Exploration of immunomodulatory mechanism of caprine Wharton's jelly derived mesenchymal stem cells.

The present study was aimed to explore the possible mechanisms by which caprine Wharton's jelly-derived MSCs (WJ-MSCs) perform their immunomodulatory function. WJ-MSCs were isolated through explants culture and characterized as per ISCT criteria using culture behavior, expression of surface markers by PCR, FACS and immunocytochemical localization (ICC), trilineage differentiation potential etc. Secretory behavior for important biomolecules (IDO, TGFß1, VEGF, IL6) was evaluated by ICC and western blot assay. Cell-to-cell communication was studied by culturing cells in cell-cell contact and trans-well system. The MSCs when co-cultured with activated Tc and Th cells, down-regulation of T cell cytokine as well as upregulation of immunomodulatory factors (VEGF A, IL10, IL6, IDO, iNOS, PTGS2, HGF, TGFß, CXCL10, CXCL11) was noticed in both cell-cell contact and trans-well culture system which was significantly higher in cell-cell contact system. Trilineage differentiation of MSCs showed significant upregulation of MHC I (CAHI) and MHC II (CLA DRB3) molecules suggesting better clinical applications of MSCs without differentiation to avoid immune rejection. It can be concluded that WJ-MSCs perform their immunomodulation through the secretion of a battery of biomolecules and work in both cell-cell contact manner and through their secretome.

Isolation and propagation of classical swine fever virus in porcine Wharton's Jelly mesenchymal stem cells.

Classical Swine Fever (CSF) is an extremely infectious and deadly disease of pigs and wild boars caused by the CSF virus (CSFV) which is a member of the Pestivirus genus and the family Flaviviridae. This study was designed to detect the permissibility and replication of CSFV in mesenchymal stem cells (MSCs) monolayer derived from Porcine Wharton's jelly. Porcine Wharton's jelly MSCs (pWJ-MSCs) were ex vivo expanded and propagated for more than 81 generations and third passage pWJ-MSCs were characterized as per standard criteria i.e., growth characteristics, trilineage differentiation potential and molecular characterization for pluripotency and stem cell surface markers. Porcine WJ tissue samples found negative for CSFV by RT-PCR test were processed further for the isolation of pWJ-MSCs and CSFV was propagated over the characterized pWJ-MSCs monolayer. No cytopathic effect was observed, which was consistent with non-cytopathic nature of CSFV. The replication of CSFV in pWJ-MSCs was affirmed by RT-PCR and demonstration of viral antigen in the cytoplasm of virus infected cells by immuno-staining technique. In total, three different CSFV isolates were propagated in pWJ-MSCs. Primary pWJ-MSCs permitted CSFV replication to good titer. To the best of our information, this is the first ever report of isolation of CSFV in pWJ-MSCs.

Comparative analysis of the immunomodulatory potential of caprine fetal adnexa derived mesenchymal stem cells.

The caprine mesenchymal stem cells (MSCs) derived from fetal adnexa are highly proliferative. These cells possess tri-lineage differentiation potential and express MSC surface antigens and pluripotency markers with a wound-healing potential. This present study was conducted to compare the immunomodulatory potential of caprine MSCs derived from the fetal adnexa. Mid-gestation caprine uteri (2-3 months) were collected from the abattoir to isolate MSCs from amniotic fluid (cAF), amniotic sac (cAS), Wharton's jelly (cWJ) and cord blood (cCB), which were expanded and characterized at the 3rd passage. These MSCs were then stimulated with inflammatory cytokines (IFN-γ and TNF-α) to assess the percentage of inhibition produced on peripheral blood mononuclear cells (PBMCs) proliferation. The percentage of inhibition on activated PBMCs proliferation produced by cWJ MSCs and cAS MSCs was significantly higher than cCB and cAF MSCs. The relative mRNA expression profile and immunofluorescent localization of different immunomodulatory cytokines and growth factors were conducted upon stimulation. The mRNA expression profile of a set of different cytokines and growth factors in each caprine fetal adnexa MSCs were modulated. Indoleamine 2, 3 dioxygenase appeared to be the major immunomodulator in cWJ, cAF, and cCB MSCs whereas inducible nitric oxide synthase in cAS MSCs. This study suggests that caprine MSCs derived from fetal adnexa display variable immunomodulatory potential, which appears to be modulated by different molecules among sources.

Mesenchymal stem cell-conditioned media: A novel alternative of stem cell therapy for quality wound healing.

Mesenchymal stem cells-conditioned media (MSCs-CM) contains several growth factors and cytokines, thus may be used as a better alternative to stem cell therapy, which needs to be elucidated. The present study was conducted to evaluate the therapeutic potential of caprine, canine, and guinea pig bone marrow-derived MSCs-CM in excision wound healing in a guinea pig model. MSCs were obtained from bone marrow, expanded ex vivo and characterized as per ISCT criteria. CM was collected assayed by western blot to ascertain the presence of important secretory biomolecules. Quantitative estimation by enzyme-linked immunosorbent assay was done for a vascular epidermal growth factor (VEGF) and interleukin-6 (IL-6) in caprine MSCs-CM and optimum time for collection of CM was decided as 72 hr. CM from all the species was lyophilized by freeze-drying method. Full-thickness (2 × 2 cm2 ) excision skin wounds were created in guinea pigs (six animals in each group) and respective lyophilized CM mixed with laminin gel was applied topically at weekly interval. On Day 28, histopathological examinations of healed skin were done by hemotoxylin and eosin staining. MSCs were found to secrete important growth factors and cytokines (i.e., VEGF, transforming growth factor-ß1, fibroblast growth factor-2, insulin-like growth factor-1, stem cell factor, and IL-6) as demonstrated by immunohistochemistry and western blot assay. It was found that allogenic and xenogenic application of CM significantly improved quality wound healing with minimal scar formation. Thus, MSCs-CM can be used allogenically as well as xenogenically for quality wound healing.

An allogenic therapeutic strategy for canine spinal cord injury using mesenchymal stem cells.

This study was conducted to characterize canine bone marrow-derived mesenchymal stem cells (BMSCs); in vivo tracking in mice, and therapeutic evaluation in canine clinical paraplegia cases. Canine BMSCs were isolated, cultured, and characterized in vitro as per International Society for Cellular Therapy criteria, and successfully differentiated to chondrogenic, osteogenic, and adipogenic lineages. To demonstrate the homing property, the pGL4.51 vector that contained luciferase reporter gene was used to transfect BMSCs. Successfully transfected cells were injected around the skin wound in mice and in vivo imaging was done at 6, 12 and 24 hr post MSCs delivery. In vivo imaging revealed that transfected BMSCs migrated and concentrated predominantly toward the center of the wound. BMSCs were further evaluated for allogenic therapeutic potential in 44 clinical cases of spinal cord injuries (SCI) and compared with conventional therapy (control). Therapeutic potential as evaluated by different body reflexes and recovery score depicted significantly better results in stem cell-treated group compared to control group. In conclusion, allogenic canine BMSCs can serve as potent therapeutic candidate in cell-based therapies, especially for diseases like SCI, where the conventional medication is not so promising.

Impact of l-carnitine on lipid content and post thaw survivability of buffalo embryos produced in vitro.

The aim of the present study was to see the impact of L-Carnitine (LC) on lipid biosynthesis and metabolism of buffalo embryos, and post thaw blastocyst survivability. In vitro fertilized (IVF) embryos were derived from slaughterhouse derived COCs and cultured in different doses of LC i.e. 0, 1 mM, 1.5 mM, 2 mM starting at 48 h post IVF. Blastocyst rate was significantly (p < 0.05) higher in 1.5 mM group than control and 1.0 mM group. Lipid content was measured indirectly by fluorescent intensity of lipid droplets after Nile red staining, and it was lower (p < 0.05) in treated than control groups. CPT1B, DGAT2 and DGAT1 mRNA expression was up regulated (p < 0.05) while AMPKg1 expression was down regulated in 1.5 mM and 2 mM groups compared to other groups (p < 0.05). mRNA expression of GLUT1, OCT4 and IFN-tau was higher (P < 0.05) in 1.5 mM group than the control group. Expression of BAX was down regulated at 1.5 mM LC. Blastocyts were vitrified by a modified OPS method and post thaw survivability of blastocysts was higher (P < 0.05) in 1.5 mM LC than other groups. In post thaw blastocysts, mRNA expression of GLUT1, OCT4 and IFN-tau was higher (P < 0.05) in 1.5 mM than other groups. Thus, it can be concluded that supplementation of l-carnitine (1.5 mM) in embryo culture media improved the quality of buffalo embryo production and post thaw blastocysts survivability by reducing fatty acid synthesis, enhancing fatty acid metabolism, and reducing lipid droplet formation.

Effect of roscovitine on developmental competence of small follicle-derived buffalo oocytes.

Background & objectives: The lower recovery of competent oocytes in buffalo species limits the commercialization of in vitro embryo production technology in field condition. In this context, pre-maturation of small follicle (SF)-derived oocytes with meiotic inhibition may be a promising alternative to obtain more number of competent oocytes. Thus, the present study was conducted with an objective to enhance the developmental potential of less competent SF-derived buffalo oocytes. Methods: All the visible follicles (used for aspiration) from buffalo ovaries were divided into two categories: large follicle (LF) (follicles having diameter ≥6 mm) and SF (follicles of diameter <6 mm). The competence of LF and SF oocytes was observed in terms of brilliant cresyl blue (BCB) staining, cleavage rate, blastocyst rate and relative gene expression of oocyte and blastocyst competence markers. Thereafter, less competent SF oocytes were treated with 0, 12.5, 25, 50 and 100 mM doses of roscovitine (cyclin-dependent kinase inhibitor) to enhance their developmental potential. Results: Based on parameters studied, LF oocytes were found to be more competent than SF oocytes. Pre-maturation incubation of SF oocytes with roscovitine reversibly arrested oocyte maturation for 24 h to ensure the proper maturation of less competent oocytes. A significantly higher number of BCB-positive oocytes were noted in roscovitine-treated group than SF group. Cleavage and blastocyst rates were also higher in roscovitine-treated group. The relative messenger RNA expression of oocyte (GDF9, BMP15, GREM1, EGFR, PTGS2 and HAS2) as well as blastocyst (INF-τ, GLUT1 and POU5F1) competence markers was significantly greater in roscovitine-treated group relative to SF group. Again, on comparison with LF group, these parameters depicted a lower value in the treatment group. Interpretation & conclusions: The findings of this study has revealed that pre-maturation incubation of SF-derived oocytes with 25 µM roscovitine can improve its developmental competence and thus can be utilized to get maximum number of competent oocytes for better commercialization of in vitro embryo production technology in buffalo.

Impact of Cryopreservation on Caprine Fetal Adnexa Derived Stem Cells and Its Evaluation for Growth Kinetics, Phenotypic Characterization, and Wound Healing Potential in Xenogenic Rat Model.

This study was conducted to know the impact of cryopreservation on caprine fetal adnexa derived mesenchymal stem cells (MSCs) on the basic stem cell characteristics. Gravid caprine uteri (2-3 months) were collected from local abattoir to derive (amniotic fluid [cAF], amniotic sac [cAS], Wharton's jelly [cWJ], and cord blood [cCB]) MSCs and expanded in vitro. Cells were cryopreserved at 3rd passage (P3) using 10% DMSO. Post-thaw viability and cellular properties were assessed. Cells were expanded to determine growth kinetics, tri-lineage differentiation, localization, and molecular expression of MSCs and pluripotency markers; thereafter, these cells were transplanted in the full-thickness (2 × 2cm2 ) rat skin wound to determine their wound healing potential. The post-thaw (pt) growth kinetics study suggested that cWJ MSCs expanded more rapidly with faster population doubling time (PDT) than that of other fetal adnexa MSCs. The relative mRNA expression of surface antigens (CD73, CD90, and CD 105) and pluripotency markers (Oct4, KLF, and cMyc) was higher in cWJ MSCs in comparison to cAS, cAF, and cCB MSCs post-thaw. The percent wound contraction on 7th day was more than 50% for all the MSC-treated groups (pre and post-thaw), against 39.55% in the control group. On day 28th, 99% and more wound contraction was observed in cAF, cAF-pt, cAS-pt, cWJ, cWJ-pt, and cCB, MSCs with better scores for epithelization, neovascularization, and collagen characteristics at a non-significant level. It is concluded that these MSCs could be successfully cryopreserved without altering their stemness and wound healing properties. J. Cell. Physiol. 232: 2186-2200, 2017. © 2016 Wiley Periodicals, Inc.

Impact of oocyte-secreted factors on its developmental competence in buffalo.

Oocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus-oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.

Effect of cryopreservation on therapeutic potential of canine bone marrow derived mesenchymal stem cells augmented mesh scaffold for wound healing in guinea pig.

The objective of this study was to compare the therapeutic potential of canine bone marrow derived mesenchymal stem cells (BM MSCs) augmented mesh scaffold for wound healing potential in guinea pig before and after cryopreservation. Bone marrow aspirate was obtained from healthy dogs and culture was expanded in vitro. MSCs augmented mesh scaffold were cryopreserved for 30 days and then used for therapeutic purposes. Both fresh and frozen thaw MSCs augmented mesh scaffold along with fresh MSCs were used for therapeutic purposes in guinea pig. No significant (P > 0.05) difference was observed in population doubling time (PDT) among fresh and frozen thawed BM MSCs. Both fresh and frozen thawed BM MSCs expressed cell surface markers (CD73, CD90, and CD105), and did not express CD34 as was confirmed by Immunocytochemistry and Real-Time Polymerase Chain Reaction. The fresh and frozen thawed BM MSCs successfully differentiated into osteogenic, chondrogenic and adipogenic lineages. Therapeutic results revealed that the percent wound contraction on day 14 was more than 65 % for the mesh augmented with MSCs as well as freshly injected MSCs group as against 33-34 % in the control group. Healed wound quality parameters viz. surface epithelium, neovascularization, and collagen characteristics were better for the mesh augmented with MSCs as well as freshly injected MSCs group compared to the control group. No significant difference was noted among fresh and frozen thawed BM MSCs group and fresh MSCs injected group. Thus, it is concluded from this study that canine BM MSCs augmented mesh scaffold both fresh and frozen thaw can be used for quality wound healing.

Profile of patients presenting with sustained ventricular tachycardia in a tertiary care center.

BACKGROUND AND AIM: Ventricular tachycardia (VT) represents the most frequent cause of sudden cardiac death. Information on clinical characteristics, acute management and outcome of patients with sustained VT is limited in our part of world. The aim of this study was to analyze the demography, hemodynamics, ECG features, underlying disease, mode of termination and outcome of patients presenting with VT. METHODS: This single center cohort study represents total of 107 patients of VT enrolled over 45 months. RESULTS: Mean age was 45 years and 59 of the patients were males. Thirty three of these patients were hemodynamically unstable (31%) and 74 were stable (69%) Coronary artery disease was the most common etiological factor accounting for 39% of patients followed by non-ischemic cardiomyopathy. Determinants of hemodynamic instability were VT in course of acute myocardial infarction (8 out of fourteen) and polymorphic pattern of VT (13 out of 26). Spontaneous termination of VT occurred in seven patients, antiarrythmic drugs terminated VT in 53 of 67 patients and in remaining 45patients VT was terminated with direct current (DC) cardioversion. Total of twenty three patients died during the hospital stay. Factors that contributed to mortality were old age, hemodynamic instability and low ejection fraction. CONCLUSION: Ischemic heart disease remains the leading cause of VT. Hemodynamically unstable VT occurs more frequently in acute myocardial infarction and polymorphic VT. Most effective method of VT termination is DC cardioversion. Old age, hemodynamic instability and ejection fraction contribute to overall mortality in VT.

Insertion/deletion polymorphism of ACE gene in females with peripartum cardiomyopathy: A case-control study.

BACKGROUND: The role of polymorphism of Angiotensin converting enzyme (ACE) gene and ACE activity in etiopathogenesis, prognosis, and many other clinical parameters in the various form of the cardiovascular disease has been established to some degree of certainty. The pathophysiology of Peripartum cardiomyopathy (PPCM) remains an area of active research. The main aim of our study was to see pattern of ACE- Insertion/Deletion (I/D) allele in PPCM and its implications on left ventricular performance indices. METHODS: This single-center case-control study included 45 cases and 70 controls. The diagnosis of PPCM was established clinically and echocardiographically. ACE genotyping was done by polymerase chain reaction (PCR) method in all subjects. RESULTS: The II, ID, and DD genotype was present in 16, 18 and 11 of subjects with PPCM and 48, 19 and 3 of controls respectively. The odds ratio for ACE-II genotype in cases vs. controls was 0.253 (95% CI=0.114-0.558; p=0.007), for that of II genotype was 1.93 (95% CI=0.86-4.3; p=0.107) and for DD genotype was 7.225 (95% CI; 1.88-27.6; p=0.0039). Overall frequency of D allele in cases was significantly higher than controls (odds=4.25; 95% CI=2.01-6.7; p=0.0001). Moreover, ejection fraction, left ventricular volume and linear dimensions were worse in patients with DD genotype. CONCLUSION: ACE DD genotype and overall frequency of D allele is significantly higher in patients with PPCM. Also, the presence of DD genotype is associated with worse systolic performance indices measured echocardiographically.

Leptin supplementation in vitro improved developmental competence of buffalo oocytes and embryos.

The aim of the present study was to determine potential role of leptin on in vitro developmental competence of buffalo oocytes and embryos. Slaughterhouse derived culture grade buffalo cumulus oocyte complexes (COCs) were matured in vitro (IVM) with leptin (10 ng/ml) or without leptin (control). In each experiment, a pool of matured COCs was used for further in vitro embryo production and another pool of COCs was used for cumulus cells and mature oocytes isolation to study the relative mRNA expression of developmentally important genes. Presumptive zygotes were cultured in embryo culture (IVC) media supplemented with leptin (10 ng/ml) or without leptin (control). Cleavage rate was higher (p < 0.05) when leptin was supplemented during IVM + IVC, both, as compared to other groups. Higher cleavage rate was observed in leptin-treated groups, though it was non-significant. Blastocyst rate was higher (p < 0.05) in all the leptin treated groups. The relative mRNA expression of LEPR (Ob-Rb), HAS2 and EGFR was significantly (p < 0.05) up-regulated and the expression of CASPASE3 was down-regulated in cumulus cells of leptin-treated groups. The expression of GDF9, BMP15, GLUT1, LEPR and CASPASE3 transcripts in leptin and non-treated oocytes did not differ. The relative mRNA expression of POU5F1and LEPR transcripts in blastocysts was higher (p < 0.05) in leptin-treated groups; the change in expression of GLUT1, INF-τ and CASPASE3 transcripts was not significant (p > 0.05). Thus, it is concluded that leptin promotes developmental competence of bubaline oocytes by modulating cumulus enabling factors and genes regulating pluripotentcy in the blastocysts.

A Comparative Study of Growth Kinetics, In Vitro Differentiation Potential and Molecular Characterization of Fetal Adnexa Derived Caprine Mesenchymal Stem Cells.

The present study was conducted with an objective of isolation, in vitro expansion, growth kinetics, molecular characterization and in vitro differentiation of fetal adnexa derived caprine mesenchymal stem cells. Mid-gestation gravid caprine uteri (2-3 months) were collected from abattoir to derive mesenchymal stem cells (MSCs) from fetal adnexa {amniotic fluid (cAF), amniotic sac (cAS), Wharton's jelly (cWJ) and cord blood (cCB)} and expanded in vitro. These cultured MSCs were used at the 3rd passage (P3) to study growth kinetics, localization as well as molecular expression of specific surface antigens, pluripotency markers and mesenchymal tri-lineage differentiation. In comparison to cAF and cAS MSCs, cWJ and cCB MSCs showed significantly (P<0.05) higher clonogenic potency, faster growth rate and low population doubling (PDT) time. All the four types of MSCs were positive for alkaline phosphatase (AP) and differentiated into chondrogenic, osteogenic, and adipogenic lineages. These stem cells expressed MSC surface antigens (CD73, CD90 and CD105) and pluripotency markers (Oct4, Sox2, Nanog, KLF, cMyc, FoxD3) but did not express CD34, a hematopoietic stem cell marker (HSC) as confirmed by RT-PCR, immunocytochemistry and flow cytometric analysis. The relative mRNA expression of MSC surface antigens (CD73, CD90 and CD105) was significantly (P<0.05) higher in cWJ MSCs compared to the other cell lines. The mRNA expression of Oct4 was significantly (P<0.05) higher in cWJ, whereas mRNA expression of KLF and cMyc was significantly (P<0.05) higher in cWJ and cAF than that of cAS and cCB. The comparative assessment revealed that cWJ MSCs outperformed MSCs from other sources of fetal adnexa in terms of growth kinetics, relative mRNA expression of surface antigens, pluripotency markers and tri-lineage differentiation potential, hence, these MSCs could be used as a preferred source for regenerative medicine.

Immediate effect of percutaneous transvenous mitral commissurotomy on atrial electromechanical delay and P-wave dispersion in patients with severe mitral stenosis.

BACKGROUND: Mitral stenosis (MS) is associated with prolonged inter- and intra-atrial electromechanical delays and increased P-wave dispersion, which are markers of atrial fibrillation (AF) risk. This study was conducted to assess the immediate effect of successful percutaneous transvenous mitral commissurotomy (PTMC) on these parameters. METHODS: This single center observational study included 25 patients with severe MS (aged 34.1 ± 7.1 years, with mean mitral valve area (MVA) of 0.74 ± 0.13 cm(2)), in sinus rhythm, who underwent successful PTMC at our hospital. P-wave dispersion (PWD) was calculated by subtracting minimum P-wave duration (P min) from maximum P-wave duration (Pmax), measured on a 12-lead surface ECG obtained from each patient in supine position at a paper speed of 50mm/s and 20mm/mV. Inter-atrial (AEMD), left intra-atrial (L-IAEMD), and right intra-atrial (R-IAEMD) electromechanical delays were measured on tissue Doppler imaging. PTMC was performed using the standard Inoue Balloon technique. All these parameters were evaluated and compared before and 24-48 h after PTMC. RESULTS: Successful PTMC led to significant reduction in AEMD (p < 0.001), L-IAEMD (p < 0.001), and R-IAEMD (p < 0.001). There were no changes in Pmax, Pmin, and PWD immediately after PTMC. CONCLUSIONS: Successful PTMC has a favorable early impact on inter- and intra-atrial electromechanical delays, which are considered as novel parameters of atrial electromechanical remodeling in MS patients. Prospective large-scale studies are required to confirm whether improvement in these markers translates into reduced long-term AF risk.

Lymphoma presenting as severe left ventricular systolic dysfunction: a case report.

Lymphoma involving the heart is rare. This is a case report on non-Hodgkin lymphoma where the patient presented for the first time with heart failure and severe left ventricular systolic dysfunction due to lymphoma infiltrating the heart muscle and had simultaneous bilateral involvement of kidneys. This type of presentation has never been described in world literature and is the first reported case.