Interactive effect of carbendazim and imidacloprid on buffalo bone marrow derived mesenchymal stem cells: oxidative stress, cytotoxicity and genotoxicity.

The effect of a combination of two pesticides, carbendazim (CBZ) and imidacloprid (IMI), was investigated on mesenchymal stem cells derived from the bone marrow of buffalo (bMSCs). The bMSCs were exposed to the CBZ (2.25 µM, 4.49 µM, and 8.98 µM) and IMI (0.81 mM, 1.61 mM, and 3.22 mM) alone as well as in combinations. The bMSCs were found to be positive for the stem cell markers, AP, CD73, and OCT4. The bMSCs showed a significant reduction (p ≤ 0.05) in cell viability, and status of anti-oxidants while a significant increase (p ≤ 0.05) in the level of LDH, ALP, and CK-MB in CBZ and IMI-treated groups. A significant increase (p ≤ 0.05) was noticed in LPO, O2─ radical, total ROS, loss of ΔΨm, apoptotic index, and DNA damage in CBZ and IMI-treated groups. A low-dose combination group showed an elevated effect compared to the groups treated with the single pesticide. The interaction index was calculated for CBZ-IMI combined treatment groups on various parameters that showed the majority of antagonist effects. Present findings confirmed that CBZ and IMI-induced cytotoxicity in bMSCs was mediated via ROS production, altered ΔΨm and LPO along with depressed antioxidant status which was responsible for cell apoptosis and cell damage. This study suggested that CBZ and IMI had a dose-dependent toxic effect when the pesticides were used alone, while, co-exposure to both the pesticides simultaneously had an antagonist or non-additive effect on buffalo bMSCs at lower dose combinations and they induced a potentiating effect at high-dose combination.

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices.

BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

In vivo differentiation potential of buffalo (Bubalus bubalis) embryonic stem cell.

Embryonic stem cells (ESCs) derived from inner cell mass (ICM) of mammalian blastocyst are having indefinite proliferation and differentiation capability for any type of cell lineages. In the present study, ICMs of in vitro-derived buffalo blastocysts were cultured into two different culture systems using buffalo fetal fibroblast as somatic cell support and Matrigel as synthetic support to obtain pluripotent buffalo embryonic stem cell (buESC) colonies. Pluripotency of the ESCs were characterised through pluripotency markers whereas, their differentiation capability was assessed by teratoma assay using immuno-compromised mice. Cumulus ooccyte complexes from slaughter house-derived ovaries were subjected to in vitro maturation, in vitro fertilization and in vitro culture to generate blastocysts. Total 262 blastocysts were derived through IVEP with 11.83 % (31/262) hatching rate. To generate buESCs, 15 ICMs from hatched blastocysts were cultured on mitomycin-C-treated homologous fetal fibroblast feeder layer, whereas the leftover 16 ICMs were cultured on extra-cellular matrix (Matrigel). No significant differences were observed for primary ESCs colony formation between two culture systems. Primary colonies as well as passaged ESCs were characterised by alkaline phosphatase staining, karyotyping and expression of transcription-based stem cell markers, OCT-4 and cell surface antigens SSEA-4 and TRA-1-60. Batch of ESCs found positive for pluripotency markers and showing normal karyotype after fifteenth passage were inoculated into eight immuno-compromised mice through subcutaneous and intramuscular route. Subcutaneous route of inoculation was found to be better than intramuscular route. Developed teratomas were excised surgically and subjected to histological analysis. Histological findings revealed presence of all the three germinal layer derivatives in teratoma sections. Presence of germinal layer derivatives were further confirmed by reverse transcriptase-polymerase chain reaction for the presence of differentiation markers like nerve cell adhesion molecule, fetal liver kinase-1 and alpha-feto protein for ectoderm, mesoderm and endoderm, respectively.

Expression and quantification of Oct-4 gene in blastocyst and embryonic stem cells derived from in vitro produced buffalo embryos.

The POU-domain transcription factor Pou5f1 (Oct-4) is involved in transcriptional regulation during early embryonic development and cell differentiation. Despite highly conserved genomic organization of Oct-4 gene in mammals, expression pattern of Oct-4 is highly variable in different species. In the present study, expression pattern of Oct-4 in buffalo blastocyst, trophoectoderm (TE), and embryonic stem cells (ESCs) was investigated. For the derivation and characterization of buffalo ESCs, inner cell masses (ICMs) were isolated from 18 hatched and 21 expanded in vitro produced buffalo blastocyst and cultured over mitomycin-C-treated buffalo fetal fibroblast feeder layer. Alkaline phosphatase (AP) activity, SSEA-1 and 4, TRA 1-60 and 1-81, and Oct-4 proteins were localized in ICM, TE, and ESCs. Quantification of Oct-4 was done by amplifying a transcript of 125 base pairs by real-time polymerase chain reaction. Primary cell colony formation was higher (P < 0.05) in hatched blastocyst (83.33%, 15/18) compared to mechanically isolated ICMs from expanded blastocyst (52.38%, 11/21). Undifferentiated buffalo ESCs were positive for AP and expressed Oct-4, SSEA-1 and 4, TRA-1-60, and TRA-1-81 proteins. Oct-4 transcripts and proteins were detected in the ICM, TE cells and were invariably present in ESCs; however, expression level of Oct-4 transcript were significantly higher in ICM and ESCs as compared to TE cells. In conclusion, expression of Oct-4 is not only restricted to the ICM and ESCs but its expression was also detected in TE cells suggesting that instead of using Oct-4 as a single marker, it is better to have other flanking molecular markers for the identification of buffalo pluripotent embryonic stem cells.