSCs into neurons in a better way than unbound NGF supplementation. Further, it was also noticed that NGF-CNPs were able to transdifferentiate cBM-MSCs without any chemical based preinduction. In conclusion, our findings propose that NGF-CNPs are capable of releasing bioactive NGF with the ability to transdifferentiate mesenchymal stem cells into neurons, suggesting its potential future application in nerve tissue regeneration.

No abstract

Generation of pluripotent stem cells by reprogramming somatic cells of quality animals has numerous potential applications in agricultural and biomedical sciences. Unfortunately, till now, reprogramming of buffalo fetal fibroblast cells (bFFs) has been very ineffient despite intensive efforts. Here, we attempted to enhance reprogramming efficiency by using the HDAC inhibitor valproic acid (VPA) in bFFs transfected with pLentG-KOSM pseudo virus carrying mouse specific pluripotent genes. FACS analysis revealed that VPA treatment significantly increased (p < 0.05) GFP cells in comparison to VPA untreated control. Further, among different concentrations, 1.5 mM VPA was found to be optimal, increasing about 5 fold GFP cells and 2.5-fold GFP+ colonies with significantly (P < 0.05) larger size as compared to control. These colonies were further propagated and characterised. The colonies displayed embryonic stem cell (ESC)-like morphology, normal karyotype, and were positive for alkaline phosphatase staining as well as immune-positive for the ESC specific markers Oct4, Nanog, SSEA1, TRA-1-60 and TRA-1-81. The primary colonies revealed significantly higher (P < 0.05) expression of pluripotent genes than control, which declined gradually on subsequent passages. The reprogrammed cells readily formed embryoid bodies in vitro and cells of all three germ layers. These results indicated that VPA treatment of viral transducted cells can improve the generation of induced pluripotent stem cells and help their long term maintenance in buffalo.

No abstract