Transplantation of stem cells, such as mesenchymal stem cells (MSCs), is a promising strategy for treating several types of intractable disorders. Mechanistically, it could not only replace damaged cells by direct contribution, but also establish an anti-inflammatory or immunomodulatory microenvironment. However, the cellular mechanisms underlying molecular and biological properties of stem cells during ex vivo expansion and also after transplantation in pathological environments remain largely elusive. We recently developed the cyanoacrylamide-based coumarin derivatives (Fluorescent real-time thiol tracer; FreSHtracer*) reversibly react with glutathione for monitoring of glutathione levels in living stem cells. These probes revealed that glutathione levels are heterogeneous among subcellular organelles and among individual cells and show dynamic changes and heterogeneity in repopulating stem cells depending on oxidative-stress or culture conditions. Importantly, a subpopulation of stem cells with high-glutathione levels exhibited increased self-renewal and migration activities in vitro and showed improved therapeutic efficiency in treating asthma. Furthermore, employing a novel combination of longitudinal intravital confocal fluorescence imaging and microcystoscopy in living animals, we investigated the distributions and properties of transplanted multipotent MSCs derived from human embryonic stem cells at single-cell resolution in real-time by performing confocal imaging of bladder tissues in a rat model of IC/BPS for up to 6 months post-transplantation. These novel real-time monitoring strategies demonstrate the novel molecular insight for maintaining stem cell functions and also enhance understanding of the in vivo behaviors of the engrafted stem cells, which is crucial to determine the efficacy and safety of stem cell-based therapies. This strategy may facilitate the translation of various stem cell-based approaches into clinical practice.

The trans-differentiation potential of mesenchymal stem cells (MSCs) is employed, but there is little understanding of the cell source-dependent trans-differentiation potential of MSCs into corneal epithelial cells. In the present study, we induced trans-differentiation of MSCs derived from umbilical cord matrix (UCM-MSCs) and from dental tissue (D-MSCs), and we comparatively evaluated the in vitro trans-differentiation properties of both MSCs into corneal epithelial-like cells. Specific cell surface markers of MSC (CD44, CD73, CD90, and CD105) were detected in both UCM-MSCs and D-MSCs, but MHCII and CD119 were significantly lower (P < 0.05) in UCM-MSCs than in D-MSCs. In UCM-MSCs, not only expression levels of Oct3/4 and Nanog but also proliferation ability were significantly higher (P < 0.05) than in D-MSCs. In vitro differentiation abilities into adipocytes and osteocytes were confirmed for both MSCs. UCM-MSCs and D-MSCs were successfully trans-differentiated into corneal epithelial cells, and expression of lineage-specific markers (Cytokeratin-3, -8, and -12) were confirmed in both MSCs using immunofluorescence staining and qRT-PCR analysis. In particular, the differentiation capacity of UCM-MSCs into corneal epithelial cells was significantly higher (P < 0.05) than that of D-MSCs. In conclusion, UCM-MSCs have higher differentiation potential into corneal epithelial-like cells and have lower expression of CD119 and MHC class II than D-MSCs, which makes them a better source for the treatment of corneal opacity.

The mesenchymal stem cells (MSCs) that reside in dental tissues hold a great potential for future applications in regenerative dentistry. In this study, we used human dental pulp cells, isolated from the molars (DPCs), in order to establish the organoid culture. DPCs were established after growing pulp cells in an MSC expansion media (MSC-EM). DPCs were subjected to organoid growth media (OGM) in comparison with human dental pulp stem cells (DPSCs). Inside the extracellular matrix in the OGM, the DPCs and DPSCs readily formed vessel-like structures, which were not observed in the MSC-EM. Immunocytochemistry analysis and flow cytometry analysis showed the elevated expression of CD31 in the DPCs and DPSCs cultured in the OGM. These results suggest endothelial cell-prone differentiation of the DPCs and DPSCs in organoid culture condition.

Insulin/IGF signaling (IIS) regulates different physiological processes such as metabolism, trehalose level, growth, and reproduction. Four IIS components are identified from the bean pod borer, Maruca vitrata (Lepidoptera: Crambidae). RNA interference (RNAi) of insulin receptor (InR), Forkhead Box O (FOXO), Target of Rapamycin (TOR) or Akt led to ovary dysfunction. Especially, the RNAi treatment significantly reduced the stem cell division in the germarium. However, an addition of a porcine insulin stimulated the cell division. Immature diets significantly influenced on the ovarian stem cell development.

Induced pluripotent stem cells (iPSCs) can be generated from adult cells. Somatic cells can be reprogrammed to form iPSCs by overexpressing transcription factors such as Oct4, Sox2, cMyc, and Klf4. To maintain undifferentiated state of iPSCs in vitro, cells have traditionally been maintained on mouse embryonic fibroblast feeders and passaged by enzymatic or mechanical dissociation methods. In this study, we compared the morphology and pluripotency of porcine iPSCs (piPSCs) after subsequent passaging using enzymatic and mechanical dissociation methods. Enzymatically and mechanically passaged piPSCs showed embryonic stem cell-like morphologies with compact cell adhesion and clear colony borders. In addition, alkaline phosphatase staining was positive for both enzymatically and mechanically passaged piPSCs. However, visual observation revealed that some colonies of enzymatically passaged piPSCs were spontaneously differentiated more than those of piPSCs mechanically passaged from 5 passage. Quantitative real-time RT-PCR demonstrated that enzymatically and mechanically passaged piPSCs expressed pluripotent genes such as Oct4, Sox2 and Nanog well at early passage. Immunofluorescent staining also confirmed that pluripotent markers such as Oct4, Sox2, and Nanog were positively expressed at early passage. However, expression levels of pluripotent genes in mechanically passaged piPSCs were also higher than those in enzymatically passaged piPSCs at early passage. Collectively, we found that mechanical passage method was better than enzymatic passage in terms of morphology and pluripotency of piPSCs at early passage. Further studies are needed to compare these dissociation methods with those obtained after more passages of piPSCs.

The estrogen-mediated effect of mesenchymal stem cells (MSCs) is a highly critical factor for the clinical application of MSCs. However, the present study is conducted on MSCs derived from adult donors, which have different physiological status with steroid hormonal changes. Therefore, we explores the important role of 17β-estradiol (E2) in MSCs derived from female and male newborn piglets (NF- and NM-pBMSCs), which are non-sexually matured donors with steroid hormones. The results revealed that in vitro treatment of MSCs with E2 improved cell proliferation, but the rates varied according to the gender of the newborn donors. Following in vitro treatment of newborn MSCs with E2, mRNA levels of Oct3/4 and Sox2 increased in both genders of MSCs and they may be correlated with both estrogen receptor α (ERα) and ERβ in NF-pBMSCs, but NM-pBMSCs were only correlated with ERα. Moreover, E2-treated NF-pBMSCs decreased in β-galactosidase activity but no influence on NM-pBMSCs. In E2-mediated differentiation capacity, E2 induced an increase in the osteogenic and chondrogenic abilities of both pBMSCs, but adipogenic ability may increased only in NF-pBMSCs. These results demonstrate that E2 could affect both genders of newborn donor-derived MSCs, but the regulatory role of E2 varies depending on gender-dependent characteristics even though the original newborn donors had not been affected by functional steroid hormones.

The purpose of this study is to confirm whether spontaneous adipocyte generation during chondrogenic induction culture affects the chondrogenic differentiation of porcine skin-derived stem cells (pSSCs). For this purpose, chondrogenic differentiation characteristics and specific marker gene expression were analyzed using cell lines showing different characteristics of spontaneous adipocyte formation. Of the four different lines of pSSCs, the pSSCs-IV line showed higher Oil red O (ORO) and glycosaminoglycan (GAG) extraction levels. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the levels of adipogenic markers peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and adipocyte Protein 2 (aP2) mRNAs were significantly higher in pSSCs-IV than those of the other pSSC lines (P<0.05). Among three chondrogenic markers, collagen type II (Col II) and sex determining region Y-box (Sox9) mRNAs were strongly expressed in pSSCs-IV (P<0.05), but not in aggrecan (Agg), which was significantly higher in pSSCs-II (P<0.05). These results demonstrate that the spontaneous adipocyte generation during chondrogenic differentiation has a positive effect on the chondrogenesis of pSSCs. More research is needed on the correlation between adipocyte generation and cartilage formation.

Mesenchymal stem cells (MSCs) have restricted life spans in vitro and can therefore only be expanded for a limited number of cell divisions before entering a senescent state and unequivocally stopping proliferation. Several types of cell culture systems have been used for large-scale expansion of MSCs. A recent trend in cell culture has been the change from serum-use to serum-supplement media. This study was conducted to investigate the proliferative effects of vegetable resources (VR) on equine adipose tissue-derived mesenchymal stem cells (eAD-MSCs) in the absence of serum and their possible mechanisms of action. Regulation of cell cycling is a key process involved in the fate of stem cells, including renewal and differentiation. In this study, we observed that the viability of eAD-MSCs was increased significantly when treated with VR under serum-free conditions. We also observed that expression levels of cell cycling-related proteins such as p53 and p21 were decreased, and proliferating cell nuclear antigen increased significantly in response to treatment with VR in eAD-MSCs under serum-free conditions. Furthermore, expression levels of cell survival-related proteins were increased in response to treatment with VR in eAD-MSCs under serum-free conditions. Therefore, our results suggest that VR promotes proliferation of eAD-MSCs under serum-free conditions.

Type1 diabetes mellitus (DM) is generally known to be caused by destruction of insulin-producing pancreatic β cells or an immune-related problem. Polydipsia is a representative symptom of DM, and it has been reported that this condition is closely related to xerostomia and is considered that hyposalivation from the salivary gland results in this phenomenon. Although various studies have reported that induction of diabetes reduces endogenous stem cells in other organs (heart, brain etc.), diabetes-related changes in endogenous stem cells in the salivary gland have not yet been well established. Therefore, in this study, to verify the change in salivary gland stem cells after diabetes, salivary gland tissues in the control and diabetes-induced groups were processed by histochemistry (Masson’s trichrome staining) for morphological analysis, TUNEL assay for cell death, and immunohistochemistry (Ki-67 and c-Kit) for cell proliferation and maturation. Diabetes induced by STZ leads to vacuolization, apoptosis, and reduction in proliferating cells/salivary gland stem cells in salivary glands of rats. This result suggests that diabetes may be associated with reduction in salivary gland function such as degeneration and inhibition of regeneration in the salivary gland.

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several cell lineages, which has implications for cell therapy and reproductive biotechnologies. Although MSCs have been isolated from many species, including humans and animals, there is limited data on MSCs from large ruminants, such as bovines. In this study, we tried to isolate and characterize bovine tongue tissue-derived MSCs (boT-MSCs) by investigating phenotype morphology, performing proliferation properties, and determining cell surface marker expression patterns, self-renewal, and differentiation potentials. As a result, the boT-MSCs were successfully isolated by collagenase digestion and maintained proliferative capacity until 20 passages. Moreover, the boT-MSCs expressed pluripotency markers (OCT3/4, SOX2, and NANOG) and MSC-specific surface markers including CD44, CD90, and CD105, but not CD45 and MHC-II. The boT-MSCs could also differentiate into mesodermal (adipocyte, osteocyte, and chondrocyte) cell lineages. Our results suggest that the tongues of bovines could be used as a source of MSCs.

BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at ED50 of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.

Mesenchymal stem cells (MSCs) have been considered an alternative source of neuronal lineage cells, which are difficult to isolate from brain and expand in vitro. Previous studies have reported that MSCs expressing Nestin (Nestin+ MSCs), a neuronal stem/progenitor cell marker, exhibit increased transcriptional levels of neural development-related genes, indicating that Nestin+ MSCs may exert potential with neurogenic differentiation. Accordingly, we investigated the effects of the presence of Nestin+ MSCs in bone-marrow-derived primary cells (BMPCs) on enhanced neurogenic differentiation of BMPCs by identifying the presence of Nestin+ MSCs in uncultured and cultured BMPCs. The percentage of Nestin+ MSCs in BMPCs was measured per passage by double staining with Nestin and CD90, an MSC marker. The efficiency of neurogenic differentiation was compared among passages, revealing the highest and lowest yields of Nestin+ MSCs. The presence of Nestin+ MSCs was identified in BMPCs before in vitro culture, and the highest and lowest percentages of Nestin+ MSCs in BMPCs was observed at the third (P3) and fifth passages (P5). Moreover, significantly the higher efficiency of differentiation into neurons, oligodendrocyte precursor cells and astrocytes was detected in BMPCs at P3, compared with P5. In conclusion, these results demonstrate that neurogenic differentiation can be enhanced by increasing the proportion of Nestin+ MSCs in cultured BMPCs.

Introduction Porcine embryonic stem cells (pESCs) derived from cloned embryos might be a useful animal model in biomedical research, however, establishment of cloned pESCs is difficult by its incomplete nuclear reprogramming. Here, we report the improved development competence of porcine cloned embryos by vitamin C (VC) supplement to establish the pESCs. Materials and Methods Slaughterhouse-derived oocytes were in vitro matured for 44h and parthenogenetic and cloned embryos were produced using matured oocytes. Both of embryos were cultured for 6 days in PZM-5 media and development rates were examined. Four different concentration of VC (0, 25, 50, 100, and 200 μg/ml) was supplemented in IVM and IVC media and preimplantation developments in the 5 groups were compared in both of embryos Results and Discussion In the cleavage rates of IVM group, significantly higher rate was shown in 50 mg/ml group than other groups (84.5 ± 0.6% vs. 69.8 ± 5.5, 75.7 ± 1.8, 80.4 ± 0.2, 72.4 ± 0.1%; P<0.05), respectively. Significantly higher rates of blastocyst development also were shown in 50 mg/ml group than other groups (27.0 ± 2.0% vs. 20.4 ± 1.4, 22.1 ± 1.3, 23.7 ± 1.2, 19.6 ± 1.3%; P<0.05), respectively. In the cleavage rate of IVC group, non-significantly different with each group (84.0 ± 1.3, 86.7 ± 1.0, 88.4 ± 1.4, 76.7 ± 3.0, 64.6 ± 4.4; P<0.05). In the blastocyst rate of IVC group, significantly higher rate was shown in 25mg/ml and 50 mg/ml group than other groups (22.3 ± 1.7, 23.8 ± 1.7% vs. 19.1 ± 1.3, 15.9 ± 1.0, 5.8 ± 1.5%; P<0.05) In conclusion, supplement of 50μg/ml of VC in IVM and IVC media enhanced the development of porcine parthenogenetic embryos and these results will be a helpful information in the development of porcine cloned embryos and derivation of its embryonic stem cells.

The production of feline induced pluripotent stem cells (iPSCs) can solve the problems that are related with existing unstable supply and demand of eggs as well as ethical aspects about embryonic stem cell at the same time. On the basis of excellent proliferation, it is to facilitate the researches about human disease like FIV and Allergen at the level of cells, not experimental animals. But, a lot of advanced researches are lean too much towards on the transduction using DNA type virus that have the risk of tumorigenesis during reprogramming and on the mLIF-dependent culture condition for the production of feline iPSCs. This being so, this study shows the reprogramming results using Sendai virus vector that is RNA type virus and have no the footprint after transduction. In addition, the feline iPSCs were stably cultured in bFGF-dependent culture condition during the reprogramming step and culture step. In conclusion, we found the bFGF-dependent culture condition in feline iPSCs and suggested the approach using Sendai virus vector as an alternative for reprogramming without concern about tumorigenesis. These methods can be universally applicable to not only the researches about reconstruction and conservation of feline species, but also to a lot of deep studies related with iPSCs or LIF, bFGF to find new approaches.

The use of pigs in neuroscience has increased over the past years because the pigs are closely related to humans in terms of anatomy and physiology. Especially, the blood-brain barrier (BBB) maintains the homeostatic microenvironment in the central nervous system (CNS) and they can provide a valuable tool for studying the neurobiology. However, only a few putative blood-brain barrier (BBB) models have been generated by co-culture of porcine primary cells. The fundamental problem is that they lose some of their phenotypes when maintained in vitro for long-term culture. To establish improved in vitro porcine BBB models, we differentiated novel brain microvascular endothelial cells (BMECs) from porcine induced pluripotent stem cells (iPSCs) using a modified human-based protocol. Briefly, the dissociated single cells from iPSCs were seeded in Geltrex. For differentiation, cells were maintained for 3 days of expansion and then switched to unconditioned medium (UM) lacking bFGF for 6-7 days. Then, we subcultured cells onto collagen/fibronectin coated plates and changed BMEC medium for 2-3 weeks. About two weeks later, we observed a cluster of round cells surrounded by spindle shaped adherent cells termed as colony-forming units (CFU) of putative BMECs. Over time, the cluster of cells disappears and remained adherent spindle-shaped cells showed properties of endothelial cells. Although further studies will be needed, this study would be a great comparative analysis of the porcine and human in vitro BBB model.

Little is known to date about neural development of pig and directed differentiation of porcine pluripotent stem cells (PSCs) to neuronal cells remains elusive. To determine whether soluble factors from glioblastoma multiforme (GBM) promoted the neural differentiation from porcine induced PSCs (iPSCs), cells were treated cultured media of GBM cells. First of all, we isolated and established primary GBM cell line (WHO grade IV). The cellular morphology of GBM cancer cell line are dendritic-like with positive expression in NESTIN, SOX2, VIMENTIN and GFAP using immunofluorescence analysis. G-banded karyotype from primary GBM cell line revealed severe numerical chromosomal aberrations. GBM-cultured medium (CM) treated iPSC-NPCs survive well in vitro when supplemented with a combination of growth factors, including EGF and bFGF. The GBM-CM treated differentiated cells showed an increased mRNA expression level of astrocyte marker, GFAP and the dopaminergic neuron marker, tyrosine hydroxylase (TH). However, there was no significant difference in mRNA expression level of oligodendrocyte marker, MBP. The protocol developed in the present study for large animal models might provide an exciting tool to bridge the present gaps in neuroscience studies between rodents and humans.

Although stem cells are used as important cell therapies in regenerative medicine, the electrophysiological problems that arise in the expansion of cells have not been known much. This study was conducted to investigate the functional expression of inward rectifying K+ current (IKir) using a patch-clamp technique, and the change in the resting membrane potential and the membrane capacitance were investigated in mesenchymal stem cells derived from human umbilical vein (hUC-MSC). The IKir plays an important role in regulating the resting membrane potential in many cells and is known to contribute to the maintenance of intracellular K+ concentration. In this study, electrophysiologically recorded current exhibited typical IKir characteristics. The current shifted along the K+ equilibrium potential (Ek) with the extracellular K+ concentration change. In addition, IKir was blocked by the divalent Ba2+ in a dose-dependent manner. The frequency of functional expression of IKir changed with number of passages (P2: 5.3% vs P8: 77.8% vs P12: 34.5%). There was no significant change in the resting membrane potential of hUC-MSC (P2: -21.0 mV, P8: -20.1 mV and P12: -21.9 mV). However, the capacitance of the cell membrane was significantly changed after P9 (P2: 8.9 pF vs P9: 16.9 pF) compared to P2. All the results suggest that changes in electrophysiological distribution of IKir as the passages increase may cause changes in K+ permeability even in cell proliferation and differentiation, suggesting a possible physiological role in maintaining cell homeostasis and resting membrane potential (RMP).

Mesenchymal stem cells (MSCs) are primary candidates for cell therapy and tissue engineering applications. A two-dimensional (2D) culture system is typically used for cell growth, but that method affects the characteristics of stem cells. The physiological cell environment connects cells not only to each other, but also to the extracellular matrix providing mechanical support, exposing the entire cell surface, and opening signaling pathways. The hanging drop method is the most widely used 3D culture method for spheroid formation. In this study, we investigated the relationship between spheroid size and changes in gene expression to determine the optimum spheroid size for use in tissue engineering. The expression levels of stemness factors such as NANOG, OCT4, and SOX2, angiogenic factors such as VEGF and IL-8, and osteogenic factors such as COX2 and TGF-β1 increased with spheroid size in the respective spheroid formation groups unlike the responses in their monolayer groups. Therefore, our results indicate that spheroid formation through the hanging drop method can increase the efficiency of MSCs-based tissue engineering over that obtained via traditional 2D cell culture systems.

Mesenchymal stem cells (MSCs) have been researched for use in biomedical applications, particularly for cell-based therapies and regenerative medicine due to their self-renewing capacity and ability to differentiate into multiple cell types such as adipose, bone, and tendon tissues. Cryopreservation of MSCs is a common preservation method that is advantageous for cellular therapies in human and veterinary medicine. Adipose tissue-derived cells have been shown to maintain their properties after cryopreservation. In this study, we investigated the morphology, proliferation (cumulative population doubling level and doubling time), cell surface markers (CD34, CD90, and CD105), and ability to differentiate into adipose, bone, and cartilage tissues in vitro of equine adipose tissue-derived MSCs (eAD-MSCs) and miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with and without long-term cryopreservation. The eAD-MSCs and mpAD-MSCs were analyzed immediately and after being frozen in liquid nitrogen for 3 years and 2 years, respectively. Cryopreserved eAD-MSCs maintained their morphology, proliferation rate, and cell surface markers compared with fresh cells. With the exception of proliferation rate, cryopreserved mpAD-MSCs also maintained their fresh cell characteristics. The proliferation rate of cryopreserved mpAD-MSCs was higher than that for fresh cells. Cryopreservation did not change the adipogenic, chondrogenic, or osteogenic differentiation potentials of eAD-MSCs and mpAD-MSCs. In summary, long-term cryopreservation maintains the cell phenotype and differentiation ability of eAD-MSCs and mpAD-MSCs. These results might be useful when developing veterinary medicine and clinical applications.

RNA Sendai virus (SeV) vector system has no risk of being integrated into the host genome. Sendai virus (SeV) vectors expressing pluripotent factors have been used to produce integration-free induced pluripotent stem cells (iPSCs) with high efficiency from various cell types in human and mouse. In this study, we generated iPSCs from pig ear fibroblast cells using the SeV vector expressing 4 human factors (POU5F1, SOX2, C-MYC, and KLF4). Colonies were emerged at Day 14 of transduction and expressed the classical pluripotency markers (POU5F1, NANOG, and SOX2) and surface marker (SSEA1). Furthermore, they showed a domed shape and could passage over 40 times under 2i (CHIR99021 and PD0325901)-LIF and MEF feeder culture condition having in vitro differentiation ability into 3 germ layers. Next, we examined the ability of six feeder free culture conditions to maintain piPSCs in a pluripotent state. piPSCs were plated on Matrigel coated dishes in different media: 1. CM: control media (LIF culture media); 2. CM-F: CM+100 ng Fetuin-A; 3. CM-N: CM+100 ng Nanog-TAT; 4. CM-2i: CM+3 uM CHIR99021+1 uM PD0325901; 5. CM-2iN: CM-2i+100 ng Nanog-TAT; 6. CM-2iN+100 ng Fetuin-A. However, piPSC could not maintain the typical self-renewal morphology on feeder free conditions regardless of culture media tested here. Further, expression of pluripotency-related genes (Oct4, Nanog and Klf4) of piPSCs cultured on feeder free conditions could not be compared with that of iPSCs cultured on MEF feeder plate. Our results suggest that integration free pluripotent stem cell from pigs could be generated by SeV vector system and maintained their pluripotency under 2i-LIF and MEF feeder culture condition, but further optimization of culture conditions may be required.