• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.23-31
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• 2011

Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

• BMB Reports
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• v.47 no.6
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• pp.354-359
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• 2014

The types of glia in the central nervous system (CNS) of the Drosophila embryo include longitudinal glia (LG), cell body glia (CBG), and peripheral glia (PG). Transcription factors, such as glial cell missing and reverse polarity, are well-established general glial cell markers. Only a few glial cell-specific markers have been identified in the Drosophila embryonic CNS, thus far. In the present study, we employed the glial cell-specific markers for LG (vir-1/CG5453 and CG31235), CBG (fabp/CG6783 and CG11902), and PG (CG2310 and moody/CG4322), and comprehensively analyzed their expression patterns, during the embryonic CNS development. Our study validated the specificity of a set of glial markers, and further revealed their spatio-temporal expression patterns, which will aid in the understanding of the developmental lineage, and investigating their role in the development and homeostasis of the Drosophila CNS in vivo.

• International Journal of Molecular Medicine
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• v.43 no.5
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• pp.2230-2240
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• 2019

Hair follicles (HFs) are a well-characterized niche for adult stem cells (SCs), and include epithelial and melanocytic SCs. HF cells are an accessible source of multipotent adult SCs for the generation of the interfollicular epidermis, HF structures and sebaceous glands in addition to the reconstitution of novel HFs in vivo. In the present study, it was demonstrated that HF cells are able to be induced to differentiate into cardiomyocyte-like cells in vitro under specific conditions. It was determined that HF cells cultured on OP9 feeder cells in KnockOut-Dulbecco's modified Eagle's medium/B27 in the presence of vascular endothelial growth factors differentiated into cardiomyocyte-like cells that express markers specific to cardiac lineage, but do not express non-cardiac lineage markers including neural stem/progenitor cell, HF bulge cells or undifferentiated spermatogonia markers. These cardiomyocyte-like cells exhibited a spindle- and filament-shaped morphology similar to that presented by cardiac muscles and exhibited spontaneous beating that persisted for over 3 months. These results demonstrate that SC reprogramming and differentiation may be induced without resulting in any genetic modification, which is important for the clinical applications of SCs including tissue and organ regeneration.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.6
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• pp.463-472
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• 2020

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods. In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

• International Journal of Oral Biology
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• v.49 no.3
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• pp.69-78
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• 2024

Sestrin 2 (SESN2) is a member of the sestrin family of stress-induced proteins that negatively regulate aging-associated biological processes. This study aims to investigate the role of SESN2 in regulating the differentiation potential and senescence of mesenchymal stem cells (MSCs) derived from young and elderly donors. Bulk RNA sequencing revealed a common decline in the SESN2 mRNA levels in MSCs from elderly individuals, which was confirmed via reverse transcription-polymerase chain reaction and western blot analyses. SESN2 knockdown in MSCs from young donors resulted in phenotypic changes similar to those in MSCs from elderly donors, including an enhanced expression of senescence and adipogenic markers and diminished expression of osteogenic markers. To confirm the effect of decreased SESN2 expression on osteogenic and adipogenic differentiation, we induced Sesn2 knockdown in mouse bone marrow-derived MSCs. Sesn2 knockdown suppressed the mRNA expression of osteogenic marker genes, alkaline phosphatase activity, and matrix mineralization. Furthermore, Sesn2 knockdown enhanced mRNA expression of the adipogenic marker genes and intracellular lipid accumulation. These results suggest that a decline in SESN2 expression during aging contributes to the shift of MSC differentiation from osteogenic to adipogenic lineage.

• IMMUNE NETWORK
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• v.22 no.5
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• pp.37.1-37.16
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• 2022

Autoimmune diseases are caused by a dysfunction of the acquired immune system. In a subset of autoimmune diseases, B cells escaping immune tolerance present autoantigen and produce cytokines and/or autoantibodies, resulting in systemic or organ-specific autoimmunity. Therefore, B cell depletion with monoclonal Abs targeting B cell lineage markers is standard care therapy for several B cell-mediated autoimmune disorders. In the last 5 years, genetically-engineered cellular immunotherapies targeting B cells have shown superior efficacy and long-term remission of B cell malignancies compared to historical clinical outcomes using B cell depletion with monoclonal Ab therapies. This has raised interest in understanding whether similar durable remission could be achieved with use of genetically-engineered cell therapies for autoimmunity. This review will focus on current human clinical trials using engineered cell therapies for B cell-associated autoimmune diseases.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.1-8
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• 2011

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.10
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• pp.1447-1460
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• 2009

Myogenic satellite cells have been isolated and identified by several recently elucidated molecular markers. Furthermore, knowledge about the precise function of these markers has provided insight into the early and terminal events of satellite cells during proliferation, differentiation, transdifferentiation, specification and activation. Recently, quiescent myogenic satellite cells have been associated with possession of Pax 3 and 7 that represent pluripotent stem cells capable of differentiating into other lineages. However, the mechanism by which myogenic satellite cells attain pluripotent potential remain elusive. Later, transdifferentiating ability of these cells to another lineage in the absence or presence of certain growth factor/ or agents has revolutionized the scope of these pluripotent myogenic satellite cells for manipulation of animal production (in terms of quality and quantity of muscle protein) and health (in terms of repair of skeletal muscle, cartilage or bone).

• Development and Reproduction
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• v.15 no.1
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• pp.41-52
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• 2011

In the present study, embryoid bodies (EBs) obtained from induced pluripotent stem cells (iPSCs) were induced to differentiate into germ lineage cells by treatment with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results were compared to the results for embryonic stem cells (ESCs) and multipotent spermatogonial stem cells (mSSCs) and quantified using immunocytochemical analysis of germ cell-specific markers (integrin-${\alpha}6$, GFR-${\alpha}1$, CD90/Thy1), fluorescence activating cell sorting (FACS), and real time-RT-PCR. We show that the highest levels of germ cell marker-expressing cells were obtained from groups treated with 10 ng/$m{\ell}$ BMP4 or 0.01 ${\mu}M$ RA. In the BMP4-treated group, GFR-${\alpha}1$ and CD90/Thy-1 were highly expressed in the EBs of iPSCs and ESCs compared to EBs of mSSCs. The expression of Nanog was much lower in iPSCs compared to ESCs and mSSCs. In the RA treated group, the level of GFR-${\alpha}1$ and CD90/Thy-1 expression in the EBs of mSSCs Induced pluripotent stem cells, Mouse embryonic stem cells, Multipotent spermatogonial stem cells, Germ cell lineage, Differentiation potential. was much higher than the levels found in the EBs of iPSCs and similar to the levels found in the EBs of ESCs. FACS analysis using integrin-${\alpha}6$, GFR-${\alpha}1$, CD90/Thy1 and immunocytochemistry using GFR-${\alpha}1$ antibody showed similar gene expression results. Therefore our results show that iPSC has the potential to differentiate into germ cells and suggest that a protocol optimizing germ cell induction from iPSC should be developed because of their potential usefulness in clinical applications requiring patient-specific cells.

• International Journal of Oral Biology
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• v.38 no.4
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• pp.161-167
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• 2013

Human dental pulp stem cells (DPSCs) are multi-potent mesenchymal stem cells that have several differentiation potentials. An understanding of thetissues that differentiate from these cells can provide insights for future regenerative therapeutics and tissue engineering strategies. The mesiodens is the most frequent form of supernumerary tooth from which DPSCs can differentiate into several lineages similar to cells from normal deciduous teeth. Recently, it has been shown that nanoscale structures can affect stem cell differentiation. In our presentstudy, we investigated the effects of a 250-nm nanoscale ridge/groove pattern array on the osteogenic and adipogenic differentiation of dental pulp cells from mesiodenscontaining human DPSCs. To this end, the expression of lineage specific markers after differentiation induction was analyzed by lineage specific staining and RT-PCR. The nanoscale pattern arrayed surface showed apositive effect on the adipogenic differentiation of DPSCs. There was no difference between nanoscale pattern arrayed surface and conventional surface groups onosteogenic differentiation. In conclusion, the nanoscale ridge/groove pattern arrayed surface can be used to enhance the adipogenic differentiation of DPSCs derived from mesiodens. This finding provides an improved understanding of the effects of topography on cell differentiation as well as the potential use of supernumerary tooth in regenerative dental medicine.