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‐β1, 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.
Mesenchymal stem cells‐conditioned media (MSCs‐CM) contains important biomolecules.
Lyophilized MSCs‐CM mixed with gel can be applied allogenically as well as xenogenically for quality wound healing.
It has better application than MSCs like; can be used fresh, lyophilized and stored at 4°C or −20°C, can be used in the form of gel‐like formulations, and at field level, it can be used by semiskilled personnel also.
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.
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 NFkβIA, NFkβ2, TNFα and IL1B, with no significant change in TGFβ1 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, TGFβ1, 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.
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.
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 3
rd
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.
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.
Canine bone marrow‐derived mesenchymal stem cells (BMSCs) successfully tracked in excision wounds.
MSCs can be used allogenically for regenerative medicine.
MSCs found very effective for nerve injury cases which are otherwise difficult to treat.
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.
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.
•l-carnitine (LC) reduced lipid content in the embryos.•LC reduced expression of lipid biosysnthesis related genes and increased expression of β-oxidation related genes.•LC supplementation improved quality embryo production and post thaw survivability.•Modified OPS used in this study is a novel cryodevice.
PDF
