Yeonsil Yu

Tonsil-derived Mesenchymal Stem Cells Ameliorate CCl4–induced Liver Fibrosis in Mice via Autophagy Activation

Liver transplantation is the treatment of choice for chronic liver failure, although it is complicated by donor shortage, surgery-related complications, and immunological rejection. Cell transplantation is an alternative, minimally invasive treatment option with potentially fewer complications. We used human palatine tonsil as a novel source of mesenchymal stem cells (T-MSCs) and examined their ability to differentiate into hepatocyte-like cells in vivo and in vitro. Carbon tetrachloride (CCl4) mouse model was used to investigate the ability of T-MSCs to home to the site of liver injury. T-MSCs were only detected in the damaged liver, suggesting that they are disease-responsive. Differentiation of T-MSCs into hepatocyte-like cells was confirmed in vitro as determined by expression of hepatocyte markers. Next, we showed resolution of liver fibrosis by T-MSCs via reduction of TGF-β expression and collagen deposition in the liver. We hypothesized that autophagy activation was a possible mechanism for T-MSC-mediated liver recovery. In this report, we demonstrate for the first time that T-MSCs can differentiate into hepatocyte-like cells and ameliorate liver fibrosis via autophagy activation and down-regulation of TGF-β. These findings suggest that T-MSCs could be used as a novel source for stem cell therapy targeting liver diseases.

3D Culture of Tonsil-Derived Mesenchymal Stem Cells in Poly(ethylene glycol)-Poly(l-alanine-co-l-phenyl alanine) Thermogel

Poly(ethylene glycol)-poly(L-alanine-co-L-phenyl alanine) (PEG-PAF) aqueous solutions undergo sol-to-gel transition as the temperature increases. The transition is driven by the micelle aggregation involving the partial dehydration of the PEG block and the partial increase in β-sheet content of the PAF block. Tonsil-tissue-derived mesenchymal stem cells (TMSCs), a new stem cell resource, are encapsulated through the sol-to-gel transition of the TMSC-suspended PEG-PAF aqueous solutions. The encapsulated TMSCs are in vitro 3D cultured by using induction media supplemented with adipogenic, osteogenic, or chondrogenic factors, where the TMSCs preferentially undergo chondrogenesis with high expressions of type II collagen and sulfated glycosaminoglycan. As a feasibility study of the PEG-PAF thermogel for injectable tissue engineering, the TMSCs encapsulated in hydrogels are implanted in the subcutaneous layer of mice by injecting the TMSC suspended PEG-PAF aqueous solution. The in vivo studies also prove that TMSCs undergo chondrogenesis with high expression of the chondrogenic biomarkers. This study suggests that the TMSCs can be an excellent resource of MSCs, and the thermogelling PEG-PAF is a promising injectable tissue engineering scaffold, particularly for chondrogenic differentiation of the stem cells.

Characterization of long-term in vitro culture-related alterations of human tonsil-derived mesenchymal stem cells: role for CCN1 in replicative senescence-associated increase in osteogenic differentiation

Although mesenchymal stem cells (MSC) isolated from bone marrow and adipose tissues are known to be subjected to in vitro culture‐related alterations in their stem cell properties, such data have not been reported in human tonsil‐derived MSC (T‐MSC). Here, we investigated the culture‐related changes of phenotypes, the senescence, and the differentiation potential of T‐MSC. T‐MSC were serially passaged by a standard protocol, and their characteristics were assessed, including MSC‐specific surface antigen profiles, the senescence, and the differentiation potentials into adipocytes, chondrocytes and osteocytes. Up to at least passage 15, we found no alterations in either MSC‐specific surface marker, CD14, CD34, CD45, CD73 and CD90, or the mRNA expression of embryonic stem cell gene markers, Nanog, Oct4‐A and Sox‐2. However, the expression of CD146, recently identified another MSC marker, dramatically decreased with increasing passages from ~ 23% at passage 3 to ~ 1% at passage 15. The average doubling time increased significantly from ~ 38 h at passage 10 to ~ 46 h at passage 15. From passage 10, the cell size increased slightly and SA‐β‐gal staining was evident. Both Alizarin Red S staining and osteocalcin expression showed that the osteogenic differentiation potential increased up to passage 10 and decreased thereafter. However, the adipogenic and chondrogenic differentiation potential decreased passage‐dependently from the start, as evidenced by staining of Oil Red O and Alcian Blue, respectively. Consistent with a passage‐dependent osteogenic differentiation, the expression of CCN1, an angiogenic protein known to be related to both senescence and osteogenesis, also increased up to passage 10. Furthermore, ectopic expression of small interfering RNA against CCN1 at passage 10 significantly reversed Alizarin Red S staining and osteocalcin expression. Altogether, our study demonstrates the characterization of long‐term in vitro cultured T‐MSC and that CCN1 may be involved in mediating a passage‐dependent increase in osteogenic potential of T‐MSC.

Myogenic differentiation potential of human tonsil-derived mesenchymal stem cells and their potential for use to promote skeletal muscle regeneration

Stem cells are regarded as an important source of cells which may be used to promote the regeneration of skeletal muscle (SKM) which has been damaged due to defects in the organization of muscle tissue caused by congenital diseases, trauma or tumor removal. In particular, mesenchymal stem cells (MSCs), which require less invasive harvesting techniques, represent a valuable source of cells for stem cell therapy. In the present study, we demonstrated that human tonsil-derived MSCs (T-MSCs) may differentiate into myogenic cells in vitro and that the transplantation of myoblasts and myocytes generated from human T-MSCs mediates the recovery of muscle function in vivo. In order to induce myogenic differentiation, the T-MSC-derived spheres were cultured in Dulbeccos modified Eagles medium/nutrient mixture F-12 (DMEM/F-12) supplemented with 1 ng/ml transforming growth factor-β, non-essential amino acids and insulin-transferrin-selenium for 4 days followed by culture in myogenic induction medium [low-glucose DMEM containing 2% fetal bovine serum (FBS) and 10 ng/ml insulin-like growth factor 1 (IGF1)] for 14 days. The T-MSCs sequentially differentiated into myoblasts and skeletal myocytes, as evidenced by the increased expression of skeletal myogenesis-related markers [including α-actinin, troponin I type 1 (TNNI1) and myogenin] and the formation of myotubes in vitro. The in situ transplantation of T-MSCs into mice with a partial myectomy of the right gastrocnemius muscle enhanced muscle function, as demonstrated by gait assessment (footprint analysis), and restored the shape of SKM without forming teratomas. Thus, T-MSCs may differentiate into myogenic cells and effectively regenerate SKM following injury. These results demonstrate the therapeutic potential of T-MSCs to promote SKM regeneration following injury.

CCN1 Secreted by Tonsil‐Derived Mesenchymal Stem Cells Promotes Endothelial Cell Angiogenesis via Integrin αvβ3 and AMPK

CCN1 is highly expressed in cancer cells and has been identified in the secretome of bone marrow‐derived mesenchymal stem cells (BM‐MSC). Although secreted CCN1 is known to promote angiogenesis, its underlying mechanism remains unclear. Here, we examined whether our recently‐established tonsil‐derived MSC (T‐MSC) secrete CCN1 and, if any, how CCN1 promotes the angiogenesis of human umbilical vein endothelial cells (HUVEC). Compared with untreated control T‐MSC, a higher level of CCN1 was secreted by T‐MSC treated with activin A and sonic hedgehog, drugs known to induce endodermal differentiation. Expectedly, conditioned medium collected from differentiated T‐MSC (DCM) significantly increased HUVEC migration and tube formation compared with that from control T‐MSC (CCM), and these stimulatory effects were reversed by neutralization with anti‐CCN1 antibody. Treatment with recombinant human CCN1 (rh‐CCN1) alone also mimicked the stimulatory effects of DCM. Furthermore, treatment with either DCM or rh‐CCN1 increased the phosphorylation of AMP kinase (AMPK), and ectopic expression of siRNA of the AMPK gene inhibited all observed effects of both DCM and rh‐CCN1. However, no alteration of intracellular ATP levels or phosphorylation of LKB1, a well‐known upstream factor of AMPK activation, was observed under our conditions. Finally, the neutralization of integrin αvβ3 with anti‐integrin αvβ3 antibody almost completely reversed the effects of CCN1 on AMPK phosphorylation, and EC migration and tube formation. Taken together, we demonstrated that T‐MSC increase the secretion of CCN1 in response to endodermal differentiation and that integrin αvβ3 and AMPK mediate CCN1‐induced EC migration and tube formation independent of intracellular ATP levels alteration. J. Cell. Physiol. 230: 140–149, 2015.

Tonsil-Derived Mesenchymal Stem Cells Differentiate into a Schwann Cell Phenotype and Promote Peripheral Nerve Regeneration

Schwann cells (SCs), which produce neurotropic factors and adhesive molecules, have been reported previously to contribute to structural support and guidance during axonal regeneration; therefore, they are potentially a crucial target in the restoration of injured nervous tissues. Autologous SC transplantation has been performed and has shown promising clinical results for treating nerve injuries and donor site morbidity, and insufficient production of the cells have been considered as a major issue. Here, we performed differentiation of tonsil-derived mesenchymal stem cells (T-MSCs) into SC-like cells (T-MSC-SCs), to evaluate T-MSC-SCs as an alternative to SCs. Using SC markers such as CAD19, GFAP, MBP, NGFR, S100B, and KROX20 during quantitative real-time PCR we detected the upregulation of NGFR, S100B, and KROX20 and the downregulation of CAD19 and MBP at the fully differentiated stage. Furthermore, we found myelination of axons when differentiated SCs were cocultured with mouse dorsal root ganglion neurons. The application of T-MSC-SCs to a mouse model of sciatic nerve injury produced marked improvements in gait and promoted regeneration of damaged nerves. Thus, the transplantation of human T-MSCs might be suitable for assisting in peripheral nerve regeneration.

Selective osteogenesis by a synthetic mineral inducing peptide for the treatment of osteoporosis

Mineralization in mammalian cells is accomplished by concerted regulation of protein-based extracellular matrix (ECM) components, such as non-collagenous proteins and collagen fibrils. In this study, we investigated the ability of a collagen-binding motif (CBM) peptide derived from osteopontin to selectively affect osteogenic or adipogenic differentiation in vitro and in vivo. In particular, increased osteogenic differentiation and decreased adipogenic differentiation were observed in human mesenchymal stem cells (hMSCs). Osteocalcin (OCN) protein expression in MC3T3-E1 cells without osteogenic inducers was then investigated following treatment with the CBM peptide. In ovariectomized (OVX) mice, estrogen deficiency induced osteoporosis and increased fat tissue deposition. However, after the CBM peptide or estradiol was injected into the OVX mice for 2 months, the increased serum OCN concentration and alkaline phosphate (ALP) activity were decreased in the estradiol-treated group (OVX-E) and the high-concentration CBM peptide-treated group (OVX-HP). Significant bone loss was also observed in the ovariectomized mice (OVX-PBS). In particular, the bone volume per total volume (BV/TV) and bone mineral density (BMD) were significantly decreased in the OVX mice; however, both of these markers were restored in the OVX-HP group, which also had significantly well-developed bone structure and bone formation. In contrast to the bone structural change, adipose tissue was increased in the OVX-PBS. However, a significant decrease in total fat and subcutaneous fat was observed in the low-concentration CBM peptide-treated group (OVX-LP) and the estradiol-treated group (OVX-E). Taken together, these results suggest that the CBM peptide could be an effective therapeutic agent for osteoporosis due to its selective stimulation of osteogenic differentiation, rather than adipogenesis.

Expression of tenocyte lineage-related factors from tonsil-derived mesenchymal stem cells

Human palatine tonsil-derived mesenchymal stem cells (TMSCs) are known to be a new source of progenitor cells. Using waste tissue after tonsillectomy as a cell provider can be the biggest benefit of TMSCs, compared with other stem cells. The purpose of this study was to investigate tenogenic differentiation of TMSCs and to access the differential effects of transforming growth factor beta 3 (TGF-β3) on the tenogenesis of TMSCs. Human tonsil was obtained after tonsillectomy. Using a cytometric analysis, we were able to find that the TMSCs had typical mesenchymal stem cell markers: positive for CD73, CD90, and CD105, and negative for CD14, CD34, and CD45. Using TGF-β3, the expressions of tenocyte-specific genes and proteins, such as collagen type 1 (COL1), tenomodulin (TNMD), and scleraxis (SCX), were measured by a quantitative polymerase chain reaction (PCR), immunofluorescence staining, immunohistochemistry and Western blot analyses. Quantitative PCR assay showed that TGF-β3 significantly increased the expressions of tenocyte lineage marker genes, including COL1, TNMD, and SCX, at a 3-day treatment, compared with control. However, these increases were not found at long-term exposures (7 or 10 days), except that TNMD expression was maintained at 50 ng/mL at a 7-day exposure to TGF-β3. Like genes, the protein expression levels of COL1, TNMD, and SCX were also induced in TGF-β3-treated TMSCs in a 3-day treatment, which were maintained for 10 days, as evidenced by immunofluorescence staining, immunohistochemistry and Western blot analyses. This study demonstrated that TMSCs in tenogenic stimulation with TGF-β3 have a high tenogenic differentiation potential.

Cyanidin-3-glucoside suppresses Th2 cytokines and GATA-3 transcription factor in EL-4 T cells

Allergic disease is dominated by Th2 immune responses. Interleukin (IL)-4 and IL-13, representative Th2 cytokines, play pivotal roles in the pathogenic activation of the Th2 immune response. In this study, we found that cyanidin-3-glucoside chloride (C3G), an anthocyanin suppressed IL-4 and IL-13 produced in activated EL-4 T cells but not Th1 cytokines including IL-2, interferon-γ, or IL-12. IL-4 and IL-13 mRNA levels and luciferase activation in cells transiently transfected with IL-4 and IL-13 promoter reporter plasmids were significantly inhibited by C3G, suggesting that suppression might be, at least in part, regulated at the transcriptional level. Data from western blot and reverse transcription-polymerase chain reaction analyses of transcription factors involved in cytokine expression suggested that expression of GATA-3, but not T-bet, was downregulated in the nucleus by C3G. Taken together, our data indicate that C3G may has potential as an anti-allergic agent suppressing Th2 activation by downregulating Th2 cytokines and the GATA3 transcription factor in allergies. Graphical Abstract Cyanidin-3-glucoside chloride, an anthocyanin, suppresses IL-4 and IL-13 expression via down regulation of GATA3 transcription factor in EL-4 T cells.

Autophagy induction in the skeletal myogenic differentiation of human tonsil-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation and are thus a valuable source for the replacement of diseased or damaged organs. Previously, we reported that the tonsils can be an excellent reservoir of MSCs for the regeneration of skeletal muscle (SKM) damage. However, the mechanisms involved in the differentiation from tonsil-derived MSCs (T-MSCs) to myocytes via myoblasts remain unclear. To clarify these mechanisms, we analyzed gene expression profiles of T-MSCs during differentiation into myocytes compared with human skeletal muscle cells (hSKMCs). Total RNA was extracted from T-MSCs, T-MSC-derived myoblasts and myocytes, and hSKMCs and was subjected to analysis using a microarray. Microarray analysis of the three phases of myogenic differentiation identified candidate genes associated with myogenic differentiation. The expression pattern of undifferentiated T-MSCs was distinguishable from the myogenic differentiated T-MSCs and hSKMCs. In particular, we selected FNBP1L, which among the upregulated genes is essential for antibacterial autophagy, since autophagy is related to SKM metabolism and myogenesis. T-MSCs differentiated toward myoblasts and skeletal myocytes sequentially, as evidenced by increased expression of autophagy-related markers (including Beclin-1, LC3B and Atg5) and decreased expression of Bcl-2. Furthermore, we reconfirmed that autophagy has an effect on the mechanism of skeletal myogenic differentiation derived from T-MSCs by treatment with 5-azacytidine and bafilomycin A1. These data suggest that the transcriptome of the T-MSC-derived myocytes is similar to that of hSKMCs, and that autophagy has an important role in the mechanism of myogenic differentiation of T-MSCs.