Substance P, a Neuropeptide, Promotes Wound Healing via Neurokinin-1 Receptor.

Substance P (SP), an endogenous neuropeptide, mediates intracellular signaling, mainly through a tachykinin receptor. The tachykinin receptors family consists of neurokinin-1 (NK-1), neurokinin-2 (NK-2), and neurokinin-3 receptors. Our previous studies on SP have shown its wound healing potentials. But the exact mechanism of wound healing by SP is not exactly known. In view of this, the present study was aimed at evaluating the in vitro wound healing effect of SP alone and in the presence of NK-1, NK-2, and both receptor antagonists. Scratch assay, transwell assay, and tumor growth factor-beta 1 (TGF-ß1) assay were performed on buffalo fetal fibroblast culture. The cotreatment of fibroblast cultures with SP alone during the 24 h caused the significant proliferation and migrations of cells in both horizontal and vertical directions. The SP in the presence of spantide II (NK-1 antagonist) failed to stimulate this migration. The treatment of cells with SP in the presence of NK-2 antagonist treatment also showed a significant reduction of migration of cells with respect to SP treatment alone. The SP in the presence of both NK-1 and NK-2 antagonists failed to stimulate the horizontal migration of cells and most of the ineffectiveness of SP was observed in this combination. The TGF-ß1 levels were significantly higher in the supernatants of cells that were exposed to SP alone. All other treatments have significantly lower TGF-ß1 levels than SP alone treatment. It is concluded that different actions on fibroblast cells by SP were mainly mediated through the NK-1 receptor.

Valproic acid assisted reprogramming of fibroblasts for generation of pluripotent stem cells in buffalo (Bubalus bubalis).

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.

Reprogramming of buffalo (Bubalus bubalis) foetal fibroblasts with avian egg extract for generation of pluripotent stem cells.

An attempt was made to study the efficiency of chicken (Gallus gallus domesticus) egg extract (EE) to reprogram buffalo (Bubalus bubalis) foetal fibroblasts (bFFs) without incorporation of ectopic transcription factors. The isolated bFFs were cultured in media supplemented with 2%, 4%, 6% and 10% EE to induce reprogramming. It was observed that fewer but larger sized alkaline phosphatase positive (AP(+ve)) colonies developed in culture system containing 2% EE whereas, more but smaller sized colonies developed in 4%, 6% and 10% EE. The developed colonies expressed pluripotency markers like Oct4, Nanog, SSEA1, TRA-1-60, TRA-1-81 and RT-PCR study revealed relative expression of genes indicating pluripotency (Oct4, Sox2, Nanog and FoxD3) increased as the concentration of EE increased in culture systems confirming the reprogramming capability of chicken EE.