• Asian-Australasian Journal of Animal Sciences
• /
• v.29 no.10
• /
• pp.1407-1415
• /
• 2016

Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study, buffalo spermatogonial stem-like cells were isolated, cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubules were enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin, were determined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining in buffalo testes and buffalo spermatogonial stem-like cells, respectively. In the following, when the isolated buffalo buffalo spermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derived neurotrophic factor medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-like cells, and accelerating the generation of genetic modified buffaloes.

• Asian-Australasian Journal of Animal Sciences
• /
• v.23 no.9
• /
• pp.1152-1158
• /
• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.79-79
• /
• 2003

Embryonic stem cells are capable of differentiating into a variety of cell lineages. However, the ultimate results of differentiation in vitro greatly depend on the duration of treatment and kinds of differentiating inducers added. In order to investigate the efficiencies of various differentiation inducers and the methods of treatment, we examined differentiation patterns of human embryonic stem cell (hESC, MB03) according to several different protocols. Exp. I) Upon differentiation using retinoic acid and ascorbic acid (RA/AA), embryoid bodies (EB, for 4days) derived from hESC was exposed to Rh (10$^{-6}$ M) and AA (50 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14, 21, or 28 days. Exp. II) When bFGF was used, neuronal precursor cells were selected for 8 days in N2 medium after EB formation. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14, 21 or 28 days. Exp. III) In addition, to examine the effects of neurotrophic factors in the production of mature neurons, groups of cells were exposed to either BDNF (5 ng/ml) or TGF-$\alpha$(10 ng/ml) during the 28 days of final differentiation. Differentiation patterns of RA/AA or bFGF treated groups were very similar; approximately 82% and 83% of the cells, respectively, were positive for anti-NF200 antibody, while it was about 10% and 11%, respectively, for anti-NF160 antibody in 28 days in N2 medium. Alsor, cells expressing TH were as low as 5%, while the cells doubled when matured at the presence of either BDNF or TGF-$\alpha$. Cells immunoreactive to anti-GAD antibody were approximately 20%. These results suggest that a maturation step rather than differentiation induction step, which is formation of EB, effects more decisively to the ultimate differentiation pattern.

• Journal of Genetic Medicine
• /
• v.12 no.1
• /
• pp.31-37
• /
• 2015

Purpose: We investigated the neurogenic potentials of amniotic fluid-derived stem cells (AFSCs) according to the expression levels of stem cell markers and ingredients in the neural induction media. Materials and Methods: Four samples of AFSCs with different levels of Oct-4 and c-kit expression were differentiated neurally, using three kinds of induction media containing retinoic acid (RA) and/or a mixture of 3-isobutyl-1-methylxanthine/indomethacin/insulin (neuromix), and examined by immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR) for their expression of neurospecific markers. Results: The cells in neuromix-containing media displayed small nuclei and long processes that were characteristic of neural cells. RT-PCR analysis revealed that the number of neural markers showing upregulation was greater in cells cultured in the neuromix-containing media than in those cultured in RA-only medium. Neurospecific gene expression was also higher in Oct-4 and c-kit double-positive cells than in c-kit-low or -negative cells. Conclusion: The stem cell marker c-kit (rather than Oct-4) and the ingredient neuromix (rather than RA) exert greater effects on neurogenesis of AFSCs.

• Journal of Animal Reproduction and Biotechnology
• /
• v.38 no.2
• /
• pp.54-61
• /
• 2023

Background: Germ cells undergo towards male or female pathways to produce spermatozoa or oocyte respectively which is essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the potential of trans-differentiation to the multiple cell lineages. Methods: Herein, rat MSCs were isolated from bone marrow and characterized by their morphological features, expression of MSC surface markers, and in vitro differentiation capability. Results: Thereafter, we induced these cells only by retinoic acid supplementation in MSC medium and, could able to show that bone marrow derived MSCs are capable to trans-differentiate into male germ cell-like cells in vitro. We characterized these cells by morphological changes, the expressions of germ cell specific markers by immunophenotyping and molecular biology tools. Further, we quantified these differentiated cells. Conclusions: This study suggests that only Retinoic acid in culture medium could induce bone marrow MSCs to differentiate germ cell-like cells in vitro. This basic method of germ cell generation might be helpful in the prospective applications of this technology.

• Molecules and Cells
• /
• v.38 no.7
• /
• pp.643-650
• /
• 2015

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.

• Reproductive and Developmental Biology
• /
• v.39 no.3
• /
• pp.89-95
• /
• 2015

The addition of growth factors and cytokines to in vitro culture (IVC) media could affect embryo development and the quality of the resulting blastocysts. The present study was performed to investigate the effect of porcine induced pluripotent stem cell (piPSC)-culture conditioned medium (CM) on the in vitro maturation (IVM) and development of parthenogentic embryos (parthenotes) in pigs. Cumulus-oocyte complexes (COCs) or activated oocytes were cultured in IVM or IVC medium supplemented with 0 (control), 25, or 50% of stem cell medium (SM) or CM, respectively. The maturation rate of CM-25% group was significantly improved when compared with control group (p<0.05), but that was not different among SM or CM groups. Blastocyst formation rate was significantly higher in CM-25% group (29.2%) than that of control (20.7%), SM-50% (19.6%) and CM-50% (23.66%, p<0.05). Cell number and the apoptotic cell index in blastocysts was significantly lower in SM-25% than in CM-25% group (p<0.05). The embryo quality related genes, OCT4, KLF4, TERT and ZFP42, were significantly increased in CM-25% group compared with control (p<0.05). In conclusion, the addition of 25% of CM to IVM and IVC medium positively influences not only the developmental potential also quality of parthenotes in pig.

• Development and Reproduction
• /
• v.21 no.3
• /
• pp.335-342
• /
• 2017

While adipose-derived stem cell-conditioned medium (ADSC-CM) has been demonstrated to promote skin wound healing, the mechanism regulating this effect remains unelucidated. In this study, we aimed to investigate the role of Ell3 in the wound healing activity of ADSC-CM. In vitro analysis revealed that Ell3 suppression in ADSCs impairs the promotive activity of ADSC-CM on the proliferation and migration of mouse embryonic fibroblasts (MEF) and normal human dermal fibroblasts (NHDF). Consistently, the expression of MMP family genes, which regulate cell proliferation and migration, was significantly suppressed in MEF and NHDF treated with siEll3-transfected ADSC-CM. Proinflammatory cytokines, such as interleukin-1 and interleukin-6, were highly expressed in MEF treated with siEll3-transfected ADSC-CM. The wound healing activity of siEll3-transfected ADSC-CM was significantly lower than that of the control in vivo. Our results suggest that Ell3 may contribute to the inhibition of inflammatory response during skin wound healing.

• Reproductive and Developmental Biology
• /
• v.36 no.4
• /
• pp.291-294
• /
• 2012

Embryonic stem cell classically cultured on feeder layer with FBS contained ES medium. Feeder-free mouse ES cell culture systems are essential to avoid the possible contamination of nonES cells. First we determined the difference between ES cell and MEF by Oct4 population. We demonstrate to culture and to induce differentiation on feeder free condition using a commercially available mouse ES cell lines.

• Journal of Animal Reproduction and Biotechnology
• /
• v.35 no.1
• /
• pp.21-27
• /
• 2020

Somatic cell nuclear transfer derived embryonic stem cells (NT-ESCs) have significant advantages in various fields such as genetics, embryology, stem cell science, and regenerative medicine. However, the poor establishment of NT-ESCs hinders various research. Here, we applied fasudil, a Rho-associated kinase (ROCK) inhibitor, to develop somatic cell nuclear transfer (SCNT) embryos and establish NT-ESCs. In the study, MII oocytes were isolated from female B6D2F1 mice and performed SCNT with mouse embryonic fibroblasts (MEFs). The reconstructed NT-oocytes were activated artificially, and cultured to blastocysts in KSOM supplemented with 10 μM fasudil. Further, the blastocysts were seeded on inactivated MEFs in embryonic stem cell medium supplemented with 10 μM fasudil. A total of 26% of embryos formed into blastocysts in the fasudil treated group, while this ratio was 44% in the fasudil free control group. On the other hand, 30% of blastocysts were established NT-ESCs after exposure of fasudil, which was significantly higher than the control group (10%). The results suggest that fasudil reduced blastocyst development after SCNT due to inhibition of 2 cell cleavage while improved the establishment of NT-ESCs through the anti-apoptotic pathway.