Case report: Spontaneous abortion of monoamniotic twins at the third trimester of pregnancy in Camelus dromedarius.

Monoamniotic twins develop when a blastocyst spontaneously splits its progenitor cells, and each group of progenitor cells independently grows to become an individual. It is the rarest type of twin pregnancy and usually has significant developmental or congenital abnormalities, a higher rate of abortion, perinatal morbidity, and mortality. There is no information regarding monoamniotic twins in livestock species. Here, we reported a spontaneous abortion of monoamniotic twins in a dromedary camel at 278 days of gestation. Gonadorelin acetate (100 µg) was injected intramuscularly to induce ovulation in the recipient. A 7 days-old embryo produced by somatic cell nuclear transfer was transferred transcervically to the recipient. Early pregnancy was confirmed by an elevated level of serum progesterone followed by ultrasonography at 22 and 44 days after embryo transfer. A single sac was observed on 22 days while twins were evident 44 days after embryo transfer. Pregnancy was periodically monitored by the tail-up phenomenon. A ruptured fetal sac was observed on the ground having two fetuses. On autopsy, full-grown fetuses were found. Their bodies were separated. There was no congenital anomaly or any malformation in the fetuses. According to the reported chronology in human twins, we hypothesized that the blastocyst splitted before 13 days as it was monoamniotic and not conjoined. If the embryo splits within 4 to 8 days, it develops two amniotic sacs, and splitting after 13 days develops conjoined fetuses. To the authors' knowledge, this is the first reported case of monoamniotic twin abortion in dromedary camels. This report will increase awareness among practicing veterinarians and camel breeders about twin abortions.

Production of transgenic first filial puppies expressing mutated human amyloid precursor protein gene.

Propagation of transgenic animals by germline transmission using assisted reproductive technologies such as in vitro fertilization (IVF) is the most efficient way to produce transgenic colonies for biomedical research. The objective of this study was to generate transgenic puppies from a founder dog expressing the mutated human amyloid precursor protein (mhAPP) gene. Experiment I assessed the characteristics of the semen prepared by freshly diluted, swim-up, and Percoll gradient methods using a computer-assisted semen analyzer (CASA). Motile and progressively motile sperm counts were higher in the Percoll gradient samples (p < 0.05) than in the swim-up and freshly diluted samples. In Experiment II, a total of 59, 70, and 65 presumptive zygotes produced by fresh, Percoll gradient, and swim-up methods, respectively, were transferred to surrogates (5 for each group); the Percoll gradient (27.27%) and swim-up samples (14.29%) showed the highest blastocyst formation rates, while fresh diluted semen did not produce any blastocyst. Experiment III examined the full-term developmental ability of embryos. Among the 5 surrogates in the Percoll gradient group, one (20.0%) became pregnant; it had 4 (6.15%) sacs and delivered 4 (6.15%; 2 males and 2 females) live puppies. Among the 4 puppies, 2 (50.0%) were found to transmit the transgene on their nail and toe under GFP fluorescence. Furthermore, the integration and expression of the mhAPP transgene were examined in the umbilical cords of all the IVF-derived puppies, and the presence of the transgene was only observed in the GFP-positive puppies. Thus, semen prepared by the Percoll method could generate transgenic puppies by male germline transmission using the IVF technique. Our result will help propagate transgenic dogs efficiently, which will foster human biomedical research.

Insights from one thousand cloned dogs.

Animal cloning has been popularized for more than two decades, since the birth of Dolly the Sheep 25 years ago in 1996. There has been an apparent waning of interest in cloning, evident by a reduced number of reports. Over 1500 dogs, representing approximately 20% of the American Kennel Club's recognized breeds, have now been cloned, making the dog (Canis familiaris) one of the most successfully cloned mammals. Dogs have a unique relationship with humans, dating to prehistory, and a high degree of genome homology to humans. A number of phenotypic variations, rarely recorded in natural reproduction have been observed in in these more than 1000 clones. These observations differ between donors and their clones, and between clones from the same donor, indicating a non-genetic effect. These differences cannot be fully explained by current understandings but point to epigenetic and cellular reprograming effects of somatic cell nuclear transfer. Notably, some phenotypic variations have been reversed through further cloning. Here we summarize these observations and elaborate on the cloning procedure.

Case Report: Spontaneous Fetal Demises at Third Trimester of Pregnancy Due to a Double Lopped Nuchal Cord in Camelus dromedarius.

The umbilical cord acts as the critical lifeline of the developing fetus by providing nutrients and oxygen to it. Umbilical cord abnormalities are considered the leading cause of stillbirth in humans, but information on stillbirths associated with umbilical cord abnormalities is very scant in the clinical practice of animals. Here, we described a case of fetal demise in camels indicated to be caused by fetal death from strangulation by its umbilical cord, which is commonly known as the nuchal cord. A pregnant camel at its 36 weeks of gestation spontaneously aborted a single fetus. The camel was 5 years old and nullipara. A 6-day-old cloned embryo was transferred transcervically to the recipient. Pregnancy was confirmed 50 days after embryo transfer by ultrasonography, and the pregnant camel was maintained under a standard nutritional plan. The neck of the aborted fetus was strangulated tightly by a double loop of the umbilical cord. There was no congenital anomaly or other malformation in the fetus. We concluded that the nuchal cord was tightly coiled around the neck of the fetus and interfered with the blood flow in the fetus by collapsing the umbilical vein and subsequently causing fetal death and abortion. To the authors' knowledge, this is the first reported case of a nuchal cord in camels.

Impact of co-transfer of embryos produced by somatic cell nuclear transfer using two types of donor cells on pregnancy outcomes in dogs.

OBJECTIVE: The present study analyzed the influence of co-transferring embryos with high and low cloning efficiencies produced via somatic cell nuclear transfer (SCNT) on pregnancy outcomes in dogs. METHODS: Cloned dogs were produced by SCNT using donor cells derived from a Tibetan Mastiff (TM) and Toy Poodle (TP). The in vivo developmental capacity of cloned embryos was evaluated. The pregnancy and parturition rates were determined following single transfer of 284 fused oocytes into 21 surrogates and co-transfer of 47 fused oocytes into four surrogates. RESULTS: When cloned embryos produced using a single type of donor cell were transferred into surrogates, the pregnancy and live birth rates were significantly higher following transfer of embryos produced using TP donor cells than following transfer of embryos produced using TM donor cells. Next, pregnancy and live birth rates were compared following single and co-transfer of these cloned embryos. The pregnancy and live birth rates were similar upon co-transfer of embryos and single transfer of embryos produced using TP donor cells but were significantly lower upon single transfer of embryos produced using TM donor cells. Furthermore, the parturition rate for TM dogs and the percentage of these dogs that remained alive until weaning was significantly higher upon co-transfer than upon single transfer of embryos. However, there was no difference between the two embryo transfer methods for TP dogs. The mean birth weight of cloned TM dogs was significantly higher upon single transfer than upon co-transfer of embryos. However, the body weight of TM dogs did not significantly differ between the two embryo transfer methods after day 5. CONCLUSION: For cloned embryos with a lower developmental competence, the parturition rate and percentage of dogs that remain alive until weaning are increased when they are co-transferred with cloned embryos with a greater developmental competence.

Influence of PMSG on Superstimulation and Embryo Development Following Somatic Cell Nuclear Transfer in Holstein Cows in the United Arab Emirates.

The present study investigated the effect of superstimulation to improve in vitro embryo production in the Gulf area, where the temperature is high. Holstein cows were classified into the control and superstimulation groups. Superstimulation was induced with a single intramuscular injection of pregnant mare serum gonadotropin (PMSG; 2500 IU) on day 14 of the estrus cycle (day 0; estrus). The development of follicles was evaluated by ultrasonography of the ovaries daily. At 40 h after the PMSG injection, oocytes were collected by the ovum pick-up (OPU) technique. OPU was performed at the same stage of the estrus cycle in the control group as in the superstimulation group. The number of follicles with a diameter of more than 6 mm and the number of retrieved cumulus-oocyte complexes were significantly higher in the superstimulation group than in the control group. Furthermore, the maturation rate was higher in the superstimulation group than in the control group. Cloned embryos were produced by somatic cell nuclear transfer using matured oocytes. The cleavage and blastocyst formation rates were significantly higher in the superstimulation group than in the control group. In conclusion, a single injection of PMSG can facilitate the efficient production of cloned cow embryos.

Comparison of pregnancy rate in dromedary camel between early-stage embryos and blastocyst transfer produced by somatic cell nuclear transfer using in vitro-matured oocytes.

We compared the pregnancy and live birth rates following transfer of early-stage embryos or blastocysts produced by somatic cell nuclear transfer using in vitro-matured oocytes. In total 102 ovaries were collected from dromedary camels at a local abattoir; from these 1048 cumulus-oocytes complexes (COCs) were aspirated and cultured for 42 h in a commercial maturation medium. Metaphase II oocytes were subjected to nuclear transfer. Somatic cell nuclear transfer-derived embryos were cultured in a commercial embryo medium for 2 or 7 days. Next, 71 early-stage embryos were surgically transferred to the left fallopian tube of 28 recipients and 47 blastocysts were transferred to the left uterine horn of 26 recipients. Early pregnancy was detected by serum progesterone (P4), and pregnancy was confirmed using ultrasonography on days 30 and 90 after embryo transfer. Pregnancy rate based on P4 level was 17.86% (5/28) and 11.54% (3/26) for early-stage embryo and blastocyst transfer, respectively. In the early-stage embryo group, out of five recipients, one recipient had lost the pregnancy by the first ultrasonography on day 30; two other recipients aborted at 14 and 24 weeks, and two recipients gave live births. In the blastocyst group, out of three recipients, one lost the pregnancy at an early stage and two recipients gave live births. Therefore, for dromedary camels, we recommend transvaginal blastocyst transfer from the standpoint of the pregnancy and live birth rate, ease of the transfer procedure, and comfort and safety of the recipients.

Development and pregnancy rates of Camelus dromedarius-cloned embryos derived from in vivo- and in vitro-matured oocytes.

OBJECTIVE: The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius. METHODS: Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method. RESULTS: The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitromatured oocytes. CONCLUSION: In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

The Resurrection of Mabrokan: Production of Multiple Cloned Offspring from Decade-Old Vitrified Tissue Collected from a Deceased Champion Show Camel.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to reproduce animals with superior genetics. Viable cell lines are usually established from tissues collected by biopsy from living animals in the SCNT program. In the present study, tissues were collected and preserved from a suddenly deceased champion camel. We established cell lines from these decade-old tissues and used them as nuclear donors. After 42 h of in vitro maturation, 68.00 ± 2.40% of oocytes reached the metaphase II (M II) stage while 87.31 ± 2.57% in vivo collected oocytes were matured at collection (p < 0.05). We observed a higher blastocyst formation rate when in vivo matured oocytes (43.45 ± 2.07%) were used compared to in vitro matured oocytes (21.52 ± 1.74%). The live birth rate was 6.45% vs. 16.67% for in vitro and in vivo matured oocytes, respectively. Microsatellite analysis of 13 camel loci revealed that all the SCNT-derived offspring were identical to each other and with their somatic cell donor. The present study succeeded in the resurrection of 11 healthy offspring from the decade-old vitrified tissues of a single somatic cell donor individual using both in vitro and in vivo matured oocytes.

Comparison of pregnancy outcomes following the transfer of early-developmental stage embryos and blastocysts produced by somatic cell nuclear transfer in Camelus dromedarius.

The embryonic stage, site of embryo transfer in the reproductive tract of the surrogate, and embryo transfer method are important for the successful production of offspring. In the present study, there was comparison of pregnancy rates in camels following the surgical transfer of early-developmental stage embryos at Day 2 and transvaginal transfer of blastocysts at Day 7. Embryos were produced by somatic cell nuclear transfer using in vivo-matured oocytes and ear fibroblasts as donor cells. A total of 305 oocytes were collected from 27 donors, among which 275 oocytes were in metaphase II. In Group A, 110 oocytes were reconstructed, 78 fused oocytes were cultured for 2 days, and 37 early-developmental stage embryos were transferred into 13 surrogates. In Group B, 165 oocytes were utilized, 117 fused oocytes were cultured for 7 days, and 24 blastocysts were trans-vaginally transferred into 12 surrogates. Pregnancy was determined when there was an increase in serum progesterone concentrations and was confirmed using real-time ultrasonography. Microsatellite analysis was performed to confirm the parentage of offspring. Two live births occurred in Groups A and B (live birth rate of 15.4% and 16.7%, respectively). Results indicate both early-developmental stage embryos and blastocysts produced by somatic cell nuclear transfer using in vivo-matured oocytes can lead to live births in camel with similar efficiency. It, therefore, is recommended that trans-vaginal blastocyst transfer be utilized for camels considering the pregnancy and live birth rates, ease of the transfer procedure and comfort and safety of surrogates.

Vitrification of Dog Skin Tissue as a Source of Mesenchymal Stem Cells.

The purpose of this study was to develop an efficient vitrification system for cryopreservation of dog skin tissues as a source of stable autologous stem cells. In this study, we performed vitrification using four different cryoprotectants, namely, ethylene glycol (EG), dimethyl-sulfoxide (Me2SO), EG plus Me2SO, and EG plus Me2SO plus sucrose, and analyzed the behaviors of cells established from warmed tissues. Tissues vitrified with 15% EG, 15% Me2SO, and 0.5 M sucrose had a normal histological appearance and the highest cell viability after cell isolation, and thus, this cocktail of cryoprotectants was used in subsequent experiments. We evaluated proliferation and apoptosis of cells derived from fresh and vitrified tissues. These cells had a normal spindle-like morphology after homogenization through subculture. Dog dermal skin stem cells (dDSSCs) derived from fresh and vitrified tissues had similar proliferation capacities, and similar percentages of these cells were positive for mesenchymal stem cell markers at passage 3. The percentage of apoptotic cell did not differ between dDSSCs derived from fresh and vitrified tissues. Real-time PCR analysis revealed that dDSSCs at passage 3 derived from fresh and vitrified tissues had similar expression levels of pluripotency (OCT4, SOX2, and NANOG), proapoptotic (BAX), and antiapoptotic (BCL2 and BIRC5) genes. Both types of dDSSCs successfully differentiated into the mesenchymal lineage (adipocytes and osteocytes) under specific conditions, and their differentiation potentials did not significantly differ. Furthermore, the mitochondrial membrane potential of dDSSCs derived from vitrified tissues was comparable with that of dDSSCs derived from fresh tissues. We conclude that vitrification of dog skin tissues using cocktail solution in combination of 15% EG, 15% Me2SO, and 0.5 M sucrose allows efficient banking of these tissues for regenerative stem cell therapy and conservation of genetic resources.

Comparison of Pluripotency, Differentiation, and Mitochondrial Metabolism Capacity in Three-Dimensional Spheroid Formation of Dental Pulp-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are valuable candidates in tissue engineering and stem cell-based therapy. Traditionally, MSCs derived from various tissues have been successfully expanded in vitro using adherent culture plates commonly called as monolayer two-dimensional (2D) cultures. Recently, many studies demonstrated that stemness and multilineage differentiation potential could be enhanced to greater extent when MSCs are cultured as suspended aggregates by means of three-dimensional (3D) culturing techniques. However, there are limited reports on changed mitochondrial metabolism on 3D spheroid formation of MSCs. Therefore, the present study was aimed at investigating the stemness, differentiation potential, and mitochondrial metabolism capacity of 3D dental pulp-derived MSC (DPSC) spheroids in comparison to monolayer cultured DPSCs. We isolated dental pulp-derived MSCs (DPSCs) and successfully developed a 3D culture system which facilitated the formation of MSC spheroids. The cell aggregation was observed after 2 hours, and spheroids were formed after 24 hours and remained in shape for 72 hours. After spheroid formation, the levels of pluripotent markers increased along with enhancement in adipogenic and osteogenic potential compared to 2D cultured control cells. However, decreased proliferative capacity, cell cycle arrest, and elevated apoptosis rate were observed with the time course of the 3D culture except for the initial 24-hour aggregation. Furthermore, oxygen consumption rates of living cells decreased with the time course of the aggregation except for the initial 24 hours. Overall, our study indicated that the short-term 3D culture of MSCs could be a suitable alternative to culture the cells.

Comparative evaluation of three different formulas for predicting the parturition date of German Shepherds following somatic cell nuclear transfer.

Several studies have reported methods to estimate the parturition date of dogs using ultrasonographic measurements. However, these prediction models were mainly determined using ultrasonographic measurements of naturally pregnant small- and medium-sized dogs, and no such studies have been performed using dogs carrying cloned fetuses produced via somatic cell nuclear transfer. The present study evaluated the abilities of three reference formulas (Luvoni and Grioni, Milani et al., and Groppetti et al.), all of which were developed using data from naturally occurring pregnancies, to accurately predict the parturition date in surrogates carrying cloned German Shepherd (GS) fetuses. All three formulas were based on the use of inner chorionic cavity diameter (ICC) measurements, obtained via ultrasonography. For evaluation, a total of 54 ICC measurements were collected from 14 pregnant bitches carrying cloned GS fetuses. We found that the clinical accuracy of the breed-specific Groppetti et al. formula was highest among those of the three formulas tested, with 87% and 100% of the estimated parturition dates (calculated based on the ICC measurements) being within 1 and 2 days, respectively, of the actual delivery date. By contrast, the Luvoni and Grioni formula showed relatively low accuracy, and the Milani et al. formula showed higher accuracy than that reported previously for natural pregnancies.

Cell Source-Dependent In Vitro Chondrogenic Differentiation Potential of Mesenchymal Stem Cell Established from Bone Marrow and Synovial Fluid of Camelus dromedarius.

Mesenchymal stem cells (MSCs) are promising multipotent cells with applications for cartilage tissue regeneration in stem cell-based therapies. In cartilage regeneration, both bone marrow (BM-MSCs) and synovial fluid (SF-MSCs) are valuable sources. However, the cellular characteristics and chondrocyte differentiation potential were not reported in either of the camel stem cells. The in vitro chondrocyte differentiation competence of MSCs, from (BM and SF) sources of the same Camelus dromedaries (camel) donor, was determined. Both MSCs were evaluated on pluripotent markers and proliferation capacity. After passage three, both MSCs showed fibroblast-like morphology. The proliferation capacity was significantly increased in SF-MSCs compared to BM-MSCs. Furthermore, SF-MSCs showed an enhanced expression of transcription factors than BM-MSCs. SF-MSCs exhibited lower differentiation potential toward adipocytes than BM-MSCs. However, the osteoblast differentiation potential was similar in MSCs from both sources. Chondrogenic pellets obtained from SF-MSCs revealed higher levels of chondrocyte-specific markers than those from BM-MSCs. Additionally, glycosaminoglycan (GAG) content was elevated in SF-MSCs related to BM-MSCs. This is, to our knowledge, the first study to establish BM-MSCs and SF-MSCs from the same donor and to demonstrate in vitro differentiation potential into chondrocytes in camels.

Comparative Study of Biological Characteristics, and Osteoblast Differentiation of Mesenchymal Stem Cell Established from Camelus dromedarius Skeletal Muscle, Dermal Skin, and Adipose Tissues.

Mesenchymal stem cells (MSCs) showed in vitro mesoderm-lineage differentiation and self-renewal capacity. However, no comparative study was reported on the biological characteristics of stem cells derived from skeletal muscle (SM-MSCs), dermal skin (DS-MSCs), and adipose tissues (A-MSCs) from a single donor in camels. The present study aimed to evaluate the influence of MSCs source on stem cell characteristics. We evaluated proliferation capacity and mesoderm-lineage differentiation potential from SM-MSCs, DS-MSCs, and A-MSCs. They showed spindle-like morphology after homogenization. The proliferation ability was not significantly difference in any of the groups. Furthermore, the portion of the cell cycle and expression of pluripotent markers (Oct4, Sox2, and Nanog) were similar in all cell lines at passage 3. The differentiation capacity of A-MSCs into adipocytes was significantly higher than that of SM-MSCs and DS-MSCs. However, the osteoblast differentiation capacity of A-MSCs was significantly lower than that of SM-MSCs and DS-MSCs. Additionally, after osteoblast differentiation, the alkaline phosphatase (ALP) activity and calcium content significantly decreased in A-MSCs compared to SM-MSCs and DS-MSCs. To the best of our knowledge, we primarily established MSCs from the single camel and demonstrated their comparative characteristics, including expression of pluripotent factors and proliferation, and in vitro differentiation capacity into adipocytes and osteoblasts.

Mitochondrial metabolism assessment of lycaon-dog fetuses in interspecies somatic cell nuclear transfer.

Many studies have reported that interspecies somatic cell nuclear transfer (iSCNT) is considered the prominent method in preserving endangered animals. However, the development rate of iSCNT embryos is low, and there are limited studies on the molecular mechanism of the iSCNT process. This study evaluated the developmental potential of interspecies lycaon (Lycaon pictus)-dog embryos and assessed the mitochondrial content and metabolism of the produced cloned lycaon-dog fetus. Of 678 collected oocytes, 516 were subjected to nuclear transfer, and 419 reconstructed embryos with male lycaon fibroblasts were transferred into 27 surrogates. Of 720 oocytes, 568 were subjected to nuclear transfer and 469 reconstructed embryos with female lycaon fibroblasts were transferred into 31 surrogates. Two recipients who received female reconstructed embryos were identified as pregnant at 30 days. However, fetal retardation with no cardiac activity was observed at 46 days. Microsatellite analysis confirmed that the cloned lycaon-dog fetus was genetically identical to the lycaon donor cell, whereas mitochondrial sequencing analysis revealed that oocyte donor dogs transmitted their mtDNA. We assessed the oxygen consumption rate and mitochondrial content of the aborted lycaon-dog fetus to shed some light on the aborted fetus's cellular metabolism. The oxygen consumption rates in the lycaon-dog fetal fibroblasts were lower than those in adult dog, lycaon and cloned dog fetal fibroblasts. Furthermore, lycaon-dog fetal fibroblasts showed decreased proportions of live and active mitochondria compared with other groups. Overall, we hypothesized that nuclear-mitochondrial incompatibility affects pyruvate metabolism and that these processes cause intrauterine fetal death.

Hematological patterns and histopathological assessment of Miniature Pigs in the experiments on human mesenchymal stem cell transplantation.

Background: Multipotent and immune privileged properties of mesenchymal stem cells (MSCs) were investigated for the treatment of various clinical diseases. For the years, many researches into the animal studies evaluated human stem cell therapeutic capacity related to the regenerative medicine. However, there were limited reports on immune privileged properties of human MSCs in animal studies. The present study investigated hematological and biochemical parameter and lymphocyte subset in mini-pigs following human MSCs transplantation as a means of validation of reliability that influence the animal test results. Methods: The miniature pigs were transplanted with human MSCs seeded with scaffold. After transplantation, all animals were evaluated by CBC, biochemistry and lymphocyte subset test. After 9 weeks, all pigs were sacrificed and organs were histologically analyzed. Results: CBC test showed that levels of RBC were decreased and reticulocyte, WBC and neutrophil were increased in transient state initially after transplantation, but returned to normal value. The proportion of B lymphocyte and cytotoxic T cell were also initially enhanced within the normal range temporarily. The female and male miniature pigs showed normal ranges for blood chemistry assessments. During the 9 weeks post-operative period, the animals showed a continuous increase in body weight and length. Furthermore, no abnormal findings were observed from the histological analysis of sacrificed pigs. Conclusions: Overall, miniature pigs transplanted with human MSCs seeded with scaffold were found to have physiologically similar results to normal animals. This result might be a reliable indicator of the animal experiments using miniature pigs with human MSCs.

Evaluation of odonto/osteogenic differentiation potential from different regions derived dental tissue stem cells and effect of 17ß-estradiol on efficiency.

BACKGROUND: The dentin is a tissue, which is formed by odontoblasts at the pulp interface of the teeth that supports the enamel. Odontoblasts, the cranial neural crest cells are derived from ectodermal mesenchymal stem cells (MSCs) and are long and polarized cells. They are present at the outer surface of dentin and play a prominent role about dentin formation. Recently, attention has been focused on induction of odontoblast using various type of MSCs and effects of the 17ß-estradiol supplementation. In this study, we establish an efficient odonto/osteoblast differentiation protocol using 17ß-estradiol supplementation while comparing the odonto/osteoblast ability of various dental MSCs. METHODS: Same donor derived four types of dental MSCs namely dental pulp stem cells (DPSCs), stem cells from apical papilla (SCAP), dental follicle stem cells (DFSCs), and periodontal ligament stem cells (PDLSCs) were evaluated for their stemness characteristics and potency towards odonto/osteoblast (Induced odonto/osteoblast) differentiation. Then 17ß-estradiol supplementation of 0 and 10 µM was applied to the odonto/osteoblast differentiation media for 14 days respectively. Furthermore, mRNA and protein levels of odonto/osteoblast markers were evaluated. RESULTS: All of the experimental groups displayed stemness characteristics by showing adipocyte and chondrocyte differentiation abilities, expression for cell surface markers and cell proliferation capacity without any significant differences. Moreover, all dental derived MSCs were shown to have odonto/osteoblast differentiation ability when cultured under specific conditions and also showed positive expression for odontoblast markers at both mRNA and protein level. Among all, DPSCs revealed the higher differentiation potential than other dental MSCs. Furthermore, odonto/osteoblast differentiation potential was enhanced by supplementing the differentiation media with 17ß-estradiol (E2). CONCLUSIONS: Thus, DPSCs possess higher odonto/osteogenic potential than the SCAPs, DFSCs, PDLSCs and their differentiation capacity can by further enhanced under E2 supplementation.

Involvement of mitochondrial biogenesis during the differentiation of human periosteum-derived mesenchymal stem cells into adipocytes, chondrocytes and osteocytes.

Due to a rapidly expanding aging population, the incidence of age-related or degenerative diseases has increased, and efforts to handle the issue with regenerative medicine via adult stem cells have become more important. And it is now clear that the mitochondrial energy metabolism is important for stem cell differentiation. When stem cells commit to differentiate, glycolytic metabolism is being shifted to mitochondrial oxidative phosphorylation (OXPHOS) to meet an increased cellular energy demand required for differentiated cells. However, the nature of cellular metabolisms during the differentiation process of periosteum-derived mesenchymal stem cells (POMSC) is still unclear. In the present study, we investigated mitochondrial biogenesis during the adipogenic, chondrogenic, and osteogenic differentiation of POMSCs. Both mitochondrial DNA (mtDNA) contents and mitochondrial proteins (VDAC and mitochondrial OXPHOS complex subunits) were increased during all of these mesenchymal lineage differentiations of POMSCs. Interestingly, glycolytic metabolism is reduced as POMSCs undergo osteogenic differentiation. Furthermore, reducing mtDNA contents by ethidium bromide treatments prevents osteogenic differentiation of POMSCs. In conclusion, these results indicate that mitochondrial biogenesis and OXPHOS metabolism play important roles in the differentiation of POMCS and suggest that pharmaceutical modulation of mitochondrial biogenesis and/or function can be a novel regulation for POMSC differentiation and regenerative medicine.