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ABSTRACT: Tissue engineering with stem cells is a fascinating approach for treating anterior cruciateligament (ACL) injuries. In our previous study, stem cells isolated from the human anteriorcruciate ligament were shown to possess extensive proliferation and differentiationcapabilities when treated with specific growth factors. However, optimal culture conditionsand the usefulness of fetal bovine serum (FBS) as a growth factor in in vitro culture systemsare yet to be determined. In this study, we compared the effects of different culture mediacontaining combinations of various concentrations of FBS and the growth factors basicfibroblastic growth factor (bFGF) and transforming growth factor β1 (TGF- β1) on theproliferation and differentiation of ligament-derived stem cells (LSCs) and bone marrowmesenchymal stem cells (BMSCs). We found that α-MEM plus 10% FBS and bFGF was ableto maintain both LSCs and BMSCs in a relatively undifferentiated state but with lower majorextracellular matrix (ECM) component gene expression and protein production, which isbeneficial for stem cell expansion. However, the differentiation and proliferation potentials ofLSCs and BMSCs were increased when cultured in MesenPRO, a commercially availablestem cell medium containing 2% FBS. MesenPRO in conjunction with TGF- β1 had thegreatest ability to induce the differentiation of BMSCs and LSCs to ligament fibroblasts,which was evidenced by the highest ligamentous ECM gene expression and proteinproduction. These results indicate that culture media and growth factors play a very importantrole in the success of tissue engineering. With α-MEM plus 10% FBS and bFGF, rapidproliferation of stem cellscan be achieved. In this study, MesenPRO was able to promotedifferentiation of both LSCs and BMSCs to ligament fibroblasts. Differentiation was furtherincreased by TGF- β1. With increasing understanding of the effects of different culture mediaand growth factors, manipulation of stem cells in the desired direction for ligament tissueengineering can be achieved.
Cell Transplantation 04/2013; · 5.13 Impact Factor
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ABSTRACT: High-fat diet (HFD)-induced glucose intolerance and insulin resistance increases the chances of developing type-2 diabetes and cardiovascular disease. To study the mechanism(s) by which a HFD impairs glucose tolerance, we used a quantitative proteomic platform that integrated pI-based OFFGEL fractionation and iTRAQ labeling to profile the temporal changes in adipose membrane protein expression in mice fed a HFD for up to 8 months. Within 2 months of starting the diet, the mice adipose and liver tissues accumulated fat droplets, which contributed to subsequent insulin resistance and glucose intolerance within 6 months. The membrane proteomics delineation of such phenotypic expression resulted in quantification of 1713 proteins with 266, 343 and 125 differentially expressed proteins in 2-, 6-, and 8-month HFD-fed versus control mice, respectively. Pathway analysis of these differentially expressed proteins revealed the interplay between up-regulation of fatty acid metabolism and down-regulation of glucose metabolism. Substantial upregulation of adipose and liver carnitine palmitoyltransferase (Cpt) 1, the rate-limiting enzyme in the transport of long-chain fatty acids into mitochondria, occurred by 2 months. The increase in hepatic Cpt 1a expression was associated with a progressive decrease in glucose uptake as evidenced by downregulation of the liver glucose transporter protein (Glut) 2. Loss of glycogen storage was found in those hepatocytes full of fat droplets. Intriguingly, skeletal muscle Cpt 1b expression was unaltered by the HFD, whereas skeletal muscle Glut 4 and tyrosine phosphoryated insulin receptor substrate 1 (p-IRS1) were substantially upregulated at the same time as abnormal glucose metabolism developed in adipose and liver tissues. This study defines some of the molecular mechanisms as well as the relationship among adipose tissue, liver and skeletal muscle during development of HFD-induced glucose intolerance in vivo and identifies Cpt 1 as a potential drug target for the control or prevention of diabetes.
Journal of Proteome Research 01/2013; · 5.11 Impact Factor
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ABSTRACT: PURPOSE: (131)I therapy is regularly used following surgery as a part of thyroid cancer management. Despite an overall relatively good prognosis, recurrent or metastatic thyroid cancer is not rare. CD133-expressing cells have been shown to mark thyroid cancer stem cells that possess the characteristics of stem cells and have the ability to initiate tumours. However, no studies have addressed the influence of CD133-expressing cells on radioiodide therapy of the thyroid cancer. The aim of this study was to investigate whether CD133(+) cells contribute to the radioresistance of thyroid cancer and thus potentiate future recurrence and metastasis. METHODS: Thyroid cancer cell lines were analysed for CD133 expression, radiosensitivity and gene expression. RESULTS: The anaplastic thyroid cancer cell line ARO showed a higher percentage of CD133(+) cells and higher radioresistance. After γ-irradiation of the cells, the CD133(+) population was enriched due to the higher apoptotic rate of CD133(-) cells. In vivo (131)I treatment of ARO tumour resulted in an elevated expression of CD133, Oct4, Nanog, Lin28 and Glut1 genes. After isolation, CD133(+) cells exhibited higher radioresistance and higher expression of Oct4, Nanog, Sox2, Lin28 and Glut1 in the cell line or primarily cultured papillary thyroid cancer cells, and lower expression of various thyroid-specific genes, namely NIS, Tg, TPO, TSHR, TTF1 and Pax8. CONCLUSION: This study demonstrates the existence of CD133-expressing thyroid cancer cells which show a higher radioresistance and are in an undifferentiated status. These cells possess a greater potential to survive radiotherapy and may contribute to the recurrence of thyroid cancer. A future therapeutic approach for radioresistant thyroid cancer may focus on the selective eradication of CD133(+) cells.
European Journal of Nuclear Medicine 10/2012; · 4.53 Impact Factor
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ABSTRACT: In solid tumors, a decreased oxygen and nutrient supply creates a hypoxic microenvironment in the central region. This hypoxic condition induces molecular responses of normal and cancer cells in the local area, including angiogenesis, metabolic changes and metastasis. In addition, other cells including mesenchymal stem cells (MSCs) have been reported to be recruited into hypoxic area of solid tumors. In our previous study, we found that hypoxic condition induces the secretion of growth factors and cytokines in MSCs and here we demonstrate that elevated secretion of transforming growth factor beta1 (TGF-β1) by MSCs under hypoxia promotes the growth, motility and invasion ability of breast cancer cells. It was found that TGF-β1 promoter activity was regulated by hypoxia and the major hypoxia regulated element was located between bp -1030 to -666 in front of the TGF-β1 promoter region. In ChIP assay, the results revealed that HIF-1 was bound to hypoxia-response element (HRE) of TGF-1 promoter. Collectively, the results indicate that hypoxia microenvironment can enhance cancer cell growth through paracrine effects of the MSCs by driving their TGF-β1 gene expression and secretion. Therefore, extra caution has to be exercised when considering hypoxia pretreatment of MSCs before cell transplantation into patients for therapeutic purposes, particularly in patients susceptible to tumor growth.
Cell Transplantation 10/2012; · 5.13 Impact Factor
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ABSTRACT: Acute paraquat (PQ) poisoning induces redox cycle and leads to fatal injury of lung. Clinical management is supportive in nature due to lack effective antidote and the mortality is very high. Mesenchymal stem cells (MSCs) process the properties of immunomodulation, anti-inflammatory and anti-fibrotic effects and oxidative stress resistance. MSCs transplantation may theoretically serve as antidote in PQ intoxication. In this study, we aimed to examine the potential therapeutic effects of MSCs in PQ-induced lung injury. The degree of PQ toxicity in rat type II pneumocyte cell line, the L2 cells, and MSCs was evaluated by examining cell viability, ultrastructural changes and gene expression. L2 cells treated with 0.5mM PQ were co-cultured in the absence or presence of MSCs. For the in vivo study, adult male SD rats were administered an intraperitoneal injection of PQ (24mg/kg body weight) and were divided into three groups. Group I : control; group II: cyclophosphamide and dexamethasone; group III: MSCs transplantation 6 hours after PQ exposure. MSCs were relatively resistant to PQ toxicity. Co-culture with MSCs significantly inhibited PQ accumulation in L2 cells and upregulated the expression of antioxidative heme-oxygenase1 and metalothionein 1a genes, reversed epithelial-to-mesenchymal transition, and increased the viability of PQ-exposed L2 cells. Treatment with MSCs resulted in a significant reduction in severity of liver and renal function deterioration, alleviated lung injury, and prolonged the life span of rats. Altogether, our results suggest that MSCs possess antidote-like effect through multifactorial protection mechanism. The results of this preclinical study demonstrate that transplantation of MSCs may be a promising therapy and should be further validated clinically.
Cell Transplantation 10/2012; · 5.13 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSCs) have been shown to improve the outcome of acute renal injury models; but whether MSCs can delay renal failure in chronic kidney disease (CKD) remains unclear. In the present study, the MSCs were cultured in media containing various concentrations of basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2-phosphate to investigate whether hepatocyte growth factor (HGF) secretion could be increased by the stimulation of these growth factors. MSCs. Then TGF-β1 treated renal interstitial fibroblast (NRK-49F), renal proximal tubular cells (NRK-52E), and podocytes were co-cultured with conditioned MSCs in the absence or presence of ascorbic acid 2-phosphate to quantify the protective effects of conditioned MSCs on renal cells. Moreover, male Sprague-Dawley rats were treated with 1×10(6) conditioned MSCs immediately after 5/6 nephrectomy and every other week through the tail vein for 14 weeks. It was found that basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2-phosphate promoted HGF secretion in MSCs. Besides, conditioned MSCs were found to be protective against TGF-β1 induced epithelial-to-mesenchymal transition of NRK-52E and activation of NRK-49F cells. Furthermore, conditioned MSCs protected podocytes from TGF-β1-induced loss of synaptopodin, fibronectin induction, cell death and apoptosis. Rats transplanted with conditioned human MSCs had a significantly increase in creatinine clearance rate, decrease in glomerulosclerosis, interstitial fibrosis and increase in CD4+CD25+Foxp3+ regulatory T cells counts in splenocytes. Together, our studies indicated that conditioned MSCs preserve renal function by their anti-fibrotic and anti-inflammatory effects. Transplantation of conditioned MSCs may be useful in treating CKD. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.
Journal of Cellular and Molecular Medicine 08/2012; · 4.13 Impact Factor
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ABSTRACT: Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used for stem cell labeling and tracking. Surface modification has been known to improve biocompatibility, biodistribution, and labeling efficiency of SPIO nanoparticles. However, the effects of amine (NH 3+)-surface-modified SPIO nanoparticles on proliferation and differentiation of human mesenchymal stem cells (hMSCs) remain unclear. The purpose of this study is to investigate how amine-surface-modified SPIO nanoparticles affected hMSCs. In this study, intracellular uptake and the contiguous presence of amine-surface-modified SPIO nanoparticles in hMSCs were demonstrated by Prussian blue staining, transmission electron microscopy and magnetic resonance imaging. Moreover, accelerated cell proliferation was found to be associated with cellular internalization of amine-surface-modified SPIO nanoparticles. The osteogenic and chondrogenic differentiation potentials of hMSCs were impaired after treating with SPIO, while adipogenic potential was relatively unaffected. Altered cytokine production profile in hMSCs caused by amine-surface-modified SPIO nanoparticles may account for the increased proliferation and impaired differentiation potentials; concentrations of the growth factors in the SPIO-labeled condition medium including amphiregulin, glial cell-derived neurotrophic factor, heparin-binding EGF-like growth factor and vascular endothelial growth factor, as well as soluble form of macrophage colony-stimulating factor receptor and SCF receptor, were higher than in the unlabeled-condition medium. In summary, although amine-surface-modified SPIO labeling is effective for cell tracking, properties of hMSCs may alter as a consequence and this needs to be taken into account when evaluating therapeutic efficacies of SPIO-labeled stem cells in vivo.
Journal of Orthopaedic Research 02/2012; 30(9):1499-506. · 2.81 Impact Factor
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ABSTRACT: Liver transplantation is the only definitive treatment for end-stage cirrhosis and fulminant liver failure, but the lack of available donor livers is a major obstacle to liver transplantation. Recently, induced pluripotent stem cells (iPSCs) derived from the reprogramming of somatic fibroblasts, have been shown to resemble embryonic stem (ES) cells in that they have pluripotent properties and the potential to differentiate into all cell lineages in vitro, including hepatocytes. Thus, iPSCs could serve as a favorable cell source for a wide range of applications, including drug toxicity testing, cell transplantation, and patient-specific disease modeling. Here, we describe an efficient and rapid three-step protocol that is able to rapidly generate hepatocyte-like cells from human iPSCs. This occurs because the endodermal induction step allows for more efficient and definitive endoderm cell formation. We show that hepatocyte growth factor (HGF), which synergizes with activin A and Wnt3a, elevates the expression of the endodermal marker Foxa2 (forkhead box a2) by 39.3% compared to when HGF is absent (14.2%) during the endodermal induction step. In addition, iPSC-derived hepatocytes had a similar gene expression profile to mature hepatocytes. Importantly, the hepatocyte-like cells exhibited cytochrome P450 3A4 (CYP3A4) enzyme activity, secreted urea, uptake of low-density lipoprotein (LDL), and possessed the ability to store glycogen. Moreover, the hepatocyte-like cells rescued lethal fulminant hepatic failure in a nonobese diabetic severe combined immunodeficient mouse model. Conclusion: We have established a rapid and efficient differentiation protocol that is able to generate functional hepatocyte-like cells from human iPSCs. This may offer an alternative option for treatment of liver diseases.
Hepatology 11/2011; 55(4):1193-203. · 11.66 Impact Factor
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ABSTRACT: Human mesenchymal stromal cells (hMSCs) are promising candidates for cell therapy and tissue regeneration. Knowledge of the molecular mechanisms governing hMSC commitment into osteoblasts is critical to the development of therapeutic applications for human bone diseases. Because protein phosphorylation plays a critical role in signaling transduction network, the purpose of this study is to elucidate the phosphoproteomic changes in hMSCs during early osteogenic lineage commitment. hMSCs cultured in osteogenic induction medium for 0, 1, 3, and 7 days were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Surprisingly, we observed a dramatic loss of protein phosphorylation level after 1 day of osteogenic induction. Pathways analysis of these reduced phosphoproteins exhibited a high correlation with cell proliferation and protein synthesis pathways. During osteogenic differentiation, differentially expressed phosphoproteins demonstrated the dynamic alterations in cytoskeleton at the early stages of differentiation. The fidelity of our quantitative phosphoproteomic analyses were further confirmed by Western blot analyses, and the changes from protein expression or its phosphorylation level were distinguished. In addition, several ion channels and transcription factors with differentially expressed phosphorylation sites during osteogenic differentiation were identified and may serve as potentially unexplored transcriptional regulators of the osteogenic phenotype of hMSCs. Taken together, our results have demonstrated the dynamic changes in phosphoproteomic profiles of hMSCs during osteogenic differentiation and unraveled potential candidates mediating the osteogenic commitment of hMSCs. The findings in this study may also shed light on the development of new therapeutic targets for metabolic bone diseases such as osteoporosis and osteomalacia.
Journal of Proteome Research 11/2011; 11(2):586-98. · 5.11 Impact Factor
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ABSTRACT: Depletion of pancreatic β-cells results in insulin insufficiency and diabetes mellitus (DM). Single transplantation of mesenchymal stem cells exhibits short-term effects in some preclinical studies. Here, we further investigated the long-term therapeutic effects of multiple intravenous MSC transplantations. In this study, multiple human MSC transplantations (4.2 × 10(7) cells/kg each time) were performed intravenously at 2-week intervals into streptozocin (STZ)-induced diabetic mice for 6 months. Blood sugar, insulin, renal function, cholesterol, and triglyceride levels were monitored. We demonstrated that compared to single intravenous transplantation, which only transiently decreased hyperglycemia, multiple MSC transplantations effectively restored blood glucose homeostasis. Systemic oxidative stress levels were reduced from the seventh week of treatment. From the 11th week, production of human insulin was markedly increased. When MSC transplantation was skipped after blood sugar level returned to normal at the end of 15th week, a sharp rebound of blood sugar occurred, and was then controlled by subsequent transplantations. At the end of 6 months, histopathology examination revealed MSCs specifically engrafted into liver tissues of the recipients. Fifty-one percent of human cells in the recipient liver coexpressed human insulin, especially those surrounding the central veins. Taken together, intravenous MSC delivery was safe and effective for blood glucose stabilization in this preclinical DM model. Multiple transplantations were essential to restore and maintain glucose homeostasis through decreasing systemic oxidative stress in the early stage and insulin production in the late stage. Liver engraftment and differentiation into insulin-producing cells account for the long-term therapeutic effects of MSCs.
Cell Transplantation 10/2011; 21(5):997-1009. · 5.13 Impact Factor
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ABSTRACT: Spinocerebellar ataxia (SCA) refers to a disease entity in which polyglutamine aggregates are over-produced in Purkinje cells (PCs) of the cerebellum as well as other neurons in the central nervous system, and the formation of intracellular polyglutamine aggregates result in the loss of neurons as well as deterioration of motor functions. So far there is no effective neuroprotective treatment for this debilitating disease although numerous efforts have been made. Mesenchymal stem cells (MSCs) possess multi-lineage differentiation potentials as well as immuno-modulatory properties, and are theoretically good candidates for SCA treatment. The purpose of this study is to investigate whether transplantation of human MSCs (hMSCs) can rescue cerebellar PCs and ameliorate motor function deterioration in SCA in a pre-clinical animal model.
Transgenic mice bearing poly-glutamine mutation in ataxin-2 gene (C57BL/6J SCA2 transgenic mice) were serially transplanted with hMSCs intravenously or intracranially before and after the onset of motor function loss. Motor function of mice was evaluated by an accelerating protocol of rotarod test every 8 weeks. Immunohistochemical stain of whole brain sections was adopted to demonstrate the neuroprotective effect of hMSC transplantation on cerebellar PCs and engraftment of hMSCs into mice brain.
Intravenous transplantation of hMSCs effectively improved rotarod performance of SCA2 transgenic mice and delayed the onset of motor function deterioration; while intracranial transplantation failed to achieve such neuroprotective effect. Immunohistochemistry revealed that intravenous transplantation was more effective in the preservation of the survival of cerebellar PCs and engraftment of hMSCs than intracranial injection, which was compatible to rotarod performance of transplanted mice.
Intravenous transplantation of hMSCs can indeed delay the onset as well as improve the motor function of SCA2 transgenic mice. The results of this preclinical study strongly support further exploration of the feasibility to transplant hMSCs for SCA patients.
Journal of Biomedical Science 08/2011; 18:54. · 2.01 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSCs), which can be isolated from bone marrow and other somatic tissues, are residing in an environment with relative low oxygen tension. The purpose of this study is to investigate the effects of hypoxia on MSCs, and we hypothesize that oxygen concentration regulates the intricate balance between cellular proliferation and commitment towards differentiation. In this study, human bone marrow-derived MSCs were cultured under hypoxia with 1% O(2). The proliferation ability of MSCs was increased after a 7-day hypoxic culture period. Migration assay showed that hypoxia enhanced the migration capabilities of MSCs. Moreover, expression of stemness genes Oct4, Nanog, Sall4 and Klf4 was increased under hypoxia. Furthermore, the differentiation ability of MSCs under hypoxia favored osteogenesis while adipogenesis was inhibited during a 4-week induction period. Cytokine antibody array analysis showed that a number of growth factors were up-regulated after a 7-day hypoxic incubation and the differential expression of growth factors may account for the increased proliferation and osteogenic potentials of MSCs under hypoxic condition. Taken together, hypoxia provides a favorable culture condition to promote proliferation as well as osteogenesis of MSCs through differential growth factor production.
Journal of Orthopaedic Research 08/2011; 30(2):260-6. · 2.81 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSCs) have been shown to possess immunomodulatory properties. Systemic lupus erythematosus is an autoimmune disease that results in nephritis and subsequent destruction of renal microstructure. We investigated whether transplantation of human umbilical cord blood-derived MSCs (uMSCs) is useful in alleviating lupus nephritis in a murine model. It was found that uMSCs transplantation significantly delayed the development of proteinuria, decreased anti-dsDNA, alleviated renal injury, and prolonged the life span. There was a trend of decreasing T-helper (Th) 1 cytokines (IFN-γ, IL-2) and proinflammatory cytokines (TNF-α, IL-6, IL-12) and increasing Th2 cytokines (IL-4, IL-10). The in vitro coculture experiments showed that uMSCs only inhibited lymphocytes and splenocytes proliferation but not mesangial cells. Long-term engraftment of uMSCs in the kidney was not observed either. Together, these findings indicated that uMSCs were effective in decreasing renal inflammation and alleviating experimental lupus nephritis by inhibiting lymphocytes, inducing polarization of Th2 cytokines, and inhibition of proinflammatory cytokines production rather than direct engraftment and differentiating into renal tissue. Therapeutic effects demonstrated in this preclinical study support further exploration of the possibility to use uMSCs from mismatched donors in lupus nephritis treatment.
Cell Transplantation 01/2011; 20(2):245-57. · 5.13 Impact Factor
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ABSTRACT: Anaplastic thyroid carcinoma (ATC) is one of the most deadly cancers. With intensive multimodalities of treatment, the survival remains low. ATC is not sensitive to (131)I therapy due to loss of sodium iodide symporter (NIS) gene expression. We have previously generated a stable human NIS-expressing ATC cell line, ARO, and the ability of iodide accumulation was restored. To make NIS-mediated gene therapy more applicable, this study aimed to establish a lentiviral system for transferring hNIS gene to cells and to evaluate the efficacy of in vitro and in vivo radioiodide accumulation for imaging and therapy. Lentivirus containing hNIS cDNA were produced to transduce ARO cells which do not concentrate iodide. Gene expression, cell function, radioiodide imaging and treatment were evaluated in vitro and in vivo. Results showed that the transduced cells were restored to express hNIS and accumulated higher amount of radioiodide than parental cells. Therapeutic dose of (131)I effectively inhibited the tumor growth derived from transduced cells as compared to saline-treated mice. Our results suggest that the lentiviral system efficiently transferred and expressed hNIS gene in ATC cells. The transduced cells showed a promising result of tumor imaging and therapy.
Journal of Oncology 01/2011; 2011:178967.
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ABSTRACT: The liver is an extraordinary organ that retains its regenerative power throughout life. The precise molecular mechanisms
regulating liver regeneration are unknown, but a number of cell types have been postulated to be involved in the process,
including (1) resident liver stem cells, (2) differentiated hepatocytes, and (3) extrahepatic stem cells. This chapter will
discuss liver regeneration from the context of extrahepatic stem cells. Recent research findings have challenged the dogma
of limited lineage commitment potency of somatic stem cells. This chapter reviews the hepatic lineage plasticity of mesenchymal
stem cells in vitro and in. In addition, it discusses developments in the application of mesenchymal stem cells for the treatment
of liver diseases in preclinical and clinical studies, as well as in possible signaling pathways and mechanisms, including
microRNAs, which are involved in the regulatory control of hepatic fate specification.
KeywordsMesenchymal stem cells-Hepatogenesis-Liver disease-miRNA-Cell therapy
12/2010: pages 155-179;
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ABSTRACT: Mesenchymal stem cells (MSCs) are of great therapeutic potentials due to their multilineage differentiation capabilities. Before transplantation, in vitro culture expansion of MSCs is necessary to get desired cell number. We observed that cell contact accelerated replicative senescence during such process. To confirm the finding as well as to investigate the underlying mechanisms, we cultured both human bone marrow- and umbilical cord blood-derived MSCs under noncontact culture (subculture performed at 60-70% of confluence), or contact culture (cell passage performed at 100% of confluence). It was found that MSCs reached cellular senescence earlier in contact culture, and the doubling time was significantly prolonged. Marked increase of senescence-associated β-galactosidase-positive staining was also observed as a result of cell contact. Cell cycle analysis revealed increased frequency of cell cycle arrest after contact culture. It was noted, however, that the telomere length was not altered during contact-induced acceleration of senescence. Moreover, cell cycle checkpoint regulator P53 expression was not affected by cell contact. Marked increase in intracellular reactive oxygen species (ROS) and a concomitant decrease in the activities of antioxidative enzymes were also observed during contact-induced senescence. Importantly, increased p16(INK4a) following Ras upregulation was found after contact culture. Taken together, cell contact induced accelerated senescence of MSCs, which is telomere shortening and p53 independent. ROS accumulation due to defective ROS clearance function together with Ras and p16(INK4a) upregulation play an important role in contact-induced senescence of MSCs. Overconfluence should therefore be avoided during in vitro culture expansion of MSCs in order to maintain their qualities for clinical application purposes. The contact-induced senescence model reported in this study will serve as a useful model system that allows further study of the molecular mechanisms of senescence in MSCs.
Cell Transplantation 12/2010; 20(8):1209-20. · 5.13 Impact Factor
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ABSTRACT: The unique self-renewal and multilineage differentiation potential of mesenchymal stem cells (MSCs) make them a promising candidate for cell therapy applications. However, during in vitro expansion of MSCs, replicative senescence may occur and will compromise the quality of the expanded cells. Because calorie restriction has been shown to effectively extend the life span of various organisms, the purpose of this study is to investigate the effect of glucose reduction on MSCs and the coordinated changes in energy utilization. It was found that the frequency of cycling cells was significantly increased, while senescence markers such as β-galactosidase activities and p16(INK4a) expression level were markedly reduced in MSCs under low-glucose culture condition. Quantitative real-time PCR analysis demonstrated the preserved trilineage differentiation potentials of MSCs after low-glucose treatment. Interestingly, the ability of osteogenic lineage commitment was improved, while the ability of adipogenic lineage commitment was delayed in MSCs after glucose reduction. In addition, we observed decreased lactate production, increased electron transport chain complexes expression, and increased oxygen consumption in MSCs after glucose reduction treatment. Increased antioxidant defensive responses were evidenced by increased antioxidant enzymes expression and decreased superoxide production after glucose reduction. Taken together, our findings suggest that MSCs utilize energy more efficiently under restricted glucose treatment and exhibit greater self-renewal and antisenescence abilities, while their differentiation potentials remain unaffected.
Cell Transplantation 11/2010; 20(6):813-25. · 5.13 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSCs) cultured on extracellular matrices with different stiffness have been shown to possess diverse lineage commitment owing to the extracellular mechanical stimuli sensed by the cells. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through the mechanotransducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MSCs. MSCs were cultured in osteogenic medium on tunable polyacrylamide hydrogels coated with type I collagen with elasticities corresponding to Young's modulus of 7.0 ± 1.2 and 42.1 ± 3.2 kPa. Osteogenic differentiation was increased on stiffer matrices, as evident by type I collagen, osteocalcin, and Runx2 gene expressions and alizarin red S staining for mineralization. Western blot analysis demonstrated an increase in kinase activities of ROCK, FAK, and ERK1/2 on stiffer matrices. Inhibition of FAK, an important mediator of osteogenic differentiation, and inhibition of ROCK, a known mechanotransducer of matrix stiffness during osteogenesis, resulted in decreased expression of osteogenic markers during osteogenic induction. In addition, FAK affects osteogenic differentiation through ERK1/2, whereas ROCK regulates both FAK and ERK1/2. Furthermore, α(2)-integrin was upregulated on stiffer matrices during osteogenic induction, and its knockdown by siRNA downregulated the osteogenic phenotype through ROCK, FAK, and ERK1/2. Taken together, our results provide evidence that the matrix rigidity affects the osteogenic outcome of MSCs through mechanotransduction events that are mediated by α(2)-integrin.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 10/2010; 26(4):730-8. · 6.04 Impact Factor
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ABSTRACT: Due to their immunomodulatory functions, mesenchymal stem cells (MSCs) have great potential for clinical applications to prevent rejection in organ transplantation and to prevent graft-versus-host disease in hematopoietic stem cell (HSC) transplantation. Since dendritic cells (DCs) play an important role in modulating diverse T cell responses, including rejection and graft-versus-host disease, the goal of this study was to investigate whether MSCs modulate DC differentiation from HSCs and if this effect could be one of the mechanisms for MSCs' immune-modulating functions. Our results demonstrate that differentiation of HSCs into mature DCs is inhibited in the presence of MSCs. Similar frequency of dendritic precursors in the cultures, either with or without MSCs, suggests that the inhibition of MSCs on the differentiation of mature DCs from HSCs could be due to the arresting of maturation at the dendritic precursor step. Reduced levels of cyclic AMP, adenosine 3',5'-cyclic monophosphate (cAMP) and beta-catenin in DC-like cells from the cocultures are detected, suggesting that induction of apoptosis and inhibition of differentiation could be the basis for the inhibition of mature DCs from HSCs by MSCs. Further, our results demonstrate that DCs derived from HSCs in the presence of MSCs are functionally impaired, especially for those after direct contact with MSCs. To investigate the basis of functional impairment, our data show downregulated tumor necrosis factor-alpha and transforming growth factor-beta1 secretion and upregulated interleukin-6 (IL6) and IL1beta secretion in the cultures with MSCs. Together, MSCs can inhibit differentiation of mature DCs from HSCs by arresting them at the precursor stage and induce their apoptosis. Further, HSC-derived DCs in the presence of MSCs are functionally impaired, which could be partly due to the upregulation of IL6 secretion.
Tissue Engineering Part A 09/2010; 16(9):2987-97. · 4.64 Impact Factor
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ABSTRACT: Osteosarcoma is the most common primary malignant bone tumor. The long-term outcome is poor for patients with metastatic disease.
From June 1989 to January 2008, 202 patients (128 males and 74 females) with high-grade osteosarcoma of the extremities were treated at our institution. Patients were divided into three groups depending on the time of identification of pulmonary metastasis: group A, identified with primary tumor diagnosis; group B, during whole treatment course; and group C, after completion of treatment. Long-term survival was calculated and factors related to metastases were analyzed.
Ninety-one patients developed pulmonary metastases; 21 in group A, 18 in group B and 52 in group C. The mean period from initial diagnosis to lung metastases in groups B and C was 22.2 months (+/-20.6). Five-year survival rates were 82.0% and 38.3% in the non-metastasis group and metastasis group, respectively (P < 0.001). The 5-year survival rate was significantly worse in group A than in group B or C (0%, 7.4%, 59.5%, P < 0.001), in patient with more than one lobe involved (27.0%, P = 0.006) and more than three pulmonary nodule metastases (21.3%, P = 0.002). Factors related to the pulmonary metastasis were: old age (65.5% in older than 27.5 years old and 41.6% in younger, P = 0.017), large tumor volume (54.4% in larger than 202.5 ml and 33.7% in smaller, P = 0.005) and elevated lactodehydrogenase (LDH; 55.1% vs.31.0% in normal, P = 0.001).
The prognosis of osteosarcoma with pulmonary metastases is dismal, especially for patients who have primary pulmonary metastases, more than three pulmonary metastatic nodules or involvement of more than one lobe. Factors such as older age, larger tumor volume and elevated LDH may reflect high metastatic rate.
Japanese Journal of Clinical Oncology 07/2009; 39(8):514-22. · 1.78 Impact Factor
