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.

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 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.

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.