It is still challenging to establish pESCs due to differences in the genetic backgrounds of mouse, human, and pig. So it is required to find pig specific pluripotency markers and cellular signaling. In this experiments, doxycycline-inducible vectors carrying OCT4, SOX2, NANOG, KLF4 and MYC known as reprogramming factors, were infected into pig stem cells for analyzing gene expression pattern. When cultured without doxycycline, pig stem cells were stably maintained in bFGF supplemented media. However, when treated with doxycycline, pig stem cells lost alkaline phosphatase activity and were differentiated within two weeks. And then, we investigated the expression of genes related to pluripotency in doxycycline-treated pig stem cells by using qRT-PCR. The qRT-PCR data revealed that expression of OCT4, CDH1 and FUT4 were significantly increased by OCT4 overexpression and OCT4 and FUT4 were also upregulated in SOX2-infected group. When infected with combination of two factors including OCT4 or SOX2, some groups could stably maintain at LIF supplemented media, having alkaline phosphatase activity. Given these data, although ectopic gene expression induced differentiation in pig stem cells, ectopic expression of OCT4 and SOX2 could upregulate pluripotent genes and overexpreession of two factors help pig stem cells adapt LIF-contained media. This study could improve understanding of pluripotent networks as well as aid in establishing bona fide pluripotent stem cells in pig.

Due to their anatomical, physiological and genetic similarities, pig is attractive animal model in biomedical research. In the recent stem cell research era, porcine derived stem cells also gain attention due to its use for the preclinical application of human. Mesenchymal stem cells (MSCs) have been studied by many researchers over decade, and their prospect for clinical application is recognized. Although porcine derived MSCs (pMSCs) have confirmed to be differentiated into various types of cells, such as osteocyte, chondrocyte, neuronal cell, cardiomyocyte and pancreatic β cell, few report has been studied regarding hepatocyte differentiation in vitro. The present study was therefore aimed for bone marrow MSCs derived from pig femur to differentiate into hepatocyte. The cells were confirmed as MSCs by characterizing their morphology, lineage differentiation capacity and surface phenotype. They showed spindle like morphology and adipocytic, osteoblastic, and chondrocytic differentiation potentials and displayed positive expression of mesenchymal markers CD29, CD44 and CD90 while lacked the expression of hematopoietic marker CD45. Under appropriate differentiation conditions, MSCs displayed hepatocyte-like morphology depending on duration of differentiation. The differentiated MSCs into hepatocyte expressed hepatocyte-specific genes including hepatocyte nuclear factor 4 (HNF4), albumin (ALB), alpha fetoprotein (AFP), alpha-1-anti trypsin (A1AT). They also showed hepatocyte-like function, glycogen storage which is identified by PAS staining. Taken together, it concluded that the bone marrow MSCs have the potential to differentiate into hepatocyte. Further studies are needed on additional hepatocytic functional assays, such as low density lipoprotein (LDL) uptake and urea synthesis of differentiated MSC.

To have a better understanding of pluripotency, whole gene expression of embryo-derived stem cells (EdSCs) in bovine species was investigated. EdSCs were established from the embryos produced by in vitro fertilization, parthenogenesis and somatic cell nuclear transfer. Then, the microarray was performed and analyzed. Differently expressed genes (DEGs) were also confirmed by Real-time PCR. Among 10,203 DEGs, little difference was found in gene expression among three kinds of EdSCs. Conversely, all EdSCs have an immensely different gene expression when compared with somatic cells, consistent with scatter plat results. To investigate shared pathways for pluripotency in all EdSCs, 2,415 co-DEGs were identified which compared with somatic cells. By KEGG database, there were 54 signaling pathways in co-DEGs and some of them were related with pluripotency maintenance such as TGFβ, WNT and JAK-STAT signaling. In TGFβ signaling, BMP family and SMAD family were involved in co-up-regulated DEGs. In WNT signaling, WNT family and receptors were included in co-up-regulated DEGs, while inhibitors of WNT signaling were associated with co-down-regulated DEGs. In JAK-STAT signaling, STAT3 belonged to co-down-regulated DEGs. These DEGs were also confirmed by Real-time PCR. Taken together, BMP and WNT pathways may be activated and paly central roles to retain pluripotency in bovine EdSCs, whereas the LIF/STAT3 pathway may not be operated well. This study was supported by a grant from the National Research Foundation of Korea (NRF-2006-2004042, and No. 2015048003 through the Oromaxillofacial Dysfunction Research Center for the Elderly at Seoul National University) and the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture, Food and Rural Affairs (MAFRA; 111160-04), Republic of Korea.

Spermatogonial stem cells (SCCs) is foundation for spermatogenesis throughout male adult life because they have ability of self-renewal and differentiation into spermatozoa. Storage of such SSCs is very important to study on male reproduction, which would contribute human male infertility to be treated. However, during cryopreservation, the most cells are damaged by cryoinjury such as apoptosis, necrosis, osmotic stress, oxidative stress and so on. For the reason, in cryopreservation technique, targeting purpose is what cells are stored stably without cryoinjury. The purpose of this study was to develop the cryoprotectant for decrease in cryoinjury of SSCs by using melatonin and necrostatin-1 as additive cryoprotectant. The SSCs with melatonin or necrostatin-1 was frozen for 1 month, and then thawed to evaluate survival, recovery and proliferation rate. The result showed that necrostatin-1 50 mM was significantly greater than DMSO control. Furthermore, we conducted the characterization of cryo-thawed SSCs with necrostatin-1 50 mM to confirm whether the SSCs could maintain the undifferentiated state. As a result, the normal expression of each marker, which is PLZF, GFRa1 and VASA, was observed except for C-kit, meaning that the cells could maintain the undifferentiated state regardless of cryopreservation. Therefore, the result indicates that the cryo-thawed SSCs have ability of proliferation and self-renewal. In conclusion, our finding verifies that cryopreservation of SSC with necrostatin-1 50 mM could be helpful to preserve the SSCs stably, contributing to various studies on male reproduction and infertility treatment

Introducing lineage-specific transcription factors (TFs) into somatic cells enables the induction of distinct cellular identities without the need to first pass through a pluripotent stem cell (PSC) state. We and others have demonstrated the direct conversion of somatic cells into adult stem cells or progenitor cells, such as angioblast-like progenitor cells, hematopoietic stem cells, and neural stem cells. The process underlying direct conversion is known to be relatively simpler and faster than that of induced pluripotent stem cell (iPSC) generation. Furthermore, directly converted cells have been shown to exhibit therapeutic potential following transplantation into respective disease models without obvious evidence for tumor formation. Thus, TF-mediated direct conversion technology has been considered as an alternative to iPSC technology for patient-specific cell- and tissue-replacement therapies. Here we show our recent findings describing the robust direct conversion of differentiated somatic cells into distinct cellular identities. Furthermore, we also show the recent 3D organoid technology for generating brain tissues from human pluripotent stem cells.

The mammalian yolk sac endoderm is an essential but understudied tissue that patterns and nourishes the embryo. This talk will present the isolation and characterization of several categories of rodent yolk sac endoderm stem cells. Specifically, we have isolated yolk sac endoderm stem cell lines from preimplantation embryos and from post-gastrulation yolk sacs. In both cases, we obtained two versions of stem cells that appear to differ in their degree of lineage maturation. I will discuss the relationship of these various isolates within the same species (rat or mouse), between species (rat vs. mouse), and with previously published isolates. I will then discuss potential applications in developmental biotechnology and toxicology as well as the human relevance of this research.

Mesenchymal stem cells (MSCs) are multipotent stem cells, which can be induced to differentiate into several cells. MSCs are also reported to possess immunomodulatory properties through secretion of inflammatory cytokines and generation of regulatory T (Treg) cells. Treg cells play an important role in allergic disorders, including atopic dermatitis. We examined the immunomodulatory effects of canine adipose tissue derived-MSCs (cAD-MSCs) in 3 groups: Group 1, untreated normal dog; Group 2, dogs with Dermatophagoides farinae ointment-induced atopic dermatitis; and Group 3, dogs with atopic dermatitis. Canine peripheral blood mononuclear cells (PBMCs) were collected from each group and co-cultured with cAD-MSCs. After co-culturing, PBMCs were separated and the expression of Treg cells was examined by flow cytometry. According to the results, the populations of Treg cells were increased in 3 ex vivo experimental groups, co-cultured with cAD-MSCs. These results would be important for the application of MSCs in clinical trials.

Mesenchymal stem cells (MSCs) are an attractive source for cell therapy, as they have the potential for differentiation into multi-lineage cells. Adipose tissue is a safe source due to its easy extraction and abundant resource, with minimal risk to the organ donor. In this study, we attempted to correlate the harvest yield and resulting multipotency of feline adipose tissue-derived mesenchymal stem cells (fAD-MSCs) in accordance with processing time. fAD-MSCs were individually isolated from the abdominal adipose tissues of 6 felines. They were divided into two groups, based on their processing times – Group 1: 0~1 day after adipose tissue harvesting; Group 2: more than 3 days after adipose tissue harvesting. In both groups, the proliferation capacity was analyzed using the cumulative population doubling level (CPDL) calculation assay. The expression levels of MSC-specific markers and differentiation potentials into mesodermal cell lineages were also evaluated. We observed that fAD-MSC isolation yields and CPDL were excellent in Group 1 compared with Group 2. We also found that the differentiation potential-specific genes (ACAN and OPN) were strongly expressed in Group 1 compared with Group 2. These results suggest that for the clinical treatments of feline diseases, fAD-MSCs should be isolated within 1 day after adipose tissue harvesting.

Bone fractures are most often seen in racetrack horses because of the high level of intensity in racing. These issues are the main cause of decreased performance in racehorses. Mesenchymal stem cells (MSCs) have been explored to improve intra-articular therapy in racehorses. MSCs are essential for the repair and regeneration of damaged tissues. In this study, the effect of intra-articular injection of MSCs in racehorses was investigated. Before accessing the MSC therapy, synovial fluids were obtained from the fracture site of racehorses, and adipose tissue was collected for MSC isolation. Using the MSC specific marker, adipose tissue-derived MSCs were identified. The racehorses received intra-articular injection of autologous MSCs (or allogeneic) (3 × 107 cells/3 mL). After 1 or 2 weeks, synovial fluids were collected from racehorses. To test the effect of MSC injection using ELISA, we analyzed inflammatory factors from the untreated samples compared to MSC-treated samples of racehorses. The level of pro-inflammatory factors (interleukin-1β and prostaglandin E2) was significantly decreased in synovial fluids of MSC-injected racehorses, compared to before accessing the MSC therapy, whereas, the level of anti-inflammatory factor (interleukin-10) was higher than prior to accessing the MSC therapy. Further studies are needed to investigate the anti-inflammatory mechanism of MSC in racehorses.

Previous reports revealed that DMfree (green tea extract) inhibited expression of the IL-6 gene in Mycobacterium lepraeinfected MSCs (mesenchymal stem cells). This study aimed to measure IL-6, IL-1β, TNF-α and PGE2 production in M. leprae-infected MSCs using ELISA. To confirm the effect of DMfree on IL-6 and signal transduction, a western blotting test was performed. DMfree inhibited the expression of IL-6 in the MSCs and the heterodimer of STAT3, which also affects the expression of multiple genes. Though DMfree pre-treatment of control MSCs produced a baseline level of IL-6, it significantly inhibited the production of IL-6 in M. leprae-infected MSCs. There was no significant difference in IL-6 production between 1 and 7 day treatment groups. M. leprae-infected MSCs produced more IL-1β, TNF-α and PGE2, but DMfree could not inhibit their production at a physiological concentration. This is different from other reports that used higher concentration of EGCG treatment, resulting in significant inhibition of the cytokines. The inhibition appears to be related to the concentration of EGCG. These results indicate that DMfree can alleviate inflammation involving IL-6.

위축돈은 정상돈에 비해 생시체중이 낮고 생후 성장과 발달도 미숙하다. 근육의 성장에 있어 muscle satellite cells(SC)의 역할에 대해 많은 연구가 진행되고 있다. 근육 내에서 줄기세포를 하는 side population(SP) 세포군이 최근 밝혀졌는데, 이 세포군은 Hoechst dye를 이용한 fluorescence-activated cell sorting(FACS)에 의해 이용하여 분리되는데, 다양한 조직(특히 근육)으로 분화되는 것으로 밝혀졌다. 위축돈과 정상돈 근육 내 SC와 SP 세포군의 숫자 및 성상을 비교분석하기 위해 semitendinosus(ST) 근육을 적출하여 SC와 SP를 분리·배양 하였다. 위축돈의 ST는 정상돈에 비해 무게가 가볍고 근섬유의 숫자와 크기도 작았다(p<0.05). 하지만 정상돈과 위축돈의 ST 근육 내 단백질과 DNA 총량은 차이가 없었다. 총 RNA 양과 DNA 농도는 정상돈의 ST 근육에서 위축돈에 비해 높게(p<0.05) 나타났다. ST 근육에서 추출된 총 세포수(yield/g)에는 정상돈과 위축돈 간 차이가 없었다. 세포의 증식률을 0, 24, 48 시간동안 두 그룹 간 차이가 없었다. 세포 배양 72시간 후 정상돈 ST 근육에서 추출된 세포의 증식률이 위축돈의 그것에 비해 높게(p<0.05) 나타났다. 정상돈 ST 근육에서 위축돈 ST 근육에 비해 SP 세포가 더 많이(p<0.05) 추출되었다. 종합해 보면, 위축돈의 근육 크기와 근섬유의 숫자가 정상돈에 비해 작고, 근육내 줄기세포의 역할을 하는 SC와 SP 세포군 또한 낮게 나타난 것은 위축자돈이 임신 기간 동안 충분한 영양을 받지 못하여 근육 내 줄기세포의 증식이 낮아졌으며, 이는 근육 내 줄기세포가 자돈의 성장과 발달에 큰 영향을 미치는 결과로 해석할 수 있다.

A tremendous increase in the human population has put poultry industry under an increased pressure to meet steep increase in the demand. Poultry is contributing 25% of the total world’s meat production and lesser cost of investment per bird makes it more suitable for the further breeding programmes. Major poultry diseases frequently lead to cardiac damage and cause huge economic losses to poultry industry due to mortality. The in vitro embryonic stem cell (ESC) technology has a futuristic approach for homogeneous populace of differentiated cells, for their further transplantations. During in vitro conditions the differentiated cell populace can be used in grafting and transplantation processes to regenerate damaged tissues. Therefore, the current study targeted the use of spermatogonial stem cells (SSCs) in the poultry production system through cardiac regeneration. The current study will also open new boulevard for the similar kind of research in other livestock species for the management of heart diseases.

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 4x105 pSSCs on mitotically in activated 2x105 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-cul-tured 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.

Fucoidan has been extensively studied as medicinal materials due to its biological activities including osteoblastic differentiation effect. However, osteoblastic effect by fucoidan is unknown in alveolar bone marrow derived mesenchymal stem cells (ABM-MSCs). The present study was undertaken to evaluate the effect of fucoidan on Osteoblastic differentiation in ABM-MSCs and explore its mechanism. Cell proliferation was analyzed by crystal violet staining. Osteoblast differentiation was determined by alkaline phosphatase activity, calcium accumulation assay and gene expression of osteoblast markers. We found that fucoidan induced cell proliferation of ABM-MSCs. Furthermore, fucoidan increased the ALP activity, calcium accumulation, and osteoblast specific genes such as Runx2, type I collagen alpha 1. Moreover, fucoidan induces the expression of asporin and bone morphogenic protein (BMP)-2 and asporin. Based on these results, these finding indicate that fucoidan induces osteoblast differentiation in ABM-MSCs and partially enhanced the mRNA expression of BMP-2 and asporin.

This study found antibacterial activity of DMfreeⓇ [green tea extract] on facultative bacteria by direct petri dish method and gene array of obligatory M. leprae infected mesenchymal stem cells (MSC). While DMfree showed DPPH radical scavenging effect and high contents of polyphenol, it did not inhibit growth of facultative bacteria such as E. coli and S. aureus on the petri dish. The result does not exclude a possible antibacterial effect of organic solvent extract of green tea rather than DMfree which comes from the water extract of green tea. Pre-treatment of DMfree appeared to have no effect on copy number of 14 genes compared with control MSC by real-time RT-PCR. However pre-treatment of DMfree on M. leprae infected MSC revealed a significant decrease of anti-inflammatory cytokine (IL-6), (P<0.038) and sharp downregulation of pro-inflammatory cytokine (IL-1). Enhanced expression of VEGFR-1 mRNA was noted in DMfree pretreated M. leprae infected MSC group (P<0.003). These results show that DMfree would stabilize M. leprae infected MSC from further inflammation by down-regulating anti-inflammatory cytokine (IL-6) and pro-inflammatory cytokine (IL-1β). This is the first report on DMfree inhibition of IL-6 and IL-1β expression in M. leprae infected MSC. Further experiments that detect protein levels of IL-1β and IL-6 may support the result of this gene array.

This study evaluated the possibility of clinical application using matrigel-based bioceramic/polymer scaffolds treated with bone morphogenetic protein, angiogenic factor, and mesenchymal stem cells (MSCs) for new bone formation. In the in vitro study, bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) containing matrigel, which is a basement membrane gel, was injected into HA/PCL scaffolds to estimate the release rates of growth factors. In the in vivo study, BMP-2, VEGF, and MSCs with matrigel-based scaffolds were implanted into rat femoral segmental defects, and new bone formation was evaluated at 4 and 8 weeks. In the results, the release rates of BMP-2 and VEGF explosively increased by day 5. For the in vivo study results, radiological evaluation revealed that the matrigel-based HA/PCL scaffolds with BMP-2 and VEGF grafted (M+B+V) and matrigel-based HA/PCL scaffolds with BMP-2, VEGF, and MSC grafted (MSC) groups showed increased bone volume and bone mineral density. Moreover, in the histological evaluation, large new bone formation was observed in the M+B+V group, and high cellularity in the scaffold was observed in the MSC group. In conclusion, grafted matrigel-based HA/PCL scaffolds with BMP-2, angiogenic factor, and MSCs increased new bone formation, and in clinical cases, it may be effective and useful to enhance healing of delayed fractures.

Bioactive peptides function effectively with a minimal amount compared to proteins. Recently SPARC related modular calcium binding 1 (SMOC1) has been implicated in regulating osteoblast differentiation and limb and eye development. In this study we synthesized a peptide covering 16 amino acids derived from the extracellular calcium binding (EC) domain of SMOC1, and its effects on proliferation and osteoblast differentiation of human bone marrow mesenchymal stem cells were examined. Treatment of SMOC1 peptide did not modulate proliferation of BMSCs. However, mineralization of BMSCs was significantly increased with a dose dependent manner. Consistently expression of osteoblast differentiation marker genes including type 1 collagen and osteocalcin was also dose dependently increased. Taken together, these results suggest that peptide derived from the EC domain of SMOC1 recapitulates at least partially osteogenic function of SMOC1.

To clarify the role of stem cells in hepatocarcinogenesis, CD44 expression was investigated in mouse livers as well as embryonic cell lineages treated with diethylnitrosamine (DEN). Liver tumors induced by DEN were analyzed by immunohistochemisty for CD44. Liver tissues were sampled at 6, 24, and 48 hr after treatment with saline or DEN. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs), and hepatocyte like cells (HCs), representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5, and 15 mM, respectively) for 24 hr, after which CD44 expression levels were examined by relative quantitative real-time PCR. CD44 expression was weakly detected in tumor cells as well as in some hepatocytes surrounding the tumor cells. However, CD44 expression was not detected in liver tissue treated with DEN at early time points. The CD44 mRNA expression level was significantly different among cells treated with 5 mM DEN at day 22 (P<0.01) as well as 1, 5, and 15 mM DEN at day 40 (P<0.01) compared with control. Taken together, CD44 expression slightly increased in mouse DEN-induced tumors. Furthermore, expression of CD44 in embryonic cell lineages treated with various doses of DEN significantly differed among embryo stem cells and derived hepatic lineage cells. This suggests that CD44 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, and its expression may increase in the tumor stage but not during early carcinogenesis.