• Biomolecules & Therapeutics
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• v.27 no.1
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• pp.25-33
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• 2019

Mesenchymal stem cells are classified as multipotent stem cells, due to their capability to transdifferentiate into various lineages that develop from mesoderm. Their popular appeal as cell-based therapy was initially based on the idea of their ability to restore tissue because of their differentiation potential in vitro; however, the lack of evidence of their differentiation to target cells in vivo led researchers to focus on their secreted trophic factors and their role as potential powerhouses on regulation of factors under different immunological environments and recover homeostasis. To date there are more than 800 clinical trials on humans related to MSCs as therapy, not to mention that in animals is actively being applied as therapeutic resource, though it has not been officially approved as one. But just as how results from clinical trials are important, so is to reveal the biological mechanisms involved on how these cells exert their healing properties to further enhance the application of MSCs on potential patients. In this review, we describe characteristics of MSCs, evaluate their benefits as tissue regenerative therapy and combination therapy, as well as their immunological properties, activation of MSCs that dictate their secreted factors, interactions with other immune cells, such as T cells and possible mechanisms and pathways involved in these interactions.

• Molecules and Cells
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• v.45 no.7
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• pp.479-494
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• 2022

Human mesenchymal stem cells (MSCs) are multipotent stem cells that have been intensively studied as therapeutic tools for a variety of disorders. To enhance the efficacy of MSCs, therapeutic genes are introduced using retroviral and lentiviral vectors. However, serious adverse events (SAEs) such as tumorigenesis can be induced by insertional mutagenesis. We generated lentiviral vectors encoding the wild-type herpes simplex virus thymidine kinase (HSV-TK) gene and a gene containing a point mutation that results in an alanine to histidine substitution at residue 168 (TK(A168H)) and transduced expression in MSCs (MSC-TK and MSC-TK(A168H)). Transduction of lentiviral vectors encoding the TK(A168H) mutant did not alter the proliferation capacity, mesodermal differentiation potential, or surface antigenicity of MSCs. The MSC-TK(A168H) cells were genetically stable, as shown by karyotyping. MSC-TK(A168H) responded to ganciclovir (GCV) with an half maximal inhibitory concentration (IC₅₀) value 10-fold less than that of MSC-TK. Because MSC-TK(A168H) cells were found to be non-tumorigenic, a U87-TK(A168H) subcutaneous tumor was used as a SAE-like condition and we evaluated the effect of valganciclovir (vGCV), an oral prodrug for GCV. U87-TK(A168H) tumors were more efficiently ablated by 200 mg/kg vGCV than U87-TK tumors. These results indicate that MSC-TK(A168H) cells appear to be pre-clinically safe for therapeutic use. We propose that genetic modification with HSV-TK(A168H) makes allogeneic MSC-based ex vivo therapy safer by eliminating transplanted cells during SAEs such as uncontrolled cell proliferation.

• Journal of Life Science
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• v.26 no.12
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• pp.1355-1359
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• 2016

Human bone marrow mesenchymal stem cells (hBM-MSCs) can differentiate into various cell types including osteoblasts, adipocytes, chondrocytes, and myocytes. Previous studies, including our own, have shown that MSCs can also differentiate into neuron-like cells. However, their rate of neuronal differentiation is not sufficient for application to stem cell therapy, which requires well-defined cell types. For this purpose, we first examined the expression of neuronal lineage markers (GFAP, MAP-2, KCNH1, Nestin, NF-M, and Tuj-1) by real-time PCR, western blot, and immunocytochemical staining. The expressions of the astrocyte marker GFAP and neuronal markers NF-M and Tuj-1 increased in neuronal differentiated MSCs (dMSCs). To improve the neuronal differentiation efficiency, PDE4, an important signaling intermediator in the progression of neuronal differentiation, was modulated using well-known inhibitors such as rolipram or resveratrol and then differentiated into neuronal cells (Roli- or RSV-dMSCs). The expressions of NF-M, Tuj-1 were increased while that of GFAP decreased in Roli- and RSV-dMSCs, which were examined by real-time PCR, western blot, and immunocytochemical staining. From these experiments, we have found that the neuronal differentiation efficiency can be ameliorated by the modulation of PDE4 activity.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.33-37
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• 2012

Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of $Tuj1$ increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous $Igf2$ may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.

• Journal of Veterinary Clinics
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• v.25 no.6
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• pp.458-465
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• 2008

The purpose of this study is to characterize canine mesenchymal stem cells (MSCs) derived from bone marrow (BM) for use in research on the applications of stem cells in canine models of development, physiology, and disease. BM was harvested antemortem by aspiration from the greater tubercle of the humerus of 30 normal beagle dogs. Canine BM-derived MSCs were isolated according to methods developed for other species and were characterized based on their morphology, growth traits, cell-surface antigen profiles, differentiation repertoire, immunocytochemistry results, and neurotrophic factor expression in vitro. The canine MSCs exhibited a fibroblast-like morphology with a polygonal or spindle-shaped appearance and long processes; further, their cell-surface antigen profiles were similar to those of their counterparts in other species such as rodents and humans. The canine MSCs could differentiate into osteocytes and neurons on incubation with appropriate induction media. RT-PCR analysis revealed that these cells expressed NGF, bFGF, SDF-1, and VEGF. This study demonstrated that isolating canine MSCs from BM, stem-cell technology can be applied to a large variety of organ dysfunctions caused by degenerative diseases and injuries in dogs. Furthermore, our results indicated that canine MSCs constitutively secrete endogenous factors that enhance neurogenesis and angiogenesis. Therefore, these cells are potentially useful for treating dogs affected with various neurodegenerative diseases and spinal-cord injuries.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5705-5711
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• 2013

Background: Embryonic stem cells (ESCs) have the potential to form teratomas when implanted into immunodeficient mice, but data in immunocompetent mice are limited. We therefore investigated teratoma formation after implantation of three different mouse ESC (mESC) lines into immunocompetent mice. Materials and Methods: BALB/c mice were injected with three highly germline competent mESCs (129Sv, BALB/c and C57BL/6) subcutaneously or under the kidney capsule. After 4 weeks, mice were euthanized and examined histologically for teratoma development. The incidence, size and composition of teratomas were compared using Pearson Chi-square, t-test for dependent variables, one-way analysis of variance and the nonparametric Kruskal-Wallis analysis of variance and median test. Results: Teratomas developed from all three cell lines. The incidence of formation was significantly higher under the kidney capsule compared to subcutaneous site and occurred in both allogeneic and syngeneic mice. Overall, the size of teratoma was largest with the 129Sv cell line and under the kidney capsule. Diverse embryonic stem cell-derived tissues, belonging to the three embryonic germ layers, were encountered, reflecting the pluripotency of embryonic stem cells. Most commonly represented tissues were nervous tissue, keratinizing stratified squamous epithelium (ectoderm), smooth muscle, striated muscle, cartilage, bone (mesoderm), and glandular tissue in the form of gut- and respiratory-like epithelia (endoderm). Conclusions: ESCs can form teratomas in immunocompetent mice and, therefore, removal of undifferentiated ESC is a pre-requisite for a safe use of ESC in cell-based therapies. In addition the genetic relationship of the origin of the cell lines to the ability to transplant plays a major role.

• Archives of Pharmacal Research
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• v.21 no.3
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• pp.298-304
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• 1998

Retinoids are applied to not only cancer prevention but also cancer chemotherapy by stimulating differentiation of cells. We studied differentiation inducing effect of all-trans retinoic acid (ATRA) by studying proportion of high dense fractions of stem-like cells and the size of S phase fraction in cell cycle. From mammary organoids obtained from 7- to 8-week old F344 female rat mammary gland, we cultured rat mammary epithelial cells (RMEC) and treated physiological doses of $10^{-6}$, $10^{-7}$, and $10^{-8}$ M ATRA from the first day and then cultured for 4, 7, and 14 days. After that, immunostaining was performed using peanut agglutinin (PNA) and anti-Thy-1.1 monoclonal antibody (Thy-1.1) that can be used as markers of differentiation. We separated four different cell subpopulations by flow cytometry: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). We observed continuous decreases of high dense fractions of stem-like cells (PNA+ subpopulations) for 14 days and as much decreases as high doses of ATRA, which were thought to be proportional to doses of ATRA. We labeled RMEC with bromodeoxyuridine and investigated cell cycle fractions that went through S phase. We observed a tendency of decrease of S phase fraction with time in culture, which, is thought to be related to continuous decreases of PNA+ subpopulations and inhibitory role of ATRA on cell cycle. These results suggest that physiological doses of ATRA could stimulate differentiation of RMEC and convert stem-like RMEC to differentiated cells in SFM for a relatively long period of 14 days.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.11
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• pp.1837-1847
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• 2020

Objective: To evaluate the pancreatic differentiation potential of α-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media. Methods: The BM-MSCs have been differentiated into pancreatic β-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media - advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated. Results: The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic β-like cells in the N2B27-based media than in the ADMEM-based media. Conclusion: 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic β-like cells.

• Molecules and Cells
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• v.40 no.8
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• pp.598-605
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• 2017

Human mesenchymal stem cells (MSCs) are currently being evaluated as a cell-based therapy for tissue injury and degenerative diseases. Recently, several methods have been suggested to further enhance the therapeutic functions of MSCs, including genetic modifications with tissue- and/or diseasespecific genes. The objective of this study was to examine the efficiency and stability of transduction using an adenoviral vector in human MSCs. Additionally, we aimed to assess the effects of transduction on the proliferation and multipotency of MSCs. The results indicate that MSCs can be transduced by adenoviruses in vitro, but high viral titers are necessary to achieve high efficiency. In addition, transduction at a higher multiplicity of infection (MOI) was associated with attenuated proliferation and senescence-like morphology. Furthermore, transduced MSCs showed a diminished capacity for adipogenic differentiation while retaining their potential to differentiate into osteocytes and chondrocytes. This work could contribute significantly to clinical trials of MSCs modified with therapeutic genes.

• Journal of Periodontal and Implant Science
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• v.41 no.1
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• pp.30-43
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• 2011

Purpose: Under different culture conditions, periodontal ligament (PDL) stem cells are capable of differentiating into cementoblast-like cells, adipocytes, and collagen-forming cells. Several previous studies reported that because of the stem cells in the PDL, the PDL have a regenerative capacity which, when appropriately triggered, participates in restoring connective tissues and mineralized tissues. Therefore, this study analyzed the genes involved in mineralization during differentiation of human PDL (hPDL) cells, and searched for candidate genes possibly associated with the mineralization of hPDL cells. Methods: To analyze the gene expression pattern of hPDL cells during differentiation, the hPDL cells were cultured in two conditions, with or without osteogenic cocktails (${\beta}$-glycerophosphate, ascorbic acid and dexamethasone), and a DNA microarray analysis of the cells cultured on days 7 and 14 was performed. Reverse transcription-polymerase chain reaction was performed to validate the DNA microarray data. Results: The up-regulated genes on day 7 by hPDL cells cultured in osteogenic medium were thought to be associated with calcium/iron/metal ion binding or homeostasis (PDE1A, HFE and PCDH9) and cell viability (PCDH9), and the down-regulated genes were thought to be associated with proliferation (PHGDH and PSAT1). Also, the up-regulated genes on day 14 by hPDL cells cultured in osteogenic medium were thought to be associated with apoptosis, angiogenesis (ANGPTL4 and FOXO1A), and adipogenesis (ANGPTL4 and SEC14L2), and the down-regulated genes were thought to be associated with cell migration (SLC16A4). Conclusions: This study suggests that when appropriately triggered, the stem cells in the hPDL differentiate into osteoblasts/cementoblasts, and the genes related to calcium binding (PDE1A and PCDH9), which were strongly expressed at the stage of matrix maturation, may be associated with differentiation of the hPDL cells into osteoblasts/cementoblasts.