Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model.

The current study was designed to study the persistence and distribution of caprine bone marrow derived mesenchymal stem cells (cBM-MSCs) when administered intra-dermally in experimentally induced cutaneous wounds in rabbits. MSC's from goat bone marrow were isolated and their differentiation potential towards adipogenic and osteogenic lineages were assayed in vitro. The isolated cells were phenotypically analysed using flow cytometry for the expression of MSC specific matrix receptors (CD73, CD105 and Stro-1) and absence of hematopoietic lineage markers. Further, these in vitro expanded MSCs were stained with PKH26 lipophilic cell membrane red fluorescent dye and prepared for transplantation into cutaneous wounds created on rabbits. Five, 2 cm linear full thickness skin incisions were created on either side of dorsal midline of New Zealand white rabbits (n = 4). Four wounds in each animal were implanted intra-dermally with PKH26 labelled cBM-MSCs suspended in 500 µl of Phosphate Buffer Saline (PBS). Fifth wound was injected with PBS alone and treated as negative control. The skin samples were collected from respective wounds on 3, 7, 10 and 14 days after the wound creation, and cryosections of 6 µM were made from it. Fluorescent microscopy of these cryosections showed that the PKH26 labelled transplanted cells and their daughter cells demonstrated a diffuse pattern of distribution initially and were later concentrated towards the wound edges and finally appeared to be engrafted with the newly developed skin tissues. The labelled cells were found retained in the wound bed throughout the period of 14 days of experimental study with a gradual decline in their intensity of red fluorescence probably due to the dye dilution as a result of multiple cell division. The retention of transplanted MSCs within the wound bed even after the complete wound healing suggests that in addition to their paracrine actions as already been reported, they may have direct involvement in various stages of intricate wound healing process which needs to be explored further.

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-ß1 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.

Molecular characterization and xenogenic application of Wharton's jelly derived caprine mesenchymal stem cells.

Aim of the present study was in vitro expansion and characterization of caprine wharton's jelly derived mesenchymal stem cells (cWJ-MSCs) to investigate their tissue healing potential in xenogenic animal model. Plastic adherent fibroblastoid cell populations with distinctive homogeneous morphology were isolated from caprine Wharton's jelly explants. These Wharton's jelly derived cells were found positive for the surface markers CD-73, STRO-1 and CD-105, whereas they were negative for hematopoetic stem cell marker CD-34. In vitro cultured cWJ-MSCs also showed differentiation properties into osteogenic, adipogenic and chondrogenic lineages as demonstrated by von Kossa, Oil Red-O and Alcian blue staining respectively, which was further confirmed and quantified by flow cytometric analysis. Furthermore, these well characterized cWJ-MSCs were evaluated for the wound-healing potential in full-thickness skin wounds in rabbit model for 28 days. Caprine WJ- MSCs treated skin wounds showed significantly (P < 0.05) higher percentage of wound contraction especially at the 21(st) day post transplantation when compared to PBS treated control group animals. Further, we observed better healing potential of cWJ-MSCs in terms of histo-morphological evaluation, epithelialisation and collagenization with matured vascularization stage by day 28 as compared to control. In conclusion, cWJ- MSCs provide an alternative inexhaustible source of mesenchymal stem cells and also unravel new perspectives pertaining to the therapeutic use of these cells in different species.

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 Dulbecco's modified Eagle's 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.

Impact of gonadotropin supplementation on the expression of germ cell marker genes (MATER, ZAR1, GDF9, and BMP15) during in vitro maturation of buffalo (Bubalus bubalis) oocyte.

The present study was designed to investigate whether gonadotropins [follicle-stimulating hormone (FSH) and luteinizing hormone (LH)] and buffalo follicular fluid (bFF) supplementation in maturation medium influences the transcript abundance of germ cell marker genes [maternal antigen that embryos require (MATER), Zygote arrest 1 (ZAR1), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15)] mRNA in buffalo (Bubalus bubalis) oocytes. Buffalo ovaries were collected from local abattoir, oocytes were aspirated from antral follicles (5-8 mm) and matured in vitro using two different maturation regimens, viz, group A: gonadotropin (FSH and LH) and group B: non-gonadotropin-supplemented maturation medium containing 20% buffalo follicular fluid (bFF). mRNA was isolated from immature (330) and in vitro matured oocytes from both the groups (A, 320; B, 340), and reverse transcribed using Moloney murine leukemia virus reverse transcriptase. Expression levels of MATER, ZAR1, GDF9, and BMP15 mRNA transcripts were analyzed in oocytes of both maturation groups as well as immature oocytes using real-time PCR. QPCR results showed that GDF9 and BMP15 transcripts were significantly (p<0.05) influenced with gonadotropins and bFF supplementation during in vitro maturation of buffalo oocyte; however, MATER and ZAR1 transcripts were not influenced with gonadotropins and bFF supplementation in vitro. These results indicated that the expression levels of MATER, ZAR1, GDF9, and BMP15 mRNA were varied differentially during in vitro maturation of buffalo oocyte and were found to be gonadotropins (FSH and LH) or bFF dependent for GDF9 and BMP15.

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.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells.

Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.