Eun-Ju Kang

Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes ☆

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of alpha-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of alpha-tocopherol (0, 100, 200, 400, 600 and 800 microM) using the straw-freezing procedure and stored at -196 degrees C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P<0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 microM alpha-tocopherol supplemented frozen-thawed groups. In alpha-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 degrees C for 3h, the expression in 200 and 800 microM alpha-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P<0.01) lower in alpha-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, alpha-tocopherol, supplemented at 200 microM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.

In vitro and in vivo osteogenesis of human mesenchymal stem cells derived from skin, bone marrow and dental follicle tissues

The present study evaluated the human mesenchymal stem cells (hMSCs) isolated from skin (hSMSC), bone marrow (hBMSC) and dental follicle (hDFMSC) tissues on their in vitro and in vivo osteogenic potential using demineralized bone matrix (DBM) and fibrin glue scaffold. Cells originated from three distinct tissues showed positive expressions of CD44, CD73, CD90, CD105 and vimentin, and differentiation ability into osteocytes, adipocytes and chondrocytes. hMSCs from all tissues co-cultured with a mixed DBM and fibrin glue scaffold in non-osteogenic induction media were positively stained by von Kossa and expressed osteoblast-related genes, such as osteocalcin (OC), osteonectin (ON), runt-related transcription factor 2 (Runx2) and osterix. For in vivo osteogenic evaluation, PKH26 labeled hMSCs were implanted into the subcutaneous spaces of athymic mice with a mixed scaffold. At 4 weeks of implantation, PKH26 labeled cells were detected in all hMSC-implanted groups. Bone formation with OC expression and radio-opacity intensity were observed around DBM scaffold in all hMSC-implanted groups. Interestingly, hDFMSCs-implanted group showed the highest OC expression and calcium content. These findings demonstrated that hDFMSCs could be a potential alternative autologous cell source for bone tissue engineering.

Peripheral nerve regeneration using autologous porcine skin-derived mesenchymal stem cells.

Porcine skin‐derived mesenchymal stem cells (pSMSCs) were evaluated on their biological MSC characterizations and differentiation into mesenchymal lineages, along with in vitro and in vivo neural inductions. Isolated pSMSCs showed plate‐adherent growth, expression of various MSC‐marker proteins and transcriptional factors, and differentiation potential into mesenchymal lineages. Neuron‐like cell morphology and various neural markers were highly detected at 6 h and 24 h after in vitro neural induction of pSMSCs, but their neuron‐like characteristics disappeared as induction time extended to 48 and 72 h. To evaluate the in vivo peripheral nerve regeneration potential of pSMSCs, a total of 5 × 106 autologous pSMSCs labelled with tracking dye, supplemented with fibrin glue scaffold and collagen tubulization, were transplanted into the peripheral nerve defected miniature pigs. At 2 and 4 weeks after cell transplantation, well‐preserved transplanted cells and remarkable in vivo nerve regeneration, including histologically complete nerve bundles, were observed in the regenerated nerve tissues. Moreover, S‐100 protein and p75 nerve growth factor receptor were more highly detected in regenerated nerve fibres compared to non‐cell grafted control fibres. These results suggest that autologous pSMSCs transplanted with fibrin glue scaffold can induce prominent nerve regeneration in porcine peripheral nerve defect sites. Copyright

Comparative Analysis of Telomere Length, Telomerase and Reverse Transcriptase Activity in Human Dental Stem Cells:

Stem cells from dental tissues have been isolated and established for tooth regenerative applications. However, basic characterization on their biological properties still needs to be investigated before employing them for effective clinical trials. In this study, we compared the telomere length, relative telomerase activity (RTA), and relative reverse transcriptase activity (RRA) as well as the surface antigen profile and mesenchymal differentiation ability in human dental papilla stem cells (DPaSCs), dental pulp stem cells (DPuSCs), and dental follicle stem cells (DFSCs) with mesenchymal stem cells (MSCs) derived from bone marrow. Dental stem cells (DSCs) were strongly positive for cell surface markers, such as CD44 and CD90. However, slightly lower expression of CD105 was observed in DPaSCs and DPuSCs compared to DFSCs and MSCs. Following specific induction, DPaSCs, DFSCs, and MSCs were successfully differentiated into adipocytes and osteocytes. However, DPuSCS, in particular, were able to differentiate into adipocytes but failed to induce into osteogenic differentiation. Further, all DSCs, MSCs, and MRC-5 fibroblasts as control were investigated for telomere length by nonradioactive chemiluminescent assay, RTA by relative-quantitative telomerase repeat amplification protocol (RQ-TRAP), and RRA by PCR-based assay. Mean telomere lengths in DPaSCs, DPuSCs, DFSCs, and MSCs was ~11 kb, and the values did not differ significantly (p < 0.05) among the cells analyzed. RTA levels in DPaSCs were significantly (p < 0.05) higher than in MSCs, DPuSCs, DFSCs, and MRC-5 fibroblasts and among DSCs, DFSCs showed a significantly (p < 0.05) lower RTA. Moreover, RRA levels were significantly (p < 0.05) higher in DPaSCs, DPuSCs, and MSCs than in DFSCs. Based on these observations, we conclude that among DSCs, DPaSCs possessed ideal characteristics on telomere length, telomerase activity and reverse transcriptase (RTase) activity, and may serve as suitable alternative candidates for regenerative medicine.

Transplantation of porcine umbilical cord matrix mesenchymal stem cells in a mouse model of Parkinson's disease.

The present study compared mesenchymal stem cells derived from umbilical cord matrix (UCM‐MSCs) with bone marrow (BM‐MSCs) of miniature pigs on their phenotypic profiles and ability to differentiate in vitro into osteocytes, adipocytes and neuron‐like cells. This study further evaluated the therapeutic potential of UCM‐MSCs in a mouse Parkinsons disease (PD) model. Differences in expression of some cell surface and cytoplasm specific markers were evident between UCM‐MSCs and BM‐MSCs. However, the expression profile indicated the primitive nature of UCM‐MSCs, along with their less or non‐immunogenic features, compared with BM‐MSCs. In vitro differentiation results showed that BM‐MSCs had a higher tendency to form osteocytes and adipocytes, whereas UCM‐MSCs possessed an increased potential to transform into immature or mature neuron‐like cells. Based on these findings, UCM‐MSCs were transplanted into the right substantia nigra (SN) of a mouse PD model. Transplantation of UCM‐MSCs partially recovered the mouse PD model by showing an improvement in basic motor behaviour, as assessed by rotarod and bridge tests. These observations were further supported by the expression of markers, including nestin, tyrosine hydroxylase (TH), neuronal growth factor (NGF), vascular endothelial growth factor (VEGF) and interleukin‐6 (IL‐6), at the site of cell transplantation. Our findings of xenotransplantation have collectively suggested the potential utility of UCM‐MSCs in developing viable therapeutic strategies for PD. Copyright