An iTRAQ proteomic study reveals an association between diet-induced enhanced fatty acid metabolism and the development of glucose intolerance in prediabetic mice.

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 proteomic 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 upregulation of fatty acid metabolism and downregulation 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.

CD133-expressing thyroid cancer cells are undifferentiated, radioresistant and survive radioiodide therapy.

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.

Systemic human orbital fat-derived stem/stromal cell transplantation ameliorates acute inflammation in lipopolysaccharide-induced acute lung injury.

OBJECTIVE: Acute lung injury results in acute respiratory distress syndrome. There is no standard therapy for acute respiratory distress syndrome but supportive care. Stem cells offer a new therapeutic potential for tissue regeneration as a result of their self-renewal, multipotency, and paracrine capabilities. The objective of this study is to investigate the effects and the mechanisms of systemic human orbital fat-derived stem/stromal cell transplantation on lipopolysaccharide-induced acute lung injury. DESIGN: Prospective, randomized, controlled study. SETTING: University-affiliated research institute. SUBJECTS: Male BALB/c mice. INTERVENTIONS: Twenty-five micrograms lipopolysaccharide in 50 µL sterile saline or 50 µL of sterile saline was delivered through intratracheal injection. Twenty mins later, the animals were further randomized into subgroups that received either a tail vein injection of 3 × 10 orbital fat-derived stem/stromal cells in 50 µL phosphate-buffered saline or 50 µL phosphate-buffered saline. MEASUREMENTS AND MAIN RESULTS: Low immunogenicity and immune-tolerated of orbital fat-derived stem/stromal cells were observed in this xenotransplanted model. Orbital fat-derived stem/stromal cells significantly reduced lipopolysaccharide-induced pulmonary inflammation, which was evidenced by a decrease in total protein concentration and neutrophil counts in alveolar fluid through bronchoalveolar lavage, reduced endothelial and alveolar epithelial permeability as well as neutrophil (Ly6G-expressing cells) and macrophage (CD68-expressing cells) infiltration. Lipopolysaccharide-induced expression of CD14, inducible nitric oxide synthase, and transforming growth factor-ß in lung tissue was significantly inhibited by orbital fat-derived stem/stromal cells. Orbital fat-derived stem/stromal cells not only reduced the circulation numbers of macrophages and neutrophils (CD11b-expressing cells), but also decreased systemic proinflammatory chemokine levels such as macrophage inflammatory protein-1-γ, B-lymphocyte chemoattractant, interleukin-12, and subsequent circulation helper T cell (CD4-expressing cells) numbers. Furthermore, few human orbital fat-derived stem/stromal cells were detectable in the recipient lung after acute inflammation subsided. CONCLUSIONS: Systemic orbital fat-derived stem/stromal cell transplantation was effective in modulating inflammation during acute lung injury. The therapeutic effect was attributed to the inhibition of acute inflammatory responses.

Mesenchymal stem cell transplantation ameliorates motor function deterioration of spinocerebellar ataxia by rescuing cerebellar Purkinje cells.

BACKGROUND: 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. METHOD: 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. RESULTS: 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. CONCLUSION: 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.

DNA Methyltransferases Modulate Hepatogenic Lineage Plasticity of Mesenchymal Stromal Cells.

The irreversibility of developmental processes in mammalian cells has been challenged by rising evidence that de-differentiation of hepatocytes occurs in adult liver. However, whether reversibility exists in mesenchymal stromal cell (MSC)-derived hepatocytes (dHeps) remains elusive. In this study, we find that hepatogenic differentiation (HD) of MSCs is a reversible process and is modulated by DNA methyltransferases (DNMTs). DNMTs are regulated by transforming growth factor ß1 (TGFß1), which in turn controls hepatogenic differentiation and de-differentiation. In addition, a stepwise reduction in TGFß1 concentrations in culture media increases DNMT1 and decreases DNMT3 in primary hepatocytes (Heps) and confers Heps with multi-differentiation potentials similarly to MSCs. Hepatic lineage reversibility of MSCs and lineage conversion of Heps are regulated by DNMTs in response to TGFß1. This previously unrecognized TGFß1-DNMTs-MSC-HD axis may further increase the understanding the normal and pathological processes in the liver, as well as functions of MSCs after transplantation to treat liver diseases.

Detection of hepatic maturation by Raman spectroscopy in mesenchymal stromal cells undergoing hepatic differentiation.

INTRODUCTION: Mesenchymal stromal cells (MSCs) are well known for their application potential in tissue engineering. We previously reported that MSCs are able to differentiate into hepatocytes in vitro. However, conventional methods for estimating the maturation of hepatic differentiation require relatively large amounts of cell samples. Raman spectroscopy (RS), a photonic tool for acquisition of cell spectra by inelastic scattering, has been recently used as a label-free single-cell detector for biological applications including phenotypic changes and differentiation of cells and diagnosis. In this study, RS is used to real-time monitor the maturation of hepatic differentiation in live MSCs. METHODS: The MSCs were cultured on the type I collagen pre-coating substrate and differentiated into hepatocytes in vitro using a two-step protocol. The Raman spectra at different time points are acquired in the range 400-3000 cm(-1)and analyzed by quantification methods and principle component analysis during hepatic differentiation from the MSCs. RESULTS: The intensity of the broad band in the range 2800-3000 cm(-1) reflects the amount of glycogen within lipochrome in differentiated hepatocytes. A high correlation coefficient between the glycogen amount and hepatic maturation was exhibited. Moreover, principle component analysis of the Raman spectra from 400 to 3000 cm(-1) indicated that MSC-derived hepatocytes were close to the primary hepatocytes and were distinct from the undifferentiated MSCs. CONCLUSIONS: In summary, RS can serve as a rapid, non-invasive, real-time and label-free biosensor and reflects changes in live cell components during hepatic differentiation. The use of RS may thus facilitate the detection of hepatic differentiation and maturation in stem cells. Such an approach may substantially improve the feasibility as well as shorten the time required compared to the conventional molecular biology methods.

Niche-dependent regulations of metabolic balance in high-fat diet-induced diabetic mice by mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) have great potential to maintain glucose homeostasis and metabolic balance. Here, we demonstrate that in mice continuously fed with high-fat diet (HFD) that developed non-insulin-dependent diabetes, two episodes of systemic MSC transplantations effectively improve glucose tolerance and blood glucose homeostasis and reduce body weight through targeting pancreas and insulin-sensitive tissues and organs via site-specific mechanisms. MSCs support pancreatic islet growth by direct differentiation into insulin-producing cells and by mitigating the cytotoxicity of interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α) in the pancreas. Localization of MSCs in the liver and skeletal muscles in diabetic animals is also enhanced and therefore improves glucose tolerance, although long-term engraftment is not observed. MSCs prevent HFD-induced fatty liver development and restore glycogen storage in hepatocytes. Increased expression of IL-1 receptor antagonist and Glut4 in skeletal muscles after MSC transplantation results in better blood glucose homeostasis. Intriguingly, systemic MSC transplantation does not alter adipocyte number, but it decreases HFD-induced cell infiltration in adipose tissues and reduces serum levels of adipokines, including leptin and TNF-α. Taken together, systemic MSC transplantation ameliorates HFD-induced obesity and restores metabolic balance through multisystemic regulations that are niche dependent. Such findings have supported systemic transplantation of MSCs to correct metabolic imbalance.

Regulation of metastatic ability and drug resistance in pulmonary adenocarcinoma by matrix rigidity via activating c-Met and EGFR.

Lung fibrosis is a poor prognostic factor for pulmonary adenocarcinoma, and the effect of a rigid microenvironment on cancer behavior is unclear. We cultured A549 cells on matrices of 0.2, 2, and 25 kPa to mimic the rigidities of normal lung parenchyma, progressive fibrotic change, and lung fibrosis, respectively. Lung tissue from patients with pulmonary adenocarcinoma was used to confirm the in vitro findings. Increased matrix rigidity promoted cell proliferation and upregulated the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-Met), and Snail expression in A549 cells. A549 cells became more resistant to the EGFR inhibitor (Erlotinib) and c-Met inhibitor (PHA-665752) when matrix rigidity increased; however, a high concentration of PHA-665752 reversed the rigidity-induced morphological pleomorphism. In human lung tissue, expression of type I collagen was more consistent with clinical fibrosis than the expression of alpha-smooth muscle antibody was. c-Met- and Snail-expressing tumor cells, rather than EGFR-experssing cells, were localized with lung parenchyma rich in type I collagen. Our findings suggest that c-Met causes the rigidity-induced biophysical reaction in pulmonary adenocarcinoma. Treatment targeting both EGFR and c-Met should be considered for patients with lung fibrosis and who are abundant type I collagen expression in the tumor mass.

Promotion of mesenchymal-to-epithelial transition by Rac1 inhibition with small molecules accelerates hepatic differentiation of mesenchymal stromal cells.

In vitro differentiation of stem cells into specific cell lineages provides a stable cell supply for cell therapy and tissue engineering. Therefore, understanding the mechanisms underlying such differentiation processes is critical for generating committed lineage-specific cell progenies effectively. We previously developed a two-step protocol to differentiate mesenchymal stromal cells (MSCs) into hepatocyte-like cells. Since hepatic differentiation involves mesenchymal-epithelial transition (MET), we hypothesize that promoting MET could further accelerate the differentiation process. Ras-related C3 botulinum toxin substrate 1 (Rac1) is involved in actin polymerization and its role in MET was investigated in the study. Our results showed that inhibition of Rac1 activation by Rac1-specific inhibitor, NSC23766, led to cells favoring epithelial morphology and being more packed during hepatic differentiation. In addition, Rac1 inhibition accelerated the upregulation of hepatic marker genes accompanied by more mature hepatic functions. Taken together, promotion of MET by inhibiting Rac1 accelerates the hepatic differentiation of MSCs. Our findings open a new prospect of directing the commitment of MSCs by manipulating cell morphology and cytoskeleton arrangement through small molecules. The results provide further insight into scaffold design for rapid production of MSC-differentiated hepatocytes.

The effects of actin cytoskeleton perturbation on keratin intermediate filament formation in mesenchymal stem/stromal cells.

F-actin plays a crucial role in composing the three-dimensional cytoskeleton and F-actin depolymerization alters fate choice of mesenchymal stem/stromal cells (MSCs). Here, we investigated differential gene expression and subsequent physiological changes in response to F-actin perturbation by latrunculin B in MSCs. Nineteen genes were down-regulated and 27 genes were up-regulated in the first 15 min after F-actin depolymerization. Functional enrichment analysis revealed that five genes involved in keratin (KRT) intermediate filaments clustering in the chromosome 17q21.2 region, i.e., KRT14, KRT19, KRT34, KRT-associated protein (KRTAP) 1-5, and KRTAP2-3, were strongly up-regulated. Transcription factor prediction identified NKX2.5 as the potential transcription factor to control KRT19, KRT34, KRTAP1-5, and KRTAP2-3; and indeed, the protein level of NKX2.5 was markedly increased in the nuclear fraction within 15 min of F-actin depolymerization. The peak of keratin intermediate filament formation was 1 h after actin perturbation, and the morphological changes showed by decrease in the ratio of long-axis to short-axis diameter in MSCs was observed after 4 h. Together, F-actin depolymerization rapidly triggers keratin intermediate filament formation by turning on keratin-related genes on chromosome 17q21.2. Such findings offer new insight in lineage commitment of MSCs and further scaffold design in MSC-based tissue engineering.

The ability to suppress macrophage-mediated inflammation in orbital fat stem cells is controlled by miR-671-5p.

INTRODUCTION: Our previous works demonstrated that systemic orbital fat-derived stem cell (OFSC) transplantation was effective in ameliorating lipopolysaccharide (LPS)-induced extensive acute lung injury (ALI) in vivo mainly through paracrine regulation of macrophage-mediated cytokine-storm. In this study, we explore the molecular mechanism(s) of OFSCs regulating macrophage activity in a cytokine-inducible fashion. METHODS: LPS (100 ng/ml)-activated macrophages were treated by conditioned medium from OFSCs (OFSCs-CM) or non-contact cultured with OFSCs for 6 hours. The potency of OFSCs on macrophage proliferation and pro-inflammation ability were determined. Expression levels of pro-inflammatory cytokines in macrophages, inducible immuno-modulatory factors in OFSCs, were investigated. Deep sequencing analysis as well as interaction between microRNA (miRNA) and genes of immuno-modulators in OFSCs induced by activated macrophages was predicted by miRTar. Transfection of miRNA inhibitor into OFSCs was performed. Real-time RT-PCR and transplantation of OFSCs into mice with LPS-induced ALI confirmed the in vitro and in vivo mechanism. RESULTS: The paracrine effect of OFSCs on inhibition of macrophage pro-inflammatory cytokine release was more potent than induction of macrophage G0/G1 cell cycle arrest. OFSCs-CM suppressed LPS-induced inducible nitric oxide synthetase and the pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 alpha, and IL-1 beta expression in macrophages. Under non-contact culture, LPS-activated macrophages effectively triggered the expression of soluble immuno-modulating factors in OFSCs, i.e., IL-10, IL-1 receptor antagonist (IL-1 RA), indoleamine 2,3-dioxygenase, and soluble TNF receptor type II (sTNF RII). Under miRTar prediction, miR-671-5p was identified as a critical microRNA in regulation of multiple immune-modulating factors in OFSCs response to macrophages. The baseline level of miR-671-5p was high in OFSCs, and down-regulation of miR-671-5p upon co-culture with activated macrophages was observed. MiR-671-5p inhibitor transfection into OFSCs selectively enhanced the IL-1 RA and sTNF RII expressions. In addition, inhibition of miR-671-5p in OFSCs enhanced the anti-inflammatory ability against LPS-induced ALI. CONCLUSION: The paracrine effect of OFSCs inhibits the pro-inflammatory ability and proliferation of macrophages. The immune-modulation capacity of OFSCs can be triggered by activated macrophages, and down-regulation of miR-671-5p enhances OFSC immuno-modulation ability by up-regulating IL-1 RA and sTNF RII expression.

In vivo fluorescence imaging reveals the promotion of mammary tumorigenesis by mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are multipotent adult stem cells which are recruited to the tumor microenvironment (TME) and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.

Amine-surface-modified superparamagnetic iron oxide nanoparticles interfere with differentiation of human mesenchymal stem cells.

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.

Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells.

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.

Ag/Au bi-metallic film based color surface plasmon resonance biosensor with enhanced sensitivity, color contrast and great linearity.

In wavelength surface plasmon resonance (SPR) biosensor, the manipulation of SPR dispersion relation by Ag/Au bi-metallic film was first time implemented. Due to the enhanced resonant wavelength shift and the sharper SPR slope of using Ag/Au bi-metallic film, the illuminated color of reflection shows one order of magnitude greater contrast than conventional SPR biosensors. Such an Ag/Au bi-metallic film based color SPR biosensor (CSPRB) allows the detail bio-interactions, for example 100 nM streptavidin, to be distinguished by directly observing the color change of reflection through naked eyes rather than the analysis of spectrometer. In addition to the enhanced sensitivity and color contrast, this CSPRB also possesses a great linear detection range up to 0.0254 RIU, which leading to the application of point-of-care tests.

Multiple intravenous transplantations of mesenchymal stem cells effectively restore long-term blood glucose homeostasis by hepatic engraftment and ß-cell differentiation in streptozocin-induced diabetic mice.

Depletion of pancreatic ß-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.

Glucose reduction prevents replicative senescence and increases mitochondrial respiration in human mesenchymal stem cells.

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 ß-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.

Development of a surface plasmon resonance biosensor for real-time detection of osteogenic differentiation in live mesenchymal stem cells.

Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.

Cell contact accelerates replicative senescence of human mesenchymal stem cells independent of telomere shortening and p53 activation: roles of Ras and oxidative stress.

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 ß-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.

Thymosin beta-4 directs cell fate determination of human mesenchymal stem cells through biophysical effects.

Change of actin filament organization at the early stage of cell differentiation directs cell fate commitment of mesenchymal stem cells (MSCs). Thymosin beta-4 (Tbeta(4)), a major G-actin sequestering peptide, is known to regulate the cytoskeleton. The study investigated the ways in which Tbeta(4) regulates cell fate determination in MSCs upon differentiation induction. It was found that Tbeta(4) decreased F-actin formation, reduced the F-actin/G-actin ratio, and inhibited osteogenic differentiation; such actin reorganization was not associated with the change of Runt-related transcription factor 2 gene expression during early osteogenic induction. Besides, Tbeta(4) reciprocally facilitated adipogenic differentiation. Tbeta(4) treatment was found to up-regulate gene as well as promote surface expression of adipocyte adhesion molecule during early adipogenic differentiation, which accompanied acceleration of adipocyte phenotypic maturation but was not associated with differential expression of peroxisome proliferator-activated receptor gamma during the first week of adipogenic induction. In summary, Tbeta(4) initiated cell fate determination of MSCs through biophysical effects exerted by cytoskeleton reorganization and altered cell-cell adhesion rather than direct regulation of lineage-determining transcriptional factors. Such findings suggest that Tbeta(4), a ubiquitous peptide, may be involved in osteoporosis when its intracellular concentration is elevated. Further investigation of targeting Tbeta(4) for future osteoporosis treatment is warranted.