B. Mohana Kumar

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.

Differential gene expression patterns in porcine nuclear transfer embryos reconstructed with fetal fibroblasts and mesenchymal stem cells

The present study compared the developmental ability and gene expression pattern at 4‐cell, 8‐cell, morula, and blastocyst stages of porcine nuclear transfer (NT) embryos from fetal fibroblasts (FFs) and mesenchymal stem cells (MSCs), in vitro fertilized (IVF), and in vivo derived embryos. MSC‐NT embryos showed enhanced blastocyst formation, higher total cell number, and a low incidence of apoptosis compared to FF‐NT embryos. Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4, Stat3, Nanog), DNA methylation (Dnmt1, Dnmt3a), histone deacetylation (Hdac2), growth factor signaling, and imprinting (Igf2, Igf2r) and apoptosis (Bax, Bcl2) regulation were observed in NT embryos. The expression of transcripts in MSC‐NT embryos more closely followed that of the in vivo derived embryos compared with FF‐NT embryos. In conclusion, MSCs with a relatively undifferentiated genome might serve as suitable donors that could be more efficiently reprogrammed to re‐activate expression of early embryonic genes in porcine NT. Developmental Dynamics 236:435–446, 2007.

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.

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor.

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy.

Neurogenic and cardiomyogenic differentiation of mesenchymal stem cells isolated from minipig bone marrow.

The present study investigated the potential of minipig bone marrow-mesenchymal stem cells (BM-MSCs) to differentiate in vitro into neuron- and cardiomyocyte-like cells. Isolated BM-MSCs exhibited a fibroblast-like morphology, expressed CD29, CD44 and CD90, and differentiated into osteocytes, adipocytes and chondrocytes. Upon induction in two different neuronal specific media, most of BM-MSCs acquired the distinctive morphological features and positively stained for nestin, neurofilament-M (NF-M), neuronal nuclei (NeuN), β-tubulin, galactocerebroside (Gal-C) and glial fibrillary acidic protein (GFAP). Expression of nestin, GFAP and NF-M was further demonstrated by RT-PCR and RT-qPCR. Following cardiomyogenic induction, MSCs exhibited a stick-like morphology with extended cytoplasmic processes, and formed cluster-like structures. The expression of cardiac specific markers α-smooth muscle actin, cardiac troponin T, desmin and α-cardiac actin was positive for immunofluorescence staining, and further confirmed by RT-PCR and RT-qPCR. In conclusion, our results showed the in vitro differentiation ability of porcine BM-MSCs into neuron-like and cardiomyocyte-like cells.

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

Characterisation and differentiation of porcine ovarian theca-derived multipotent stem cells.

In this study, the cellular properties and in vitro differentiation capacity of porcine ovarian theca-derived multipotent stem cells (TSCs) were examined. Isolated TSCs were expanded into a homogeneous population that had a typical fibroblast-shaped morphology and was positive for alkaline phosphatase activity. Cell cycle analysis indicated that TSCs had high proliferative potential. Flow cytometry analysis demonstrated expression of mesenchymal cell surface markers (CD29, CD44 and CD90) on TSCs. Among three pluripotent markers tested (OCT4, NANOG and SOX2), only SOX2 was expressed in TSCs at protein and mRNA levels. Cytochemical staining demonstrated that TSCs differentiated in vitro into osteocytes and adipocytes. Lineage specific transcripts expressed by differentiated osteocytes including osteonectin, osteocalcin and RUNX2. Lineage specific transcripts expressed by differentiated adipocytes included adipocyte fatty acid binding protein-2 (aP2) and peroxisome proliferator-activated receptor-γ2. Following induction in oogenesis media, TSCs exhibited sequential changes in morphology, resembling oocyte-like cells (OLCs), and expressed transcription factors (OCT4, NANOG and SOX2), oocyte-specific marker genes (GDF9B, C-MOS, DAZL, VASA, ZPC, SCP3 and STELLA) and the folliculogenesis marker follicular stimulating hormone receptor. These results indicated that TSCs derived from ovarian follicles are capable of differentiating into mesenchymal lineages and OLCs.

Donor-matched functional and molecular characterization of canine mesenchymal stem cells derived from different origins.

Canine mesenchymal stem cells (cMSCs) have generated a great interest as a promising source for cell-based therapies. To understand the basic biological properties of cMSCs derived from bone marrow (cBM-MSCs), adipose tissue (cA-MSCs), and dermal skin (cDS-MSCs) from a single donor, the present study compared their alkaline phosphatase (AP) activity, expression of CD markers and stem cell transcription factors, differentiation ability into osteogenic, adipogenic, and chondrogenic lineages, in vivo ectopic bone formation, chromosomal stability, cell cycle status, telomere length, and telomerase activity. Expressions of AP activity and transcription factors (Oct3/4, Nanog, and Sox2) were either absent or extremely weak in all cMSCs. CD marker profile (CD45-, CD90+, and CD105+) and differentiation capacity were exhibited by all cMSCs, although cA-MSCs had enhanced cytochemical staining associated with expression of lineage-specific markers. In vivo bone formation of cMSCs was performed with demineralized bone matrix (DBM) by transplanting into the subcutaneous spaces of 9-week-old BALB/c-nu mice, followed by radiographic and histological analysis after 1 and 2 months. cA-MSCs and cDS-MSCs, in contrast to the in vitro observations, also displayed higher in vivo osteogenic abilities than cBM-MSCs. Ploidy analysis showed that cells were diploid and contained no noticeable chromosomal abnormalities. Furthermore, a relatively low percentage of cells was found at the G1 phase in all cMSCs, especially in DS-MSCs. Regardless of the different tissue sources, cMSCs from a single donor showed no differences in telomere lengths (~18–19 kbp) but exhibited varied telomerase activity. The above results suggest that tissue-specific cMSCs derived from a single donor possess slight differences in stem cell properties.

Developmental expression of lineage specific genes in porcine embryos of different origins

PurposeThis study compared the expression of genes involved in pluripotency, segregation of inner cell mass (ICM) and trophectoderm (TE), and primitive endoderm (PE) formation in porcine embryos produced by in vitro fertilization (IVF), parthenogenetic activation (PA), and nuclear transfer (NT) using either fetal fibroblasts (FF-NT) or mesenchymal stem cells (MSC-NT).MethodsBlastocyst formation and total cell number were analyzed. The expression patterns of transcripts, including SRY-related HMG-box gene 2 (SOX2), reduced expression gene 1 (REX1/ZFP42), LIN28, caudal type homeobox 2 (CDX2), TEA domain family member 4 (TEAD4), integrin beta 1 (ITGB1) and GATA6 were assessed at the 4–8 cell and blastocyst stage embryos by real-time PCR.ResultsDevelopmental rates to blastocyst stage and total cell number were higher in IVF and PA embryos than in NT embryos. But MSC-NT embryos had increased blastocyst formation and higher total cell number compared to FF-NT embryos. The relative expressions of transcripts were higher in blastocysts than in 4–8 cell stage embryos. The mRNA expression levels of SOX2 and REX1 were largely similar in embryos of different origins. However, the genes such as LIN28, CDX2, TEAD4, ITGB1 and GATA6 showed the differential expression pattern in PA and NT embryos compared to IVF embryos. Importantly, the transcript levels in MSC-NT embryos were relatively less variable to IVF than those in FF-NT embryos.ConclusionMSCs seem to be better donors for porcine NT as they improved the developmental competency, and influenced the expression pattern of genes quite similar with IVF embryos than that of FFs.