Characterization of Stem Cells from Human Exfoliated Deciduous Anterior Teeth with Varying Levels of Root Resorption.

BACKGROUND: Deciduous teeth undergo the physiologic process of resorption, during which the remnant pulp undergoes activation. However, the quality of stem cells obtained at various stages of root resorption has not been documented. OBJECTIVE: To isolate and characterize stem cells from deciduous teeth with varying levels of root resorption. STUDY DESIGN: Healthy primary anterior teeth were extracted according to the treatment needs of the patient. The teeth were categorized into SHED(1/3)- teeth with 0 to 1/3rd root resorption, SHED(2/3)- teeth with 1/3rd to 2/3rd root resorption, and SHED(COMP)- teeth with more than 2/3rd root resorption. SHED were characterized based on their morphology, viability, proliferation rate, population doubling time, expression of cell surface markers, and in vitro differentiation potential into osteocytes and adipocytes. RESULTS: SHED from all three groups demonstrated largely similar morphological and cellular characteristics. However, SHED(2/3) showed relatively better characteristics in terms of growth kinetics and phenotypic marker expression. Also, the differentiation ability for osteogenic and adipogenic cell lineages was slightly higher in SHED(1/3) and SHED(2/3) compared with SHED(COMP). CONCLUSION: Based on the cellular, phenotypic and biological characteristics, it is suggested that SHED (2/3) could be a useful source for tissue regeneration, and warrants further investigations.

Effect of electron-beam irradiation on antimicrobial, antibiofilm activity, and cytotoxicity of mouth rinses.

BACKGROUND: Oral health diseases are common in all regions of the world. Mouth rinses are widely used generally by population as a port of daily oral care regimen. In addition to antimicrobial activity, mouth rinses possess certain cytotoxic effects. Electron-beam (E-beam) radiation is a form of ionizing energy known to induce structural, physical, and chemical changes in irradiated products. In this study, the modulatory effects of E-beam in irradiated mouth rinses were evaluated for its biological activities. MATERIALS AND METHODS: The antimicrobial activities of nonirradiated and irradiated mouth rinses were evaluated for its antimicrobial and antibiofilm activities against oral pathogens, Enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, and Candida albicans. The antimicrobial activity was evaluated by disc diffusion method and antibiofilm activity was evaluated by O'Toole method. The cytotoxicity was evaluated against human gingival fibroblast (HGF) cells by 3-(4, 5 Dimethythiazol-yl)-2,5-Diphenyl-tetrazolium bromide assay. RESULTS: Colgate Plax (CP) exhibited the antimicrobial activity against the tested pathogens, and a significant (P< 0.05) increase was observed against S. aureus at 750 Gy irradiation. Further, CP significantly (P< 0.05) suppressed S. mutans, S. aureus, and C. albicans biofilm. Listerine (LS) inhibited S. mutans and C. albicans biofilm. Whereas irradiated CP and LS significantly (P< 0.05) suppressed the biofilm formed by oral pathogens. The suppression of biofilm by irradiated mouth rinses was dose- and species-dependent. There was no significant (P > 0.05) difference in the cytotoxicity of irradiated and nonirradiated mouth rinses on HGF cells. However, an increased percentage viability of HGF cells was observed by mouth rinses irradiated at 750 Gy.xs CONCLUSION: The E-beam irradiation enhanced the antibiofilm activity of mouth rinses without modifying the cytotoxicity.

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.

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.

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 Parkinson's 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.

Epigenetic modification of fetal fibroblasts improves developmental competency and gene expression in porcine cloned embryos.

Treatment of somatic cells with DNA methylation and histone deacetylation inhibitors has been hypothesized to improve the potential reprogramming after nuclear transfer (NT). The objective of this study was to investigate the developmental competence and gene expression during the porcine preimplantation development of in vitro fertilized (IVF) and embryos cloned with porcine fetal fibroblasts (pFF) (pFF-NT), and pFF treated by 0.5 µM 5-azacytidine (5-azaC) (pFF+5-azaC-NT) or 1.0 mM sodium butyrate (NaB) (pFF+NaB-NT). IVF embryos had significantly (P < 0.05) higher blastocyst rates (27.7 ± 2.6 %) and total cell numbers (46.7 ± 3.9). However, NT embryos from pFF+5-azaC and pFF+NaB showed enhanced developmental potential with significantly (P < 0.05) higher rates of blastocysts (21.3 ± 2.9 % and 22.4 ± 1.7 %, respectively) than those from pFF (15.1 ± 2.5 %). Further, NT embryos from pFF+5-azaC and pFF+NaB (33.8 ± 4.1 and 35.7 ± 5.2, respectively) had significantly (P < 0.05) higher total cell numbers than those from pFF (24.6 ± 3.5). Differential expression pattern of genes involved in DNA methylation (DNA methyltransferases- DNMT1, DNMT2, DNMT3A and DNMT3B), histone acetylation (histone acetyltransferase 1- HAT1) and histone deacetylation (histone deacetylases- HDAC1, HDAC2 and HDAC3) were observed in NT embryos when compared to IVF counterparts. However, the relative expressions of genes in pFF+5-azaC-NT and pFF+NaB-NT groups were largely comparable to those of IVF embryos than pFF-NT embryos. In conclusion, modification of the epigenetic status by reducing DNA methylation or enhancing histone acetylation levels in pFF improved the developmental rates, total cell number and the transcription profile of porcine NT embryos. Thus, somatic cells with relatively hypomethylated or hyperacetylated genome may enhance reprogramming efficiency in porcine NT.

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.

PURPOSE: This 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). METHODS: Blastocyst 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. RESULTS: Developmental 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. CONCLUSION: MSCs 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.

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), ß-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.

Differential cytotoxic effects of sodium meta-arsenite on human cancer cells, dental papilla stem cells and somatic cells correlate with telomeric properties and gene expression.

We investigated the effects of sodium meta-arsenite (NaAsO(2)) on human cancer cells (MDA-MB-231, MCF-7 and U-87 MG), dental papilla tissue stem cells (DPSCs) and somatic cells [MRC-5 fetal fibroblasts and adult muscle cells (MCs)] by examining telomeric properties, endogenous reverse transcriptase (RT) activity and the expression of tumorigenesis-linked genes. Half maximal inhibitory concentration (IC(50)) values were higher in DPSCs and MCs, possessing longer telomere lengths when compared to cancer cells. Levels of telomerase and RT activity, and the expression of protein 53 (p53), B-cell lymphoma 2 (BCL2), nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB), transforming growth factor beta (TGFß) and vascular endothelial growth factor (VEGF) were significantly lower in cancer cells following sodium meta-arsenite treatment, whereas the effect was absent or marginally detected in DPSCs and somatic cells. Collectively, sodium meta-arsenite effectively induced cellular cytotoxicity by inhibiting telomerase and RT activity, and down-regulating transcript levels in cancer cells with shorter telomere lengths, whereas more tolerance was evident in DPSCs and somatic cells possessing longer telomere lengths.

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.

Porcine mesenchymal stem cells--current technological status and future perspective.

Similarities of porcine mesenchymal stem/progenitor cells (MSCs) with human counterpart allow them to be considered as a valuable model system for in vitro studies and preclinical assessments. Effective isolation and expansion of porcine MSCs from different origins, namely bone marrow, umbilical cord Wharton's jelly, amniotic fluid, umbilical cord blood and peripheral blood has been reported. The differentiation of porcine MSCs into mesenchymal lineages under in vitro conditions is consistent and growing evidence has also suggested their transdifferentiation abilities. Results of preclinical studies unveil a time dependent retention, engraftment, migration, ex vivo and in vivo differentiation characteristics and possibility for genetic modification of MSCs. Findings on immunogenicity and the immunomodulatory capacity of porcine MSCs are encouraging and valuable to understand the host compatibility following transplantation. Furthermore, suitability of porcine MSCs as donors in nuclear transfer offers a greater potential to medicine and biopharming. Here, we highlight recent findings in the areas of porcine MSC sources, differentiation ability, transplantation applications and their potential as nuclear donors for somatic cell nuclear transfer.

Effect of alpha-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.

Expression pattern of oxygen and stress-responsive gene transcripts at various developmental stages of in vitro and in vivo preimplantation bovine embryos.

The present study examined the expression pattern of oxygen (O(2)) and stress-responsive gene transcripts at various preimplantation developmental stages of in vitro produced (IVP) and in vivo derived (IVD) bovine embryos. Embryos were produced in vitro from oocytes matured, fertilized and cultured in synthetic oviductal fluid (SOF) medium under low (5%) and high (20%) O(2) concentrations. In vivo embryos were derived from 18 superovulated and artificially inseminated cows. In IVP and IVD groups, embryos were collected at 2-, 4-, 8-, 16-cell morula and blastocyst stages at specific time points for gene expression analysis. The cleavage rates (69.8+/-4.8%) did not differ significantly, but blastocyst rates were significantly higher (28.5+/-3.7%) in low O(2) than those in high O(2) group (18.7+/-3.9%). Mean cell number in low O(2) (145+/-12) and high O(2) (121+/-73) IVP blastocyst were lower (P<0.05) than those of IVD blastocyst (223+/-25). The ICM ratio of IVD blastocyst (26+/-4) was lower (P<0.05) than that of IVP embryos under 5% O(2) (33+/-5) and 20% O(2) (34+/-4) concentrations, respectively. Using real time PCR, for the set of target transcripts (Glut1, Glut5, Sox, G6PD, MnSOD, PRDX5, NADH and Hsp 70.1) analyzed, there were differences in the mRNA expression pattern at 2-, 4-, 8-, 16-cell morula and Day 7 blastocyst stages between the two embryo sources. It can be concluded that, although in vitro bovine embryo culture in SOF medium under low (5%) O(2) concentration provided a more conducive environment in terms of blastocyst formation; differences in the total cell number and gene expression pattern between the IVP and IVD embryos reflected the effect of O(2) concentration.

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.

Enhanced development of porcine embryos cloned from bone marrow mesenchymal stem cells.

In the present study, we have characterized an isolated population of porcine bone marrow mesenchymal stem cells (MSCs) for multilineage commitment and compared the developmental potential of cloned embryos with porcine MSCs and fetal fibroblasts (FFs). MSCs exhibited robust alkaline phosphatase activity and later transformed into mineralized nodules following osteoinduction. Furthermore, MSCs underwent adipogenic and chondrogenic differentiation by producing lipid droplets and proteoglycans, respectively. Primary cultures of FFs from a female fetus at ~30 day of gestation were established. Donor cells at 3-4 passage were employed for nuclear transfer (NT). Cell cycle analysis showed that the majority of MSCs in confluence were in the G0/G1 stage. Cumulus-oocyte complexes were matured and fertilized in vitro (IVF) as control. The cleavage rate was significantly (P<0.05) higher in IVF than in NT embryos with MSCs and FFs (84.54.6% vs. 52.25.4% and 50.85.2%, respectively). However, blastocyst rates in IVF and NT embryos derived from MSCs (20.62.5% and 18.43.0%) did not differ, but were significantly (P<0.05) higher than NT derived from FFs (9.52.1%). Total cell number and the ratio of ICM to total cells among blastocysts cloned from MSCs (34.45.2 and 0.380.08, respectively) were significantly (P<0.05) higher than those from FFs (22.65.5 and 0.180.12, respectively). Proportions of TUNEL positive cells in NT embryos from FFs (7.31.8%) were significantly (P<0.05) higher than in MSCs (4.61.3%) and IVF (2.50.9%). The results clearly demonstrate that multipotent bone marrow MSCs have a greater potential as donor cells than FFs in achieving enhanced production of cloned porcine embryos.

Isolation and viability of presumptive spermatids collected from bull testes by Percoll density gradient.

The objective of the present study was to develop a procedure for isolating pure populations of round spermatid(s) (RS) by Percoll density gradient from bull testes. Bull testes were de-capsulated and testicular tissues were dissociated enzymatically to recover RS. After being filtered through a 20 microm nylon mesh, the cells were centrifuged at 650 x g for 25 min through the discontinuous Percoll density gradients (20, 35, 40, 45 and 90% Percoll solution). Isolated cells were analyzed by microscopic observation for survivability and apoptosis. In Experiment 1, both microscopic observation and DNA analysis by flow cytometry showed that approximately 40% of cells collected from 35% Percoll gradient were presumptive RS, whereas in 40% Percoll gradient, mostly primary spermatocytes were observed. Experiment 2 compared the effect of 35% Percoll density isolation on the incidence of apoptosis and necrosis in fresh and frozen-thawed cells to those of untreated cells. The percentage (mean+/-S.E.M.) of necrosis in cells collected from 35% Percoll gradient was less (P<0.05) than in untreated and frozen-thawed cells from 35% Percoll gradient (11.7+/-3.1% compared with 26.3+/-2.0% and 53.5+/-1.3%, respectively), but the rate of apoptosis did not differ (1.2+/-0.49% compared with 2.5+/-0.8% and 0.9+/-0.04%, respectively). The proportional data (mean+/-S.E.M.) of live cells in Percoll treated group were greater (P<0.05) than in untreated and frozen-thawed cells from the 35% Percoll gradient (86.7+/-3.26% compared with 70.8+/-2.73% and 41.9+/-1.69%, respectively). Experiment 3 compared the development rates of embryos injected with RS isolated from fresh and frozen-thawed cells collected with the 35% Percoll gradient to those of untreated cells, and parthenotes as control. There were no significant (P>0.05) differences in the rates of cleavage and blastocyst development between untreated fresh cells and fresh cells collected from the 35% Percoll gradient (75.4 and 10.5% compared with 82.4 and 12.8%). However, there were lesser (P<0.05) cleavage and blastocyst rates in frozen-thawed cells from the 35% Percoll gradient (51.6 and 6.3%) and parthenotes (60.7 and 4.1%) were observed. These results suggest that isolation of presumptive RS by 35% Percoll density gradient is effective in eliminating apoptotic and early necrotic cells. However, the use of RS in improving the developmental potential of embryos merits further studies.

In vitro development of bovine oocytes reconstructed with round spermatids.

The timing between round spermatid(s) (RS) injection and oocyte activation are critical for spermatid remodeling and embryo development in intracytoplasmic injection of round spermatid (ROSI) procedure. The objective of the present study was to develop an appropriate oocyte activation method for producing developmentally competent bovine embryos reconstructed with RS. Embryos reconstructed by ROSI were compared with three activation treatments for the rates of pronuclear formation, development and ploidy. RS were isolated from bull testes by Percoll density gradients. Matured oocytes were divided into three activation groups. In Group 1, oocytes were activated with ionomycin (5 microM, 5 min) before ROSI. In Group 2, oocytes were activated with ionomycin after ROSI. In Group 3, oocytes were activated twice with ionomycin before and after ROSI. All the eggs were then incubated in cycloheximide (CHX, 10 microg/mL) for 5 h and cultured in CR1aa medium for up to 8 days. Three methods of oocyte activation were also compared for the activation and development of parthenotes. Activation rates among the groups were 70-79% and did not differ. Cleavage rates in parthenotes were significantly (P < 0.05) higher in Group 3 than in Groups 1 and 2, but blastocyst rates did not differ among the groups. In ROSI embryos, the rates of cleavage and development into blastocysts were significantly (P < 0.05) greater in Group 3 (82.3% and 13.1%) than in Groups 1 and 2 (53.7, 5.8% and 64.2, 1.7%, respectively). Ploidy analysis by examining the metaphase spreads of ROSI blastocysts displayed greater numbers of diploid chromosomal complements. These results suggest that intracytoplasmic RS injection combined with repeated ionomycin activation followed by CHX treatment is more efficient for producing developmentally competent embryos.

Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos.

The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene (Qiagen, Inc.), a lipid-based reagent compared to electroporation in fetal-derived fibroblast cells (FFC), cumulus-derived fibroblast cells (CFC), and adult ear skin-derived fibroblast cells (AEFC). Parameters compared were factors such as chromosome abnormality, gene expression, and the incidence of apoptosis. Further, the TG embryos with transfected donor cells generated by electroporation or Effectene were compared to IVF and SCNT embryos in terms of rates of cleavage, blastocyst formation, and blastocyst cell number. Most of the cells (>80%) at confluence were at G0/G1 and considered to be suitable nuclear donors for cloning. Transfection with a plasmid containing the enhanced green fluorescent protein (pEGFP-N1) gene into FFC did not increase the incidence of chromosomal abnormalities. The rates of apoptosis in different cell types transfected with pEGFP-N1 were 3.3%-5.0%, and the values did not differ among groups. In addition, the rates of apoptosis in various cells between 5-7 and 20-22 cell passages did not differ. However, the efficiency of gene transfecton into FFC by Effectene reagent (14.2 +/- 1.7) was significantly (P < 0.05) higher than that obtained by electroporation (5.1 +/- 1.0). Among various cell types, the efficiency of gene transfection by Effectene and eletroporation of FFC (14.2 +/- 1.7 and 5.1 +/- 1.0, respectively) was significantly (P < 0.05) higher than transfection of CFC and AEFC by either method (9.4 +/- 1.5 and 3.3 +/- 0.8, 8.8 +/- 0.7, and 2.1 +/- 0.4, respectively). In TG embryos produced by SCNT with electroporation and Effectene, the rates of cleavage and blastocyst formation were significantly lower (P < 0.05) than those of IVF controls, but rates did not differ between SCNT and TG embryos. Similarly, significantly higher (P < 0.05) total cell numbers in day-8 blastocysts were observed in IVF controls than those in SCNT and TG embryos, but did not differ between SCNT and TG (136 vs. approximately 110, respectively). The results demonstrated that, though there were no difference in the rates of chromosomal aneuploidy and the incidence of apoptosis among various cell types, transfected with or without pEGFP-N1, FFC were the cell type most effectively transfected and Effectene was a suitable agent for transfection.