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.

Progesterone modulates adhesion molecules in uterine epithelial cells and in vitro embryo production in buffalo.

This study was undertaken to evaluate the role of progesterone (P4) in modulation of the expression profile of adhesion-related molecules in uterine epithelial cells (UECs) and in vitro blastocyst production in buffalo. UECs were isolated from slaughterhouse-derived uteri by enzymatic treatment, and cells were characterized by immunocytochemistry (ICC) and PCR assays. The well-characterized UECs were exposed to different concentrations of P4 (0, 0.314, 3.14 and 6.28 ng/ml) along with the basal level of oestradiol for 6 days. Thereafter, the relative mRNA expression of different biomolecules such as mucin 1 (MUC1), osteopontin, integrin alpha (α3, α6 and αV) and beta (ß1 and ß3) subunits, progesterone receptor (PR) and oestrogen receptor, was evaluated. Further, day 2 post-insemination embryos were cultured in mSOF supplemented with or without P4. UECs were found positive for cytokeratin expression and negative for vimentin expression. Progesterone treatment significantly enhanced the mRNA expression of most of the transcripts compared with the control group, and correspondingly, the immunofluorescence depicted higher protein expression of all these molecules. Further, the long-term exposure of UECs to P4 downregulated the expression of PR and, concomitantly, MUC1. Progesterone supplementation to embryo culture medium significantly (p < .05) improved the blastocyst rate. The study demonstrates the role of P4 hormone in modulation of the expression of early implantation-related biomolecules in uterine epithelial cells; hence, adequate level of steroids is crucial for normal embryo development and its implantation.

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.

Embryo-Uterine Cross-Talk: Exploration of the Immunomodulatory Mechanism in Buffalo.

Understanding the molecular cross-talk between the embryo and uterine endometrium is crucial for the improvement of IVF outcomes. The present work was undertaken to investigate the effect of pre-implantation embryo on the expression profile of immune-related genes in uterine epithelial cells (UECs) and PBMCs in buffalo. UECs were isolated from slaughterhouse-derived non-gravid uteri, cultured ex vivo and characterized, and buffalo embryos were produced in vitro from slaughterhouse-derived ovaries. Embryos co-cultured with steroid-treated UECs significantly stimulated (p < 0.05) the relative mRNA abundance of PTGS2, ISG15, OAS1, MX2, IFNAR1 and IFNAR2 in UECs while they significantly suppressed the mRNA expression of NFkßIA, NFkß2, TNFα and IL1B, with no significant change in TGFß1 and IL10 in the co-culture of embryos with UECs. In vitro treatment of PBMCs with conditioned media (CM) derived from embryos as well as UEC−embryo co-culture upregulated the mRNA abundance of ISG15, TGFß1, PTGS2OAS1, MX2 and STAT1 while it downregulated IL17 and TNFα expression. The expression of IFNAR1 and IFNAR2 was elevated in PBMCs cultured in embryo-derived CM, but there was no significant change in PBMCs cultured in UEC−embryo co-culture CM. Thus, it can be concluded that the developing embryo and its secretions modulate the expression of immune responses by inducing an anti-inflammatory action in uterine epithelial cells for acceptance of the semi-allogenic embryo in the uterus to sustain pregnancy in buffalo.

Smad4 and γ-secretase knock-down effect on osteogenic differentiation mediated via Runx2 in canine mesenchymal stem cells.

Cell lineage determination during mesenchymal stem cell (MSCs) differentiation is a highly orchestrated process involving diverse signaling pathways and distinct classes of regulatory molecules. Bone morphogenetic protein (BMP) signaling positively influence the osteoblast lineage determination, whereas the Notch signaling may have a dimorphic action. Effective regenerative therapy for repairing bone defects requires ample knowledge of the signaling pathways responsible for the differentiation of MSCs. To elucidate the signaling pathways that drives canine bone-marrow derived MSCs towards osteogenic lineage, the current work was focused on BMP and Notch signaling. Target genes of Runx2, Smad4 and γ-secretase were silenced by short hairpin RNA (shRNA) in canine MSCs. Evaluation of the effect of gene silencing on in-vitro osteogenic differentiation potential was done by quantitative polymerase chain reaction (qPCR) for osteoblastic markers (Osteocalcin and Osteopontin) and Alizarin red S staining for the extracellular deposition of calcium. Silencing of Runx2 significantly reduced the osteocalcin and osteopontin gene expression while a similar trend was observed in the case of smad 4 silencing and their combination groups, but there was no difference found in Hey 1 expression. Runx2 and Smad4 silencing groups showed very less positive staining with Alizarin red S staining, whereas knockdown of γ-secretase and its combination groups showed reverse results as that of Runx2 and Smad4. Runx2 plays an indispensable part in directing the canine mesenchymal stem cells towards osteogenic lineage. Also, Smad-mediated BMP signaling induced the osteoblast-specific gene expression, whereas the notch pathway negatively regulated the osteogenic differentiation of canine MSCs.

Comparative evaluation of fracture healing potential of differentiated and undifferentiated guinea pig and canine bone marrow-derived mesenchymal stem cells in a guinea pig model.

BACKGROUND AND AIM: This work was conducted to compare the therapeutic potential of undifferentiated and osteogenic differentiated canine (xenogeneic) and guinea pig (allogeneic) BMSCs in fracture healing using guinea pig as a model. MATERIALS AND METHODS: A well-characterized homogenous population of third passage mesenchymal stem cells of bone marrow origin was used in all the experiments. MSCs from both the species, i.e., canine and guinea pigs, were differentiated and characterized. Expression of MHC I and II along with co-stimulatory molecules was assessed based on relative mRNA expression. The osteogenic differentiated and undifferentiated MSCs from both species were used for evaluating fracture healing in the guinea pig model. The healing potential was assessed based on radiographic, histopathology, and clinical observations. RESULTS: BMSCs from both species expressed MSC surface antigens and successfully differentiated to osteogenic, chondrogenic, and adipogenic lineages. The mRNA expression of class I and II MHC molecules in all the three lineages showed no significant (p > 0.05) differences after differentiating to adipogenic, chondrogenic, and osteogenic lineages. Radiographic and clinical examination revealed that MSCs therapy significantly improved bone fracture healing with a non-significant (p > 0.05) difference between differentiated and undifferentiated BMSCs. In addition, allogeneic MSCs therapy performed better than xenogeneic therapy. CONCLUSION: MSCs remained hypo immunogenic after differentiation and have comparable fracture healing potential though allogeneic MSCs have better therapeutic potential than xenogenic MSCs.

Improved quality and fertilizability of cryopreserved buffalo spermatozoa with the supplementation of methionine sulfoxide reductase A.

BACKGROUND: The excessive reactive oxygen species produced during semen-freezing and -thawing damage the macromolecules resulting in impairment of cellular functions. Proteins are the primary targets of oxidative damage, wherein methionine residues are more prone to oxidation and get converted into methionine sulfoxide, thus affecting the protein function. The methionine sulfoxide reductase A (MsrA) catalyzes the conversion of methionine sulfoxide to methionine and restores the functionality of defective proteins. OBJECTIVES: To establish the expression of MsrA in male reproductive organs, including semen and its effect on quality of cryopreserved semen upon exogenous supplementation, taking buffalo semen as a model. MATERIALS AND METHODS: The expression of MsrA was established by immunohistochemistry, PCR, and Western blots. Further, the effect of recombinant MsrA (rMsrA) supplementation on the quality of cryopreserved spermatozoa was assessed in three treatment groups containing 1.0, 1.5, and 2.0 µg of rMsrA/50 million spermatozoa in egg yolk glycerol extender along with a control group; wherein the post-thaw progressive motility, viability, membrane integrity, and zona binding ability of cryopreserved spermatozoa were studied. RESULTS: The MsrA was expressed in buffalo testis, epididymis, accessory sex glands, and spermatozoa except in seminal plasma. In group 2, the supplementation has resulted in a significant (p < 0.05) improvement as compared to the control group in mean progressive motility (47.50 ± 2.50 vs. 36.25 ± 2.63), viability (56.47 ± 1.85 vs. 48.05 ± 2.42), HOST (50.76 ± 1.73 vs. 44.29 ± 1.29), and zona binding ability of spermatozoa (149.50 ± 8.39 vs. 29.50 ± 2.85). DISCUSSION AND CONCLUSION: In the absence of native MsrA of seminal plasma, the supplementations of rMsrA may repair the oxidatively damaged seminal plasma proteins and exposed sperm plasma membrane proteins resulting in better quality with a fivefold increase in fertilizability of frozen-thawed spermatozoa. The findings can be extended to other species to improve the semen quality with the variation in the amounts of rMsrA supplementation.

Expression profile of adhesion molecules in blastocyst vis-a-vis uterine epithelial cells.

Models using in vitro produced buffalo embryos and in vitro cultured uterine epithelial cells (UECs) may be useful in understanding the intricacies of embryo-uterine cross talk. In the present study, buffalo UECs were obtained from slaughterhouse derived non-gravid uterus. UECs monolayer was treated with steroids (10pg/ml estradiol for 24h and 3.14 ng/ml progesterone for another 5 days). In vitro produced buffalo blastocysts were co-cultured over steroid treated UECs monolayer and at 72 h of co-culture, embryo attachment rate was higher in UECs treated with steroids (71.86% vs. 26.55%) while no attachment was observed on plastic surface. Naturally hatched or assisted hatched blastocysts were co-cultured over UECs monolayer treated with 3.14ng/ml progesterone (P4), or without any treatment for 72 h and the effect of co-culture on the expression profile of adhesion related biomolecules was analyed in UECs and blastocysts. Cultured UECs and blastocysts cultured in embryo culture media were considered as control. It was observed that the expression of MUC1 in UECs was significantly (p < 0.05) higher in control group than treatment groups. The relative mRNA abundance of integrins and osteopontin was significantly (p < 0.05) higher in UECs and blastocysts of treatment groups than control group. Expression of IFN-τ was significantly higher (p < 0.05) in embryos co-cultured with UECs than other treatment groups. It can be concluded that P4 supplementation is required for the modulation of adhesion molecules and co-culture of blastocysts and UECs together affect the expression of adhesion molecules both in blastocyts and in UECs.

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.

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.

Expression and secretory profile of buffalo fetal fibroblasts and Wharton's jelly feeder layers.

The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Wharton's jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules viz. bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs.

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.

GREM1, EGFR, and HAS2; the oocyte competence markers for improved buffalo embryo production in vitro.

Selection of competent oocytes is crucial for successful in vitro embryo production and correlating expression profile of oocyte competence markers in cumulus cells with oocyte quality is an important preamble. In the present study, expression profile of oocyte competence markers (GREM1, EGFR, HAS2, and TNFAIP6) was correlated through brilliant cresyl blue (BCB) staining and subsequently with in vitro embryo production. Excellent to good quality buffalo, cumulus oocyte complexes (COCs) were stained with BCB (26 µM) and based on the blue coloration of cytoplasm COCs were divided in two groups as BCB(+ve) and BCB(-ve). Mean percentage of BCB(+ve) oocytes was significantly higher (P < 0.05) than BCB(-ve) oocytes (56.79 ± 1.22 vs. 43.20 ± 1.22), the mean oocyte diameter was also significantly larger (P < 0.05) for the BCB(+ve) group than that of BCB(-ve) group (145.7 ± 1.8 µm vs. 132.7 ± 1.9 µm). The cleavage rate (%), blastocyst rate (%), and total cell number was significantly (P < 0.05) higher in BCB(+ve) than BCB(-ve) group (71.15 ± 2.17 vs. 52.89 ± 2.65; 31.58 ± 1.11 vs. 7.73 ± 0.97, and 93.14 ± 2.42 vs. 71.42 ± 2.09, respectively). Relative mRNA expression of marker genes in cumulus cells increased significantly (P < 0.05) after maturation viz. GREM1 (10.13 folds), EGFR (9.04 folds), HAS2 (27.91 folds), and TNFAIP6 (64.81 folds). Brilliant cresyl blue positive oocytes showed significantly higher (P < 0.05) expression of GREM1, EGFR, and HAS2 transcripts than BCB(-ve) oocytes, however, no significant change in the expression of TNFAIP6 gene was observed. It is concluded from the study that GREM1, EGFR, and HAS2 could be used as molecular markers of oocyte competence for an improved buffalo embryo production in vitro.