Nitin E. Gade

Isolation, culture and characterization of caprine mesenchymal stem cells derived from amniotic fluid

Amniotic fluid (AF) represents heterologous cell types and a specific group of these cells show high growth rate and multipotent characteristics. The aim of the present study was to culture and fully characterize the putative stem cell population isolated from caprine mesenchymal stem cells. Plastic adherent fibroblastoid cell population could be successfully isolated from the caprine amniotic fluid. In vitro expanded caprine amniotic fluid derived mesenchymal stem cells (cAF-MSCs) showed high proliferation ratio with a doubling time of 33.1h and stained positive for alkaline phosphatase. Relative transcript abundance of CD-73, CD-90 and CD-105 surface markers were analyzed by SYBR green based real time PCR and their respective proteins were localized through immunocytochemistry, however cAF-MSCs were found negative for haematopoietic marker CD-34. When exposed to corresponding induction condition, cAF-MSCs differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through von Kossa, Oil Red O and Alcian blue staining respectively. Furthermore, these cells were found positive for undifferentiated embryonic stem cell markers like Oct-4, Nanog, Sox-2, SSEA-1 and SSEA-4 which accentuate their pluripotent property. In conclusion, caprine amniotic fluid represents a promising source of mesenchymal stem cells with high proliferative and differentiation potential and these cells offer their scope for multiple regenerative therapies.

Expression of nitric oxide synthase isoforms in different stages of buffalo (Bubalus bubalis) ovarian follicles: effect of nitric oxide on in vitro development of preantral follicle.

The present study was designed to investigate the expression of nitric oxide synthase (NOS) isoforms in buffalo ovarian preantral (PFs), antral (AFs) and ovulatory (OFs) follicles (Experiment 1); effect of NO on in vitro survival and growth of PFs (Experiment 2) and NOS activity in immature oocytes by NADPH-diaphorase test (Experiment 3). In Experiment 1, NOS isoforms (neuronal, inducible and endothelial) were localized immunohistochemically; mRNA and protein expression was analyzed by semi-quantitative RT-PCR and western blot, respectively. In Experiment 2, PFs were isolated by micro-dissection method from buffalo ovaries and cultured in 0 (control), 10(-3), 10(-5), 10(-7) and 10(-9) M sodium nitroprusside (SNP). PFs were further cultured with 10(-5) M SNP + 1.0 mM N(ω)-nitro-L-arginine methyl ester (L-NAME) or 1.0 μg/ml hemoglobin (Hb) to examine the reversible effect of SNP. Immunohistochemical studies demonstrated that inducible nitric oxide synthase (iNOS) immunoreactivity was predominantly localized in granulosa and theca cells whereas, neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase in the theca, granulosa and cumulus cells of PFs, AFs and OFs. The amount of mRNA as well as protein of nNOS and eNOS was found similar between different stages of follicles. In contrast, higher level of iNOS mRNA was observed in OFs and protein in the AFs. Higher doses of SNP (10(-3), 10(-5), 10(-7) M) inhibited (P < 0.05) while, lower dose of SNP (10(-9) M) stimulated (P < 0.05) the survival, growth, and antrum formation of PFs. The inhibitory effects of SNP were reversed by Hb, while L-NAME was not found effective. In conclusion, expression of NOS isoforms mRNA and protein in PFs, AFs, and OFs and NOS enzyme activity in immature follicular oocytes suggest a role for NO during ovarian folliculogenesis in buffalo. NO plays a dual role on growth and survival of PFs depending on its concentration in the culture medium.

Molecular and cellular characterization of buffalo bone marrow-derived mesenchymal stem cells.

Immune privileged mesenchymal stem cells (MSCs) can differentiate into multiple cell types and possess great potential for human and veterinary regenerative therapies. This study was designed with an objective to isolate, expand and characterize buffalo bone marrow-derived MSCs (BM-MSCs) at molecular and cellular level. Buffalo BM-MSCs were isolated by Ficoll density gradient method and cultured in Dulbeccos modified Eagles medium supplemented with fetal bovine serum (FBS). These cells were characterized through alkaline phosphatase (AP) staining, colony-forming unit (CFU) assay, mRNA expression analysis (CD 73, CD 90, CD 105, Oct4 and Nanog), immunolocalization along with flow cytometry (Stro 1, CD 73, CD 105, Oct4, Sox2 and Nanog) and in situ hybridization (Oct4 and Sox2). Multilineage differentiation (osteogenic, adipogenic and chondrogenic) was induced in vitro, which was further assessed by specific staining. Buffalo BM-MSCs have the capacity to form plastic adherent clusters of fibroblast-like cells and were successfully maintained up to 16(th) passage. These cells were AP positive, and further CFU assay confirmed their clonogenic property. RT-PCR analysis and protein localization study showed that buffalo BM-MSCs are positive for various cell surface markers and pluripotency markers. Cytoplasmic distribution of mRNA for pluripotency markers in buffalo BM-MSCs and multilineage differentiation were induced in vitro, which was further assessed by specific staining. To the best of our knowledge, this is the first report of buffalo BM-MSCs, which suggests that MSCs can be derived and expanded from buffalo bone marrow and can be used after characterization as a novel agent for regenerative therapy.

Expression and characterization of constitutive heat shock protein 70.1 (HSPA-1A) gene in in vitro produced and in vivo-derived buffalo (Bubalus bubalis) embryos.

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA-1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA-1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo-derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA-1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA-1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8-16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA-1A revealed that it shares 91-98% identity with other mammalian sequences. It can be concluded that higher level of HSPA-1A mRNA in IVP embryos in comparison with in vivo-derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA-1A gene could be used as a stress biomarker during pre-implantation development.

Comparative study on characterization and wound healing potential of goat (Capra hircus) mesenchymal stem cells derived from fetal origin amniotic fluid and adult bone marrow

Caprine amniotic fluid (cAF) and bone marrow cells (cBM) were isolated, expanded and phenotypically characterized by mesenchymal stem cells (MSCs) specific cell surface markers. Both cell types were compared for multilineage differentiation potential by flow cytometry using specific antibodies against lineage specific markers. Furthermore, in vitro expanded cAF-MSCs showed higher expression of trophic factors viz. VEGF and TGF-β1 as compared to cBM-MSCs. Full-skin thickness excisional wounds created on either side of the dorsal midline (thoracolumbar) of New Zealand White rabbits were randomly assigned to subcutaneous injection of either fetal origin cAF-MSCs (n=4) or adult cBM-MSCs (n=4) or sterile PBS (control, n=4). The rate of wound closure was found faster (p<0.05) in cAF-MSCs treated wounds as compared with cBM-MSCs and PBS treated wounds especially on 21st day post-skin excision. Histomorphological examination of the healing tissue showed that wound healing was improved (p<0.05) by greater epithelialization, neovascularization and collagen development in cAF-MSCs as compared to cBM-MSCs and PBS treated wounds.