Balamuthu Kadalmani

Comparative investigation of the differentiation capability of bone-marrow- and adipose-derived mesenchymal stem cells by qualitative and quantitative analysis

Mesenchymal stem cells (MSCs) hold promise for cell-based therapy in regenerative medicine. To date, MSCs have been obtained from conventional bone marrow via a highly invasive procedure. Therefore, MSCs are now also isolated from sources such as adipose tissue, cord blood and cord stroma, a subject of growing interest. As the characterization and differentiation potential of adipose-derived MSCs (AD-MSCs) and bone-marrow-derived MSCs (BM-MSCs) have not been documented, we have evaluated and compared the characteristics of both MSC types by qualitative and quantitative analyses. Both cell types show similar morphology and surface protein expression, being positive for stromal-associated markers and negative for hematopoietic and endothelial markers. The colony-forming potential of AD-MSCs is distinctly higher than that of BM-MSCs. Nonetheless, similar adipogenic and osteogenic differentiation is observed in both groups of MSCs. Cytochemical qualitative analysis and calcium mineralization demonstrate higher levels toward osteogenic differentiation in BM-MSCs than in AD-MSCs. On the contrary, the percentage of Nile red oil staining for differentiated adipocytes is higher in AD-MSCs than in BM-MSCs. Quantitative real-time polymerase chain reaction shows similar patterns of osteogenic- and adipogenic-associated gene expression in both cell types. Each of the MSCs respond in functional analysis by exhibiting unique properties at the differentiation level according to their micro-environmental niche. Thus, quantitative analysis might be a valuable means of describing stem cell multipotency, in addition to qualitative investigation.

In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

BackgroundMultipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs) possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs) and adult dermal skin (hADSSCs) using explants cultures and were compared with bone marrow (hMSC-TERT) and adipose tissue-derived mesenchymal stem cells (hADMSCs) for their potential differentiation into osteoblasts, adipocytes, and endothelial cells.ResultsConcordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC), with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA) was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues.ConclusionsOur data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs represents a novel source of stem/stromal cells for tissue regeneration and the vascularization of engineered tissues. Moreover, the CD146 investigations suggested that the microenvironmental niche might contribute to direct stromal cells multipotency toward certain lineages, which warrants further investigation.

Skin-derived multipotent stromal cells – an archrival for mesenchymal stem cells

Progenitor stem cells have been identified, isolated and characterized in numerous tissues and organs. However, their therapeutic potential and the use of these stem cells remain elusive except for a few progenitor cells from bone marrow, umbilical cord blood, eyes and dental pulp. The use of bone marrow-derived hematopoietic stem cells (HSC) or mesenchymal stem cells (MSCs) is restricted due to their extreme invasive procedures, low differentiation potential with age and rejection. Thus, we need a clinical grade alternative to progenitor stem cells with a high potential to differentiate, naïve and is relatively easy in in vitro propagation. In this review, we summarize cell populations of adherent and floating spheres derived from different origins of skin, or correctly foreskin, by enzymatic digestion compared with established MSCs. The morphology, phenotype, differentiation capability and immunosuppressive property of the adherent cell populations are comparable with MSCs. Serum-free cultured floating spheres have limited mesodermal but higher neurogenic differentation potential, analogous to neural crest stem cells. Both the populations confirmed their plethora potential in in vitro. Together, it may be noted that the skin-derived adherent cell populations and floating cells can be good alternative sources of progenitor cells especially in cosmetic, plastic and sports regenerative medicine.

Verrucarin A induces apoptosis through ROS-mediated EGFR/MAPK/Akt signaling pathways in MDA-MB-231 breast cancer cells.

The present study was carried out to elucidate the mechanisms underlying Verrucarin A (VA)‐induced cytotoxicity in human breast cancer cell line MDA‐MB‐231. VA inhibited the growth of MDA‐MB‐231 cells by induction of reactive oxygen species (ROS)‐dependent mitochondrial apoptosis. Elevation of ROS production, associated with changes in Bax/Bcl‐2 ratio, led to loss of mitochondrial membrane potential (Δψm) and cytochrome c release in VA‐treated cells. Release of cytochrome c from mitochondria to cytosol triggered activation of caspase‐3, PARP cleavage, DNA fragmentation, and finally apoptotic cell death. Furthermore, VA‐induced apoptosis was accompanied by the activation of p38MAPK and inhibition of phosphorylation of EGFR as well as of Akt and ERK1/2. However, pre‐treatment with n‐acetyl cysteine, an ROS scavenger, and SB202190, a p38MAPK inhibitor, significantly inhibited VA‐induced ROS generation, EGFR inhibition, p38MAPK activation and apoptosis. Moreover, pharmacological inhibition of EGFR and ERK1/2 significantly accelerated the VA‐induced apoptosis in MDA‐MB‐231 cells. Collectively, these results indicate that VA‐induces ROS elevation in cancer cells, which results in the activation of p38MAPK and inhibition of EGFR/Akt/ERK signaling cascade and, ultimately, cancer cell death. J. Cell. Biochem. 115: 2022–2032, 2014.

Curative property of Withania somnifera Dunal root in the context of carbendazim-induced histopathological changes in the liver and kidney of rat.

The liver and kidney of rat underwent severe histopathological lesions when treated with a single bolus dose of carbendazim, a fungicide, particularly affecting the hepatocytes and the renal corpuscles, respectively. The effects appear to be manifestations of the microtubule-disrupting activity of carbendazim. Treatment of carbendazim-treated rats with the powder of tuberous root of Withania somnifera (Ashwagandha) for 48 days resulted in complete cure of these organs. The results indicate that Withania somnifera would be an effective curative for carbendazim-induced histopathological changes in the liver and kidney.

Verrucarin A, a protein synthesis inhibitor, induces growth inhibition and apoptosis in breast cancer cell lines MDA-MB-231 and T47D.

Verrucarin A (VA), a protein synthesis inhibitor, derived from the pathogen fungus Myrothecium verrucaria, inhibits growth of leukemia cell lines and activates caspases and apoptosis and inflammatory signaling in macrophages. We have investigated VA-induced growth inhibition in breast cancer cells MDA-MB-231 and T47D and, particularly, the mechanism of VA-induced apoptosis. VA treatment brought about apoptotic cell death in a dose- and time-dependent manner which was associated with chromatin condensation, cell shrinkage, nuclear fragmentation and intracellular ROS production. Mitochondrial membrane depolarization, activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax and p53 expression were observed. VA thus affects the viability of both the breast cancer cells by triggering ROS-mediated intrinsic mechanism of apoptosis.

Apolipoprotein E Induction in Syrian Hamster Testis Following Tributyltin Exposure A Potential Mechanism of Male Infertility

Tributyltin (TBT) is a common environmental contaminant used as the active ingredient in many products such as a biocides, wood preservatives, disinfecting agents, and antifouling paints. The TBT is a known endocrine disruptor. The aim of the current investigation was to determine the toxicity of TBT in the reproductive tract of adult male Syrian hamsters and to ascertain whether this compound results in untoward effects on apolipoprotein E (ApoE), a lipoprotein central to sex hormone synthesis. The TBT was administered orally to male Syrian hamsters at doses of 50, 100, and 150 ppm/kg for 65 days of treatment. We determined body weight, testis weight, sperm count, sperm morphology, testis histology, ApoE expression, serum lipid profile, testosterone level, follicle-stimulating hormone receptor (FSHR), and steroid hormone receptor expression compared to vehicle-treated controls. High doses of TBT significantly affected each of these parameters in Syrian hamsters. Weight and morphology of the testis were altered as well as sperm production. Real-time reverse-transcriptase polymerase chain reaction analysis revealed that expression of ApoE messenger RNA was upregulated in testes from TBT-treated groups compared with controls while the expression of androgen receptor, FSHR, estrogen receptor α (ESR1), and estrogen receptor β (ESR2) was decreased. We posit that exposure to TBT hinders intracellular cholesterol transport resulting in abnormal sex steroid biosynthesis and subsequent spermatogenic defects. Importantly, these effects may account for the decreased level of normal sperm observed in hamsters exposed to TBT.

Molecular mechanisms of tributyltin-induced alterations in cholesterol homeostasis and steroidogenesis in hamster testis: In vivo and in vitro studies

Tributyltins (TBT) are ubiquitous and persistent environmental contaminants that disturb normal endocrine function including gonadal function in humans and marine organisms. TBT was administered through oral route to male Syrian hamsters at daily doses of 50, 100, and 150 ppm/kg for 65 days. Changes in testis morphology, immunohistochemistry of iNOS, 3β‐HSD, and 17β‐HSD, cholesterol transport receptor, nuclear receptors, and transcription factors were analyzed. TBT treatment affected each of these parameters to significant levels in a dose‐dependent manner compared to vehicle treated control. Real‐time PCR and protein analyses revealed that expression levels of ApoE and LDL‐R mRNA were up‐regulated in the testis of TBT‐treated animals while the expression levels of SR‐B1, LXR, PPARs α, β, and γ, SCAP, SREBP 1 and 2, 3β‐HSD, 17β‐HSD, CYP17A1, and P450SCC were down‐regulated. Leydig cells were isolated and separated adopting percoll gradient centrifugation under aseptic condition. The viability of Leydig cell was affected by TBT treatment in a dose‐ and time‐dependent manner. Further, the mechanism of action of TBT was ascertained by siRNA transfection of ApoE, which was upregulated, and SREBP, which was down‐regulated. These observations led us to infer that exposure to TBT hinders intracellular cholesterol transport resulting in abnormal sex steroid biosynthesis and alteration of steroidogenic enzyme activities. Finally, we could recognize ApoE and SREBP as the major factors regulating genes that control cholesterol biosynthesis and steroidogenesis that ultimately inhibit the synthesis of testosterone. Therefore, ApoE is one of the important molecular targets that can be intercepted in context of male infertility/male contraception.

Antiproliferative and apoptosis-induction studies of 5-hydroxy 3′,4′,7-trimethoxyflavone in human breast cancer cells MCF-7: an in vitro and in silico approach

Abstract The aim of this study was to find the efficacy of 5-hydroxy 3′,4′,7-trimethoxyflavone (HTMF), a flavonoid compound isolated from the medicinal plant Lippia nodiflora, in inhibiting the proliferation and inducing apoptosis in human breast cancer cell line MCF-7. The anti-proliferative effect of the compound HTMF was confirmed using MTT cytotoxicity assay. Increased apoptotic induction by HTMF was demonstrated by acridine orange/ethidium bromide (AO/EtBr) and Hoechst 33258 staining studies. The phosphatidylserine translocation, an early feature of apoptosis and DNA damage were revealed through AnnexinV-Cy3 staining and comet assay. Moreover, the significant elevation of cellular ROS was observed in the treated cells, as measured by 2,7-diacetyl dichlorofluorescein (DCFH-DA). The mRNA expression studies also supported the effectiveness of HTMF by shifting the Bax:Bcl-2 ratio. The treatment of MCF-7 cells with HTMF encouraged apoptosis through the modulation of apoptotic markers, such as p53, Bcl-2, Bax, and cleaved PARP. In silico molecular docking and dynamics studies with MDM2-p53 protein revealed that HTMF was more potent compound that could inhibit the binding of MDM2 with p53 and, therefore, could trigger apoptosis in cancer cell. Overall, this study brings up scientific evidence for the efficacy of HTMF against MCF-7 breast cancer cells. Graphical Abstract

Atypical expression of COX-2, StAR, CYP19A1 and apoptotic regulators in CD90 positive endometrial stromal cells from women with endometriosis

Purpose Endometriosis is an invasive gynecologic disease characterized by diminished apoptosis, sustained ectopic survival of dysfunctional endometrial cells and implantation of endometriotic lesions outside of the uterus. The purpose of this investigation was to determine the expression pattern of apoptotic and steroidogenic genes in the progenitor stem cell population of eutopic tissues from women with endometriosis and compare them to controls. The expression of these genes was determined in a subpopulation of endometrial cells that displayed CD90 positivity (CD+ve90) and colony forming capacity. Methods We obtained endometrial samples from women with or without endometriosis. Endometrial stromal cells (ESCs) were isolated and cultured for 15 days. Purified ESCs were sorted by using a multipotent mesenchymal stromal cell multi-color flow cytometry kit. Single cell cloning was performed by serial dilution in 96-micro well plates. Fifteen days later, colonies were identified (CFUs). The colonies were chosen and cultured. mRNA expression of apoptotic genes, mitogen activated kinase 14 (MAPK14), nuclear factor kappa B (NFkB), steroidogenic acute regulatory protein (StAR), aromatase (CYP19A1) and cyclo-oxygenase-2 (COX-2) were determined by qRT-PCR. Protein levels of StAR, CYP19A1 and COX-2 were determined by western blotting. Results A subset of stromal cells derived from women with endometriosis were isolated and identified as progenitor stem cells based on their CD90 positivity and colony forming ability. The cells displayed increased levels of MAPK14, NFkB, COX-2, StAR and CYP19A1 both at the mRNA and protein level compared to stromal cells derived from controls. Similarly, pro-apoptotic molecules such as Bax were decreased whereas anti-apoptotic molecules such as Bcl2 were decreased at the mRNA level compared to stromal cells derived from controls. Conclusions CD90+ve ESCs derived from women with endometriosis displayed markers suggesting stem cell-like properties and aberrant expression of apoptotic and steroidogenic enzymes which may contribute to sustained survival of these cells.