Search

Background and objectives Outbred mice are widely used in toxicology, pharmacology, and fundamental biomedical research. However, there have been no reports of in vitro culture systems for spermatogonial stem cells (SSCs) derived from these mice. Methods As a step towards constructing a non-cellular niche supporting the in vitro maintenance of outbred mouse SSC self-renewal, we systematically investigated the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in SSCs derived from Imprinting Control Region (ICR) mice. Results Among the genes encoding 25 integrin subunits, integrin α1, α5, α6, α9, αV, and αE, and integrin β1 and β5 had significantly higher transcriptional levels than the other subunits. Furthermore, at the translational level, integrin α5, α6, α9, αV, and αE, and β1 were localized on the surface of SSCs, but integrin α1 and β5 not. Moreover, significantly stronger translational expression than integrin α9 and αE was observed in integrin α5, α6, αV, and β1. SSCs showed significantly increased adhesion to fibronectin, laminin, tenascin C and vitronectin, and functional blocking of integrin α5β1, α6β1, α9β1 or αVβ1 significantly inhibited adhesion to these molecules. Conclusions We confirmed that integrin α5β1, α6β1, α9β1 and αVβ1 actively function on the surface of undifferentiated SSCs derived from outbred ICR mice.

Previously, we reported that endometrial stromal (ES) and endometrial epithelial (EE) cells did not attach to tenascin C, indicating the absence of active integrin α9β1 on the surface of mouse ES and EE cells. However, that study used recombinant tenascin C without fibronectin (FN) type III repeats interacting with integrin heterodimers. Therefore, we re-evaluated the presence of integrin α9β1 actively functioning on the surface of mouse ES and EE cells using full-length native tenascin C with FN type III repeats. The functionality of integrin α9β1 was confirmed using attachment and antibody inhibition assays. Both mouse ES and EE cells showed significantly increased adhesion to native tenascin C, and functional blocking of integrin α9β1 significantly inhibited adhesion to native tenascin C. These results demonstrate that the integrin α9 and β1 subunits function as active heterodimers on the plasma membrane of mouse ES and EE cells, respectively.

Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics similar to humans have been requested for developing stem cell-based bioengineering techniques directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old male outbred ICR mice (ICRESCs). Similar to those from 129/Ola mouse blastocysts (E14ESCs), the established ICRESCs showed inherent characteristics of ESCs except for partial and weak protein expression and activity of alkaline phosphatase. Moreover, ICRESCs were not originated from embryonic germ cells or pluripotent cells that may co-exist in outbred ICR strain-derived mouse embryonic fibroblasts (ICRMEFs) used for deriving colonies from inner cell mass of outbred ICR mouse blastocysts. Furthermore, instead of outbred ICRMEFs, hybrid B6CBAF1MEFs as feeder cells could sufficiently support in vitro maintenance of ICRESC self-renewal. Additionally, ICRESC-specific characteristics (self-renewal, pluripotency, and chromosomal normality) were observed in ICRESCs cultured for 40th subpassages (164 days) on B6CBAF1MEFs without any alterations. These results confirmed the successful establishment of ESCs derived from outbred ICR mice, and indicated that self-renewal and pluripotency of the established ICRESCs could be maintained on B6CBAF1MEFs in culture.

본 연구는 그림책 부모참여 프로그램에 참여한 어머니들의 경험과 인식 변화를 알아보고자 하였다. 이를 위해 어머니 8명을 연구 참여자로 하여, 8주간 주1회 2시간씩 그림책에 대한 지식을 교육하고 도서대여를 실시하였다. 연구자료는 그룹면담과 개별면담 2회를 실시하여 녹음하고 QSR NVivo 10.0 프로그램으로 분석하였다. 분석 결과, 첫째, 그림책 부모참여프로그램에 관한 긍정적 경험은 지속적인 부모참여로써의 그림책 읽어주기로 글과 그림, 파라텍스트 읽어주기, 그림책 장르 알아가기, 좋은 그림책 찾아가기, 삶 속에 자리매김하는 그림책 읽어주기, 각자에게 적절한 그림책 읽어주기의 횟수 찾아가기로 나타났다. 둘째, 그림책 부모참여 프로그램을 통한 인식 변화에는 책 읽어주기의 진정한 가치 찾아가기, 부담감에서 벗어나 함께 책 읽는 즐거움 향유하기, 다른 사람들과 서평 공유할 기회 누리기 등이 있었다. 본 연구 결과를 통해 어머니들이 어린이문학에 대한 이해를 확장하고 그림책 읽어주기를 실천할 수 있도록 능동적인 부모참여 프로그램의 필요성이 제기되었다.

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α9 and β1 subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII-like repeats. Therefore, this study re-evaluated the existence of integrin α9 β1 actively functioning on the plasma membrane of porcine SSCs using full-length native tenascin C with FNIII-like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α9 β1 on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α9 β1 significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α9 and β1 subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.

Although somatic cell nuclear transfer (SCNT)-derived embryonic stem cells (ESCs) in pigs have great potential, their use is limited because the establishment efficiency of ESCs is extremely low. Accordingly, we tried to develop in-vitro culture system stimulating production of SCNT blastocysts with high performance in the colony formation and formation of colonies derived from SCNT blastocysts for enhancing production efficiency of porcine ESCs. For these, SCNT blastocysts produced in various types of embryo culture medium were cultured in different ESC culture medium and optimal culture medium was determined by comparing colony formation efficiency. As the results, ICM of porcine SCNT blastocysts produced through sequential culture of porcine SCNT embryos in the modified porcine zygote medium (PZM)-5 and the PZM-5F showed the best formation efficiency of colonies in α-MEM-based medium. In conclusion, appropriate combination of the embryo culture medium and ESC culture medium will greatly contribute to successful establishment of ESCs derived from SCNT embryos.

In vivo, melanocytes occupy three-dimensional (3D) space. Nevertheless, most experiments involving melanocytes are performed in a two-dimensional microenvironment, resulting in difficulty obtaining accurate results. Therefore, it is necessary to construct an artificial in vivo-like 3D microenvironment. Here, as a step towards engineering a precisely defined acellular 3D microenvironment supporting the maintenance of human epidermal melanocytes (HEMs), we examined the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in HEMs. Real-time PCR and fluorescent immunoassay analyses were used to elucidate the expression of integrin α and β subunit genes at the transcriptional and translational levels, respectively. The functionality of the presumed integrin heterodimers was confirmed using attachment and antibody-inhibition assays. Among the genes encoding 12 integrin subunits (α1, α2, α3, α4, α5, α6, α7, αV, β1, β3, β5, and β8) showing significantly higher transcription levels, proteins translated from the integrin α2, α4, α5, β1, β3, and β5 subunit genes were detected on the surface of HEMs. These HEMs showed significantly increased adhesion to collagen I, fibronectin, laminin, and vitronectin, and functional blockade of the integrin α2 subunits significantly inhibited adhesion to collagen I, fibronectin, and laminin. In addition, there was no significant inhibition of the adhesion to fibronectin or vitronectin in HEMs with functional blockade of the integrin α4, α5, or αV subunits. These results indicate that the active integrin α2β1 heterodimer and the inactive integrin α4, α5, αV, β3, and β5 subunits are all localized on the surface of HEMs.

To date, in vitro culture systems able to sufficiently expand the small population of spermatogonial stem cells (SSCs), a tool for the development of sperm-mediated gene transfer techniques in transgenic pigs, in the porcine seminiferous tubule have not been reported. Therefore, as a step toward engineering a noncellular niche to support the in vitro maintenance of porcine SSC self-renewal, we investigated the types of integrin heterodimers that are expressed and functional on their membrane. The α and β integrin subunit protein expressions were analyzed using immunocytochemistry and fluorescence immunoassay, and the function of integrin heterodimers was confirmed by attachment and antibody inhibition assays. The integrin subunits, α3, α4, α5, α6, α8, α9, αV, and β1, were identified on the surface of them. Moreover, they showed significantly increased adhesion to fibronectin, laminin, and vitronectin, and functional blocking of integrin α4β1, α6β1, or αVβ1 significantly inhibited adhesion to these molecules. They showed significantly decreased adhesion to tenascin C and functional blocking of integrin α5β1 did not significantly inhibit adhesion to fibronectin. Accordingly, we confirmed that the integrin heterodimers α4β1, α6β1, and αVβ1 actively function on the surface of undifferentiated porcine SSCs, whereas α3, α5, α8, and α9 are present in inactive forms.

We investigated microbial diversity in a manure storage tank (MST) storing untreated manure and an aeration tank (AT) during swine manure treatment process using the next-generation sequencing in order to find the aeration effect on microbial diversity. Proteobacteria were more abundant in the AT group than in the MST group and may include denitrifying bacteria contributing to nitrous oxide (N2O) emission or aerobic bacteria stimulated by oxygen. The opposite held true for the phyla Bacteroidetes and Firmicutes that may include anaerobic bacteria inhibited under aerobic conditions in the AT group.

To assess whether the follicle-stimulating hormone (FSH) subunits observed in patients with gonadotroph adenomas (GA) can cause infertility, the effects of subunits and heterodimeric FSH on the in vitro follicle development were evaluated in mice. The partial forms of FSH in follicle culture did not induce development into pseudoantral follicles, whereas follicles cultured with native FSH developed into pseudoantral follicles and produced mature metaphase II oocyte. Therefore, intact FSH is needed for folliculogenesis, implying that production of FSH with a partial structure in GA may result in infertility.

Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research.

Here, as a basic study in the construction of a non-cellular niche that supports artificial organization of three-dimensional endometrial tissue, we defined the types of integrin heterodimers that are expressed transcriptionally, translationally and functionally in endometrial stromal (ES) and endometrial epithelial (EE) cells isolated from the mouse uterus in estrus. Gene and protein expression of integrin subunits were analyzed at the transcriptional and translational level by real-time PCR and fluorescent immunoassay, respectively. Moreover, the functionality of integrin heterodimers was confirmed by attachment and antibody inhibition assays.Itga2,Itga5,Itga6,Itga9,Itgav,Itgb1,Itgb3andItgb5in ES cells, andItga2,Itga5,Itga6,Itga7,Itga9,Itgav,Itgb1,Itgb3,Itgb4,Itgb5andItga6and in EE cells showed significantly higher transcriptional levels than the other integrin subunits. Furthermore, translational expression of the total integrin α and β subunit genes that showed increased transcription was determined in ES and EE cells. ES cells showed significantly increased adhesion to collagen I, fibronectin and vitronectin, and functional blocking of integrin α2, α5or αVsignificantly inhibited adhesion to these molecules. Moreover, EE cells showed significantly increased adhesion to collagen I, fibronectin, laminin and vitronectin, and functional blocking of integrin α2, α5, α6or αVsignificantly inhibited adhesion to these molecules. Accordingly, we confirmed that integrin α2β1, α5β1, αVβ1, αVβ3and/or αVβ5, and integrin α2β1, α5β1, α6β1and/or α6β4, αVβ1, αVβ3and/or αVβ5, actively function on the surface of ES and EE cells from mouse uterus in estrus phase, respectively.

Despite the fact that porcine embryonic stem cells (ESCs) are a practical study tool, in vitro long-term maintenance of these cells is difficult in a two-dimensional (2D) microenvironment using cellular niche or extracellular matrix proteins. However, a three-dimensional (3D) microenvironment, similar to that enclosing the inner cell mass of the blastocyst, may improve in vitro maintenance of self-renewal. Accordingly, as a first step toward constructing a 3D microenvironment optimized to maintain porcine ESC self-renewal, we investigated different culture dimensions for porcine ICM-derived cells to enhance the maintenance of self-renewal. Porcine ICM-derived cells were cultured in agarose-based 3D hydrogel with self-renewal-friendly mechanics and in 2D culture plates with or without feeder cells. Subsequently, the effects of the 3D microenvironment on maintenance of self-renewal were identified by analyzing colony formation and morphology, alkaline phosphatase (AP) activity, and transcriptional and translational regulation of self-renewal-related genes. The 3D microenvironment using a 1.5% (w/v) agarose-based 3D hydrogel resulted in significantly more colonies with stereoscopic morphology, significantly improved AP activity, and increased protein expression of self-renewal-related genes compared to those in the 2D microenvironment. These results demonstrate that self-renewal of porcine ICM-derived cells can be maintained more effectively in a 3D microenvironment than in a 2D microenvironment. These results will help develop novel culture systems for ICM-derived cells derived from diverse species, which will contribute to stimulating basic and applicable studies related to ESCs.

Here, as a basic study in revealing the correlation between extracellular matrix components and spontaneous abortion, we defined the types of integrins expressed on the surface of endometrial stromal (ES) cells retrieved from the uterus of a patient experiencing spontaneous abortion. For these, the types of integrin subunits in the ES cells retrieved from a woman with spontaneous abortion were identified at the transcriptional and translational levels, and functional assay was conducted for confirming the combinations of integrin α and β subunits. Among the genes encoding 25 integrin subunits, significantly high transcription was seen in integrins α1, α2, α3, α4, α5, αV, β1, β3, and β5. Translation of integrins α1, α3, α5, αV, and β1 on the cell surface was detected in almost all ES cells, whereas integrins α2, α4, β3, and β4 were expressed translationally only in some ES cells. Subsequently, ES cells showed significantly increased adhesion to collagen I, laminin, fibronectin, and vitronectin, and functional blocking of integrin α1, α3, α5, and αV significantly inhibited adhesion to these molecules. These results demonstrated that active heterodimers composed of integrins α1β1, α3β1, α5β1, and αVβ1 were co-localized on the surface of ES cells derived from a patient experiencing spontaneous abortion.

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α

Gelatin has been reported to induce generation of mesenchymal stem cells (MSCs) with enhanced potential of differentiation into neuronal lineage cells. However, the presence of various cell types besides MSCs in bone marrow has raised doubts about the effects of gelatin. In the following report, we determined whether gelatin can directly enhance neurogenic differentiation potential in MSCs without proliferation of neural progenitor cells (NPCs). MSCs comprised a high proportion of bone marrow-derived primary cells (BMPCs) and gelatin induced significant increases in MSC proliferation during primary culture, and the proportion of MSCs was maintained at more than 99% throughout the subculture. However, NPCs comprised a low percentage of BMPCs and a decrease in proliferation was detected despite gelatin treatment during the primary culture, and the proportion of subcultured NPCs gradually decreased. In a similar manner, MSCs exposed to gelatin during primary culture showed more enhanced neurogenic differentiation ability than those not exposed to gelatin. Together, these results demonstrate that gelatin directly enhances neurogenic differentiation in bone marrow-derived MSCs without stimulating NPC proliferation.

The microbiota of human skin is influenced by host and environmental factors. To determine if chronological age influences the composition of the skin microbiota on the forehead and hands, 73 Korean women were sorted into one of three age groups: (1) 10&ndash, 29 years (n = 24), (2) 30&ndash, 49 years (n = 21), and (3) 50&ndash, 79 years (n = 28). From the 73 women, 146 skin samples (two skin sites per person) were collected. 16S rRNA gene amplicon sequencing was then conducted to analyze the skin microbiota. The overall microbial distribution varied on the forehead but was similar on the hands across the three age groups. In addition, the composition of the skin microbiota differed between the forehead and hands. Commensal microbiota, such as Streptococcus, Staphylococcus, Cutibacterium, and Corynebacterium, which contribute to maintaining skin health via dominant occupation, were affected by increasing age on forehead and hand skin. Alpha diversity indices increased significantly with age on forehead skin. This study indicates that older people may be more susceptible to pathogenic invasions due to an imbalanced skin microbiota resulting from age-related changes. The results of our study may help develop new strategies to rebalance skin microbiota shifted during aging.

Previous reports showed that fibronectin (FN) was effective in stimulating the recovery of damaged dermis. However, native FN has multifunctional domains transmitting beneficial as well as unbeneficial signals to dermal tissue cells through the mediation of integrin heterodimers. The use of a functional domain [FN type III9-10 fragments (FNIII9-10)] providing beneficial effects on the physiology of dermal tissue cells would enhance an in vitro culture system for dermal fibroblasts (DFs). We therefore investigated the FNIII9-10-derived extracellular signaling effect on the physiology of DFs during in vitro culture. Recombinant FNIII9-10 proteins were constructed and their functionality was determined by observing the adhesion of adult human DFs (aHDFs) to recombinant FNIII9-10 and of low adhesion integrin α5β1- and αvβ3-blocked aHDFs to recombinant FNIII9-10. Cellular proliferation, morphology, and senescence were measured and compared in the aHDFs cultured on native FN and recombinant FNIII9-10 for short or long periods. The results show that recombinant FNIII9-10-derived extracellular signaling stimulated increased proliferation of aHDF (both in short- and long-term cultures) and inhibited the generation of morphological abnormalities (in short- and long-term cultures) and cellular senescence (long-term culture) when compared with native FN-derived extracellular signaling. Our results suggest that, instead of native FN, recombinant FNIII9-10 better enhanced the in vitro culture of aHDFs while diminishing the adverse effects associated with the use of human-derived materials.

Spermatogonial stem cells (SSCs) are a useful tool for the generation of genetically modified transgenic sperm. As a result, the transfer of specific genes into the cytoplasm of SSCs is crucial for the successful generation of transgenic sperm. Here, we report electroporation conditions optimized for SSCs derived from the porcine testis. The highest transfection efficiency and cell viability were observed in porcine SSCs transfected with 1 μg transgenic vector with a single electric pulse from an electroporator at a voltage of 100 V and a capacitor setting of 250 μF. The transfection efficiency and cell viability were constant regardless of the size of the transgenic vector. Furthermore, we did not detect loss of spermatozoa differentiation potential in the transfected porcine SSCs. From these results, we confirm that this electroporation-based gene delivery system can effectively introduce foreign DNA into the genome of porcine SSCs without any loss of the original porcine SSC characteristics, which will be important in the generation of mosaicism-free transgenic pigs produced from transgenic porcine sperm.

This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blastocyst formation after PA was significantly increased by rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) relative to the control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM relative to untreated oocytes (20.3% vs. 14.3%, 0.05 < p < 0.1), while no improvement was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin relative to the control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs.

Although episomal vectors are commonly transported into cells by electroporation, a number of electroporation-derived problems have led to the search for alternative transfection protocols, such as the use of transfection reagents, which are inexpensive and easy to handle. Polyplex-mediated transport of episomal vectors into the cytoplasm has been conducted successfully in immortalized cell lines, but no report exists of successful transfection of primary cells using this method. Accordingly, we sought to optimize the conditions for polyplex-mediated transfection for effective delivery of episomal vectors into the cytoplasm of primary mouse embryonic fibroblasts. Episomal vectors were complexed with the commercially available transfection reagents Lipofectamine 2000, FuGEND HD and jetPEI. The ratio of transfection reagent to episomal vectors was varied, and the subsequent transfection efficiency and cytotoxicity of the complexes were analyzed using flow cytometry and trypan blue exclusion assay, respectively. No cytotoxicity and the highest transfection yield were observed when the ratio of transfection reagent to episomal vector was 4 (v/wt) in the cases of Lipofectamine 2000 and FuGENE HD, and 2 in the case of jetPEI. Of the three transfection reagents tested, jetPEI showed the highest transfection efficiency without any cytotoxicity. Thus, we confirmed that the transfection reagent jetPEI could be used to effectively deliver episomal vectors into primary cells without electroporation.

Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research.

Acquisition of sperm capacitation post-ejaculation into the female reproductive tract is an essential step in the fertilization process. Accordingly, during in vitro fertilization, successful fertilization requires the induction of capacitation in epididymis-retrieved sperm. To date, many candidate substances have been considered as capacitation inducers; however, there are no reports on the efficiency of inducing sperm capacitation among the diverse inducers. Therefore, we attempted to determine the inducer with the best capacitation in inbred mouse sperm by comparing the capacitation performance of a variety of inducers and the percentage of in vitro fertilization-generated zygotes. Calcium chloride, progesterone, bovine serum albumin (BSA), heparin, and lysophosphatidylcholine (Lyso-PC) were used as candidate capacitation inducers. Optimized concentrations of each inducer (2.7 mM calcium, 15 μM progesterone, 0.3% (w/v) BSA, 50 mM heparin, and 100 μM Lyso-PC) were determined based on the ratio of sperm showing an acrosome reaction using Coomassie G-250 blue staining. Calcium at 2.7 mM showed the strongest capacitation induction compared to the other inducers. In vitro fertilization was performed using sperm incubated in each inducer and the ratio of fertilized oocytes was determined. Calcium at 2.7 mM and 0.3% (w/v) BSA showed the highest fertilization rates compared to 15 μM progesterone, 50 mM heparin, and 100 μM Lyso-PC. From these results, we found that 2.7 mM calcium and 0.3% (w/v) BSA were the most effective sperm capacitation inducers of mouse sperm for in vitro fertilization.

Contents Although basic fibroblast growth factor (bFGF) is an essential factor supporting the maintenance of porcine embryonic stem (ES) cell self-renewal and pluripotency, its high cost has limited previous studies, and the development of a low-cost culture system is required. For these systems, in vivo blastocysts were progressively cultured under various conditions consisting of different culture mediums and/or different feeder cell numbers at a low concentration of bFGF. As the results, the sequential culture of in vivo-derived porcine blastocysts on 5.0 × 105 mouse embryonic fibroblast (MEF) feeder cells in alpha minimum essential medium-based medium for primary culture, on 2.5 × 105 MEF feeder cells in Mixture medium for the 1st subpassage, and on 2.5 × 105 MEF feeder cells in DMEM/Ham's F10-based medium for the post-2nd subpassage could support the establishment and maintenance of porcine ES-like cells at the low concentration of bFGF. The established porcine ES-like cells showed ES cell-specific characteristics such as self-renewal and pluripotency. We confirmed that porcine ES-like cells could be generated from in vivo-derived porcine blastocysts at a low concentration of bFGF.

In the present study, we examined change of ionized calcium-binding adapter molecule 1 (Iba-1) in the adult and aged gerbil spinal cords. Significant change of morphological feature and neuronal cell loss were not observed in both adult and aged spinal cords of gerbil after NeuN immunohistochemistry and Fluoro-Jade B histofluoresce staining. Iba-1-immunoreactive microglia broadly distributed in the spinal cord. Most of Iba-1-immunoreactive microglia showed ramified forms in the adult gerbil cervical and lumbar spinal cords. However, morphological changes of Iba-1-immunoreactive microglia were observed in the cervical and lumbar regions of the aged gerbil spinal cord. These microglia were showed a hypertrophied body with shortened swollen processes which was characteristic of activated microglia. In addition, Iba-1 protein level significantly higher in aged cervical and lumbar spinal cords than those in the adult gerbil. The present study showed an increase of activated forms of Iba-1-immunoreactive microglia and its protein level without marked changes in morphological features and neuronal loss in the aged spinal cord compared to those in the adult gerbil spinal cord. This result suggests that the increase of Iba-1 expression in the aged spinal cord may be closely associated with age-related changes in aged gerbil spinal cord.

Despite that porcine spermatogonial stem cells (pSSCs) have been regarded as a practical tool for preserving eternally genetic backgrounds derived from pigs with high performance in the economic traits or phenotypes of specific human diseases, there were no reports about precise definition of niche conditions promoting proliferation and maintenance of pSSCs. Accordingly, we tried to determine niche conditions supporting proliferation and maintenance of undifferentiated pSSCs for short-term. For these, undifferentiated pSSCs were progressively cultured in different composition of culture medium, seeding density of pSSCs, type of feeder cells and concentration of growth factors, and then total number of and alkaline phosphatase (AP) activity of pSSCs were investigated at post-6 day culture. As the results, the culture of 4 × 105 pSSCs on mitotically in activated 2 × 105 STO cells in the mouse embryonic stem cell culture medium (mESCCM) supplemented with 30 ng/ml glial cell line-derived neurotrophic factor (GDNF) was identified as the best niche condition supporting effectively the short-term maintenance of undifferentiated pSSCs. Moreover, the optimized short-term culture system will be a basis for developing long-term culture system of pSSCs in the following researches

Despite many researches related with in-vitro culture of porcine spematogonial stem cells (SSCs), adherent culture system widely used has shown a limitation in the maintenance of porcine SSC self-renewal. Therefore, in order to overcome this obstacle, suspension culture, which is known to have numerous advantage over adherent culture, was applied to the culture of porcine SSCs. Porcine SSCs retrieved from neonatal testes were suspension-cultured for 5 days or 20 days, and characteristics of suspension-cultured porcine SSCs including proliferation, alkaline phosphatase (AP) activity, and self-renewal-specific gene expression were investigated and compared with those of adherent-cultured porcine SSCs. As the results, the suspension-cultured porcine SSCs showed entirely non- proliferative and significantly higher rate of AP-positive cells and expression of self-renewal-specific genes than the adherent-cultured porcine SSCs. In addition, long-term culture of porcine SSCs in suspension condition induced significant decrease in the yield of AP staining-positive cells on post-day 10 of culture. These results showed that suspension culture was inappropriate to culture porcine SSCs, because the culture of porcine SSCs in suspension condition didn’t stimulate proliferation and maintain AP activity of porcine SSCs, regardless of culture periods.

A better understanding of the organ specific factors that regulate the migration of mesenchymal stem cells (MSCs) into the target organ is essential for optimization of strategies to improve the repair after injury. In the present study, we showed that the kidney injury molecule-1 (KIM-1), a well-known kidney-specific biomarker, enhanced the in vitro migration capacity of MSCs as a potent kidney-specific chemo-attractant or an inducer. The in vitro roles were verified by migration assay using KIM1-PK1 cell lines, the mouse proximal tubular epithelial cells (mPTEs) and recombinant human KIM-1 proteins (rhKIM-1). Immunofluorescence staining displayed specific ectodomain binding of KIM-1 on the surface of MSCs. Upregulation of chemokine receptor type 4 (CXCR4) protein when treated with tumor necrosis factor alpha (TNF-α) was shown. The effect of KIM-1 on migration of MSCs was augmented by TNF-α pretreatment in a dose-dependent manner, and reduced by AMD3100, an antagonist of CXCR4. These results suggest that KIM-1 is a potential chemo-ligand of CXCR4 and may play an important role in kidney-specific migration of MSCs via interaction between KIM-1 and CXCR4.

Despite the fact that dermal fibroblasts are a practical model for research related to cell physiology and cell therapy, an in vitro culture system excluding serum, which complicates standardization and specificity and induces variability and unwanted effects, does not exist. We tried to establish a CDCS that supports effective proliferation of aHDFs. KDMEM supplemented with 5% (v/v) KSR, 12 ng/ml bFGF, 5 ng/ml EGF and 1 μg/ml hydrocortisone supported sufficient proliferation of aHDFs for 1 week. However, aHDF proliferation was decreased greatly after subculture. This problem could be overcome by culturing aHDFs in CDCM in culture plates coated with 10 μg/ml FN. Long-term culture of aHDFs was achieved using CDCM and FN-coated culture plates for 7 weeks. The optimized CDCS increased the proliferation of aHDFs significantly, without any increase in the senescence rate or alteration in morphology of aHDFs, despite long-term culture. In conclusion, we established a CDCS that improved proliferation of aHDFs while inhibiting cellular senescence. The CDCS will contribute to advances in various future research related to clinical skin regeneration.

Spermatogonial stem cells (SSCs) developed into sperms through spermatogenesis have been utilized as a useful tool in the field of regenerative medicine and infertility. However, a small number of highly qualified SSCs are resided in the seminiferous tubule of testis, resulted in developing effective in-vitro culture system of SSCs for solving simultaneously quantitative and qualitative problems. Presently, SSCs can be enriched on testicular stromal cells (TSCs), but there are no systematic researches about TSC culture. Therefore, we tried to optimize culture condition of TSCs derived from mouse with different strains. For these, proliferation and viability were measured and compared by culturing ICR outbred or DBA/2 inbred mouse-derived TSCs at 35 or 37℃. In case of ICR strain, primary TSCs cultured at 37℃ showed significantly higher proliferation and viability than those at 35℃ and significant increase of proliferation and viability in sub-passaged TSCs was detected in the 35℃ culture condition. Moreover, sub-passage of primary TSCs at 35℃ induced no significant effects on proliferation and viability. In contrast, in case of DBA/2 strain, significantly improved proliferation were detected in the primary TSCs cultured at 35℃, which showed no significant difference in the viability, compared to those at 37℃. Furthermore, sub-passaged TSCs cultured at 37℃ showed no significant differences in proliferation and viability, compared to those at 35℃. However, with significant decrease of proliferation induced by sub-passage of primary TSCs at 35℃, no significant effects on proliferation and viability were resulted from sub-passage of primary TSCs at 37℃. From these results, culture temperature of primary TSCs derived from outbred and inbred strain of mouse could be separately optimized in primary culture and subculture.

In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.

To date, the methods available for isolating spermatogonial stem cells (SSCs) from porcine testicular cells have a low efficiency of cell separating. Therefore, we tried to develop a novel isolation technique with a high-yield cell separating ability to isolate SSCs from porcine testes.We confirmed the presence of SSCs by measuring alkaline phosphatase (AP) activity and SSC-specific gene expression in neonatal porcine testis-derived testicular cells. Subsequently, the isolation of SSCs from testicular cells was performed using different techniques as follows: differential plating (DP), double DP, Petri dish plating post-DP, magnetic-activated cell sorting (MACS), and MACS post-DP. Positive AP staining was used to assess and compare the isolation efficiency of each method.Petri dish plating post-DP resulted in the highest isolation efficiency. The putative SSCs isolated using this method was then further characterized by analyzing the expression of SSC-specific genes and -related proteins, and germ cell-specific genes. OCT4, NANOG, EPCAM, THY1, and UCHL1 were expressed transcriptionally, and OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and PLZF were expressed translationally in 86 % of the isolated SSCs. In contrast, no difference was observed in the percentage of cells expressing luteinizing hormone receptor (LHR), a Leydig cell-specific protein, or GATA4, a Sertoli cell-specific protein, between SSCs and negative control cells. In addition, transcriptional expression of VASA, a primordial germ cell-specific marker, and DAZL, a premeiotic germ cell-specific marker, wasn't and was detected, respectively.We successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.

Poor understanding of the differentiation of mesenchymal stem cells (MSCs) has resulted in a low differentiation yield, and has hindered their application in medicine. As a solution, priming MSCs sensitive to signaling, thus stimulating differentiation into a specific cell lineage, may improve the differentiation yield. To demonstrate this, priming MSCs were produced by using a gelatin matrix for the isolation of primary MSCs from bone-marrow-derived primary cells. Subsequently, cellular characteristics and sensitivity to specific differentiation signals were analyzed at passage five. Compared to non-priming MSCs, priming MSCs showed no significant differences in cellular characteristics, but demonstrated a significant increase in sensitivity to neurogenic differentiation signals. These results demonstrate that generation of priming MSCs by specific extracellular signaling increases the rate of differentiation into a cell-specific lineage.

Objective To evaluate the effect of antioxidant combinations on human sperm motility and morphologic normality during preparation and in vitro incubation to improve sperm quality for assisted reproductive technology (ART). Design Prospective study. Setting Laboratory. Patient(s) Six fertile males, 21 to 25 years of age, after 3 days of sexual abstinence. Intervention(s) Sperm retrieved from patients with normal fertility prepared and incubated in vitro in medium supplemented with taurine, cysteine, and/or glutathione. Main Outcome Measure(s) Motility indices, including motility (MOT), average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), and morphologic normality. Result(s) Taurine supplementation during sperm preparation had no significant effects on motility or morphologic normality. However, when sperm was incubated for 24 hours under taurine-supplemented conditions, the rates of reduction in MOT, VAP, VCL, and VSL were statistically significantly decreased compared with taurine-free conditions. Morphologic normality was maintained, regardless of taurine treatment. The optimal taurine concentration for improving sperm motility and morphologic normality during preparation and in vitro incubation was 1 mM. Subsequently, combined treatment with 1 mM taurine, 1 mM cysteine, and 1 mM glutathione induced statistically significant synergistic effects on MOT, VAP, VCL, and VSL without any alteration of morphologic normality. Conclusion(s) Combined treatment with taurine, cysteine, and glutathione statistically significantly ameliorated the reduction in sperm motility during in vitro manipulation of human sperm.