Bone marrow‐derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion‐induced lung injury

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow‐derived mesenchymal stem cells (BM‐MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM‐MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion‐induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM‐MSCs. Seventy mice were pre‐treated with BM‐MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro‐vascular endothelial cells (HPMVECs) were pre‐conditioned with BM‐MSCs by oxygen‐glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3‐kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI‐treated mice, administration of BM‐MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD‐treated HPMVECs, co‐culture with BM‐MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM‐MSCs decreased the level of PI3K class I and p‐Akt while the expression of PI3K class III was increased. Finally, BM‐MSCs‐induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM‐MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM‐MSCs and will help to develop new cell‐based therapeutic strategies in lung injury.     

Genetic correction of Werner syndrome gene reveals impaired pro‐angiogenic function and HGF insufficiency in mesenchymal stem cells

WRN mutation causes a premature aging disease called Werner syndrome (WS). However, the mechanism by which WRN loss leads to progeroid features evident with impaired tissue repair and regeneration remains unclear. To determine this mechanism, we performed gene editing in reprogrammed induced pluripotent stem cells (iPSCs) derived from WS fibroblasts. Gene correction restored the expression of WRN. WRN^+/+ mesenchymal stem cells (MSCs) exhibited improved pro‐angiogenesis. An analysis of paracrine factors revealed that hepatocyte growth factor (HGF) was downregulated in WRN^?/? MSCs. HGF insufficiency resulted in poor angiogenesis and cutaneous wound healing. Furthermore, HGF was partially regulated by PI3K/AKT signaling, which was desensitized in WRN^?/? MSCs. Consistently, the inhibition of the PI3K/AKT pathway in WRN^+/+ MSC resulted in reduced angiogenesis and poor wound healing. Our findings indicate that the impairment in the pro‐angiogenic function of WS‐MSCs is due to HGF insufficiency and PI3K/AKT dysregulation, suggesting trophic disruption between stromal and epithelial cells as a mechanism for WS pathogenesis.     

Prolyl hydroxylase inhibitor dimethyloxalylglycine enhances mesenchymal stem cell survival

Mesenchymal stem cell (MSC) transplantation is a promising approach in the therapy of ischemic heart or CNS diseases; however, the poor viability of MSCs after transplantation critically limits the efficacy of this new strategy. Prolyl hydroxylase inhibition followed by HIF‐1α up‐regulation participates in the regulation of apoptosis and cell survival, which have been shown in cancer cells and neurons. The role of prolyl hydroxylase inhibition by dimethyloxalylglycine (DMOG) in regulation of cell survival has not been investigated in MSCs. In the present investigation with MSCs, apoptosis and cell death induced by serum deprivation were assessed by caspase‐3 activation and trypan blue staining, respectively. The mitochondrial apoptotic pathway and PI3K/Akt cell survival pathway were evaluated. DMOG significantly attenuated apoptosis and cell death of MSCs, stabilized HIF‐1α and induced downstream glucose transport 1 (Glut‐1) synthesis. DMOG treatment reduced mitochondrial cytochrome c release, nuclear translocation of apoptosis inducing factor (AIF), and promoted Akt phosphorylation. A specific PI3K inhibitor, wortmannin, blocked Akt phosphorylation and abrogated the beneficial effect of DMOG. These data suggest that the DMOG protection of MSCs may provide a novel approach to promote cell survival during cell stress. J. Cell. Biochem. 106: 903–911, 2009. © 2009 Wiley‐Liss, Inc.     

PDGF-BB-induced MT1-MMP expression regulates proliferation and invasion of mesenchymal stem cells in 3-dimensional collagen via MEK/ERK1/2 and PI3K/AKT signaling

Mesenchymal stem cells (MSCs) mobilize membrane type-1 matrix metalloproteinase (MT1-MMP) to traffic through both 3-dimensional (3D) collagen as well as basement membrane barriers, but factors capable of regulating the expression and activity of the protease remain unidentified. Herein, we report that the MT1-MMP-dependent invasive activities of rat MSCs are controlled by PDGF-BB. Furthermore, PDGF-BB also stimulates MSC proliferation in 3D type I collagen via an MT1-MMP-dependent process that is linked to pericellular collagen degradation. PDGF-BB stimulates MT1-MMP expression at both the mRNA and protein levels in concert with ERK1/2 and PI3K/AKT activation. Inhibition of ERK1/2 or PI3K/AKT activity potently suppresses both MT1-MMP-dependent invasive and proliferative activities. Basement membrane invasion is likewise stimulated by PDGF-BB in an MT1-MMP-dependent manner via ERK1/2 and PI3K/AKT signaling. Taken together, these data serve to identify PDGF-BB as an important MSC agonist that controls invasive and proliferative activities via MT1-MMP-dependent processes that are regulated by the ERK1/2 and PI3K/AKT signaling pathways.     

Detection of cytokines in supernatant from hematopoietic stem/progenitor cells co-cultured with mesenchymal stem cells and endothelial progenitor cells

This study aimed to investigate the significance of cytokine expression in supernatant from hematopoietic stem/progenitor cells (HSCs/HPCs) co-cultured with mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs). Mononuclear cells (MNCs) were isolated from normal human umbilical cord blood and then cultured solely or co-cultured with MSCs or EPCs. Changes in the number of MNCs and HSCs/HPCs were observed, and MNC proliferation was tested by carboxyfluorescein diacetate succinimidyl ester. The cultured supernatants of the treated MSCs and EPCs were collected at 24 h after co-culture and used to determine the concentrations of IL-3, IL-6, stem cell factor (SCF), TPO, Flt3l, and VEGF. The total number and proliferation of MNCs increased significantly when co-cultured with MSCs or EPCs than when cultured alone, particularly when MNCs were co-cultured with EPCs. The differences in IL-3 and Flt3l concentrations between groups were not significant. However, IL-6 in the MSC group was significantly higher than that in the two other groups. The SCF and TPO concentrations were highly expressed in the EPC group. The VEGF concentrations in the MSC group and the EPC group were higher than those in the control group. These results indicated that MSCs and EPCs possibly favor the proliferation of MNCs and HSCs/HPCs. IL-6 and VEGF may be related to hematopoietic reconstitution and homing ability of HSCs/HPCs. TPO may have a specific relationship with the promotion of HSCs/HPCs differentiation.     

An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells

Magnesium (Mg²⁺) is a mineral with the ability to influence cell proliferation and to modulate inflammatory/immune responses, due to its anti-inflammatory properties. In addition, mesenchymal stem cells (MSCs) modulate the function of all major immune cell populations. Knowing that, the current work aimed to investigate the effects of Mg²⁺ enrichment, and its influence on the immunomodulatory capacity of MSCs. Murine C3H/10T1/2 MSCs were cultivated in media with different concentrations of Mg²⁺ (0, 1, 3 and 5 mM), in order to evaluate the effects of Mg²⁺ on MSC immunomodulatory properties, cell proliferation rates, expression of NFκB and STAT-3, production of IL-1β, IL-6, TGF-β, IL-10, PGE2 and NO, and TRPM7 expression. The results showed that TRPM7 is expressed in MSCs, but Mg²⁺, in the way that cells were cultivated, did not affect TRPM7 expression. Additionally, there was no difference in the intracellular concentration of Mg²⁺. Mg²⁺, especially at 5 mM, raised proliferation rates of MSCs, and modulated immune responses by decreasing levels of IL-1β and IL-6, and by increasing levels of IL-10 and PGE2 in cells stimulated with LPS or TNF-α. In addition, MSCs cultured in 5 mM Mg²⁺ expressed lower levels of pNFκB/NFκB and higher levels of pSTAT-3/STAT-3. Furthermore, conditioned media from MSCs reduced lymphocyte and macrophage proliferation, but Mg²⁺ did not affect this parameter. In addition, conditioned media from MSCs cultured at 5 mM of Mg²⁺ modulated the production profile of cytokines, especially of IL-1β and IL-6 in macrophages. In conclusion, Mg²⁺ is able to modulate some immunoregulatory properties of MSCs.     

Transplanted interleukin-4--secreting mesenchymal stromal cells show extended survival and increased bone mineral density in the murine femur

Background

Mesenchymal stromal cell (MSC)–based therapy has great potential to modulate chronic inflammation and enhance tissue regeneration. Crosstalk between MSC-lineage cells and polarized macrophages is critical for bone formation and remodeling in inflammatory bone diseases. However, the translational application of this interaction is limited by the short-term viability of MSCs after cell transplantation.

Genetic mismatch affects the immunosuppressive properties of mesenchymal stem cells in vitro and their ability to influence the course of collagen-induced arthritis

Introduction

The immunological and homing properties of mesenchymal stem cells (MSCs) provide a potentially attractive treatment for arthritis. The objective of this study was to determine effects of genetic disparity on the immunosuppressive potential of MSCs in vitro and in vivo within collagen induced arthritis (CIA).

Methods

The ability of DBA/1, FVB and BALB/c MSC preparations to impact the cytokine release profile of CD3/CD28 stimulated DBA/1 T cells was assessed in vitro. The effect of systemically delivered MSCs on the progression of CIA and cytokine production was assessed in vivo.

Results

All MSC preparations suppressed the release of TNFα and augmented the secretion of IL-4 and IL-10 by stimulated DBA/1 T-cells. However, assessment of the ratio of IFNγ to IL-4 production indicated that the more genetically distant BALB/c MSCs had significantly less immunosuppressive capacity. Systemic delivery of BALB/c MSC resulted in an exacerbation of CIA disease score in vivo and a higher erosive disease burden. This was not seen after treatment with syngeneic or partially mismatched MSCs. An increase in serum levels of IL-1β was observed up to 20 days post treatment with allogeneic MSCs. An initial elevation of IL-17 in these treatment groups persisted in those treated with fully mismatched BALB/c MSCs. Over the course of the study, there was a significant suppression of serum IL-17 levels in groups treated with syngeneic MSCs.

Conclusions

These data demonstrate a significant difference in the immunosuppressive properties of syngeneic and allogeneic MSCs in vitro and in vivo, which needs to be appreciated when developing MSC based therapies for inflammatory arthritis.     

Lipopolysaccharides can protect mesenchymal stem cells (MSCs) from oxidative stress-induced apoptosis and enhance proliferation of MSCs via Toll-like receptor(TLR)-4 and PI3K/Akt

Apoptosis of implanted mesenchymal stem cells (MSCs) limits the efficiency of MSC therapy. Recent studies showed the ligands of Toll-like receptors (TLRs) could control the function of these cells. We have investigated the effect of lipopolysaccharides (LPS), a ligand of TLR4, on the survival of MSCs and explored the roles of TLR4 and PI3K/Akt. H₂O₂/serum deprivation(H₂O₂/SD) induced apoptosis of MSCs but LPS-preconditioning (1.0 μg/ml) protected MSCs from H₂O₂/SD-induced apoptosis and promoted their proliferation. Western blotting showed that 1.0 μg/ml LPS enhanced phosphorylation of both Akt at Ser 473 and nuclear factor-kappa B (NF-κB) p65 at Ser 536. However, the protective effects of LPS on survival were not observed in TLR4^lps-del MSCs. The results suggest appropriate treatments with LPS can protect MSCs from oxidative stress-induced apoptosis and improve the survival of MSCs via the TLR4 and PI3K/Akt pathway.     

Overexpression of phosphoinositide-3-kinase class II alpha enhances mesenchymal stem cell survival in infarcted myocardium

The efficacy of mesenchymal stem cell (MSC) therapy for myocardial regeneration is limited by the poor survival of stem cells after transplantation into the infarcted heart. To improve the cell survival of MSCs in the infarcted heart, MSCs were genetically engineered to overexpress phosphoinositide-3-kinase class II alpha (PI3K-C2α). PI3K-C2α overexpression increased PI3K expression and the cell viability of MSCs. Furthermore, levels of survival-related phosphorylation were elevated in PI3K-C2α-MSCs. But, the level of apoptotic proteins downregulated and the number of PI-positive cells decreased in PI3K-C2α-MSCs compared to hypoxic MSCs. Nine rats per group had 1 × 10⁶ cells (20 μl PBS) transplanted after myocardial infarction. One week after transplantation, infarct size and area of fibrosis were reduced in the PI3K-C2α-MSC-transplanted group. The number of TUNEL positive cells declined, while the mean microvessel count per field was higher in the PI3K-C2α-MSC group than the MSC-injected group. Heart function was improved in the PI3K-C2α-MSCs group as assessed using a Millar catheter at 3 weeks after transplantation. These findings suggest that overexpression of PI3K-C2α in MSCs can assist cell survival and enhance myocardial regeneration.     

Differential effects of Akt pathway inhibitors on IL-1β-induced protein phosphorylation in human fibroblast-like synoviocytes

Context: Interleukin (IL)-1β activates various signal transduction pathways including p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt in human fibroblast-like synoviocytes (HFLS).

Objective: We investigated the effects of an Akt inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and Akt RNAi knockdown on IL-1β-induced protein phosphorylation in HFLS to clarify the role of the PI3K/Akt signaling pathway in the phosphorylation of the inhibitor of κB (IκB)α and heat shock protein 27 (HSP27).

Materials and methods: A multiplex suspension array system was used for the detection of phosphorylated proteins.

Results: IL-1β induced biphasic phosphorylation of IκBα, with the first phase occurring 10?min after IL-1β stimulation, and this was augmented by treatment with Akt inhibitor IV. However, this phenomenon was not observed after treatment with LY-294002, a PI3K inhibitor. Furthermore, Akt inhibitor IV suppressed ERK2 phosphorylation, whereas LY-294002 and Akt RNAi had no effect. In contrast, Akt inhibitor IV, LY-294002, and Akt RNAi augmented HSP27 phosphorylation.

Discussion and conclusions: Modulation of different stages of the PI3K/Akt pathway may differentially affect the phosphorylation of IκBα and HSP27 in HFLS.     

Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 × 10³, 1 × 10⁴ or 1 × 10⁵ cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-γ and TNF-α were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 × 10⁴ cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 × 10⁴ cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-γ-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-α induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes.     

Protein profiling of human lung telocytes and microvascular endothelial cells using iTRAQ quantitative proteomics

Telocytes (TCs) are described as a particular type of cells of the interstitial space ( www.telocytes.com ). Their main characteristics are the very long telopodes with alternating podoms and podomers. Recently, we performed a comparative proteomic analysis of human lung TCs with fibroblasts, demonstrating that TCs are clearly a distinct cell type. Therefore, the present study aims to reinforce this idea by comparing lung TCs with endothelial cells (ECs), since TCs and ECs share immunopositivity for CD34. We applied isobaric tag for relative and absolute quantification (iTRAQ) combined with automated 2‐D nano‐ESI LC‐MS/MS to analyse proteins extracted from TCs and ECs in primary cell cultures. In total, 1609 proteins were identified in cell cultures. 98 proteins (the 5th day), and 82 proteins (10th day) were confidently quantified (screened by two‐sample t‐test, P < 0.05) as up‐ or down‐regulated (fold change >2). We found that in TCs there are 38 up‐regulated proteins at the 5th day and 26 up‐regulated proteins at the 10th day. Bioinformatics analysis using Panther revealed that the 38 proteins associated with TCs represented cellular functions such as intercellular communication (via vesicle mediated transport) and structure morphogenesis, being mainly cytoskeletal proteins and oxidoreductases. In addition, we found 60 up‐regulated proteins in ECs e.g.: cell surface glycoprotein MUC18 (15.54‐fold) and von Willebrand factor (5.74‐fold). The 26 up‐regulated proteins in TCs at 10th day, were also analysed and confirmed the same major cellular functions, while the 56 down‐regulated proteins confirmed again their specificity for ECs. In conclusion, we report here the first extensive comparison of proteins from TCs and ECs using a quantitative proteomics approach. Our data show that TCs are completely different from ECs. Protein expression profile showed that TCs play specific roles in intercellular communication and intercellular signalling. Moreover, they might inhibit the oxidative stress and cellular ageing and may have pro‐proliferative effects through the inhibition of apoptosis. The group of proteins identified in this study needs to be explored further for the role in pathogenesis of lung disease.     

Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire

Background aimsMesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions.MethodsHuman, bone marrow derived MSCs were exposed to human plasma +/– heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry.ResultsPlasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14⁺ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells.ConclusionsThis study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.     

Effect and mechanisms of sacral nerve stimulation on visceral hypersensitivity mediated by nerve growth factor

To investigate the efficacy of sacral nerve stimulation (SNS) on nerve growth factor (NGF) mediated visceral sensitivity in normal rat and visceral hypersensitivity model rats. 120 male newborn rats were randomly divided into 6 groups: group A was normal model group; group B ~ F were all sensitized with acetic acid enema and grouped again. Group c2 was given NGF antagonist, d2 group was given NGF agonist, e2 group was given PI3K inhibitor, and f2 group was given PLC‐γ inhibitor. After treatment, the expression of NGF, TrKA, PI3K, AKT, PLC‐γ, NF‐κB, TRPV1, pTRPV1 and intracellular Ca²⁺ content were detected. The expression of protein TRPV1 and pTRPV1 was increased, and Ca²⁺ was increased in the visceral hypersensitive group. NGF, TrKA in NGF antagonist group, PI3K, AKT, NF‐κB in PI3K inhibitor group, PLC‐γ in PLC‐γ inhibitor group were all almost not expressed. The relative expression of NGF, TrKA, PI3K, AKT, PLC‐γ and NF‐κB in NGF antagonist group was lower than that in visceral hypersensitivity group and NGF activator group (P < .01). The relative expression of NGF, TrKA, PI3K and AKT mRNA in NGF antagonist group was lower than that in the normal model group (P < .01). There was no significant difference in the relative expression of PLC‐γ and NF‐κB mRNA (P > .05). The expression level of MAPK, ERK1 and ERK2 in visceral hypersensitivity group was higher than that in PI3K inhibitor group and PLC‐γ inhibitor group. The normal group Ca²⁺ curve was flat, and the NGF agonist group had the highest Ca²⁺ curve peak. Calcium concentration in visceral hypersensitivity group was higher than that in PI3K inhibitor group and that in PLC‐γ inhibitor group was higher than that in NGF antagonist group. The binding of TrkA receptor to NGF activates the MAPK/ERK pathway, the PI3K/Akt pathway and the PLC‐γ pathway, causing changes in the fluidity of intracellular and extracellular Ca²⁺, resulting in increased sensitivity of visceral tissues and organs.     

Sodium Butyrate Promotes the Differentiation of Rat Bone Marrow Mesenchymal Stem Cells to Smooth Muscle Cells through Histone Acetylation

Establishing an effective method to improve stem cell differentiation is crucial in stem cell transplantation. Here we aimed to explore whether and how sodium butyrate (NaB) induces rat bone marrow mesenchymal stem cells (MSCs) to differentiate into bladder smooth muscle cells (SMCs). We found that NaB significantly suppressed MSC proliferation and promoted MSCs differentiation into SMCs, as evidenced by the enhanced expression of SMC specific genes in the MSCs. Co-culturing the MSCs with SMCs in a transwell system promoted the differentiation of MSCs into SMCs. NaB again promoted MSC differentiation in this system. Furthermore, NaB enhanced the acetylation of SMC gene-associated H3K9 and H4, and decreased the expression of HDAC2 and down-regulated the recruitment of HDAC2 to the promoter regions of SMC specific genes. Finally, we found that NaB significantly promoted MSC depolarization and increased the intracellular calcium level of MSCs upon carbachol stimulation. These results demonstrated that NaB effectively promotes MSC differentiation into SMCs, possibly by the marked inhibition of HDAC2 expression and disassociation of HDAC2 recruitment to SMC specific genes in MSCs, which further induces high levels of H3K9^ace and H4^ace and the enhanced expression of target genes, and this strategy could potentially be applied in clinical tissue engineering and cell transplantation.     

Genetic comparison of mouse lung telocytes with mesenchymal stem cells and fibroblasts

Telocytes (TCs) are interstitial cells with telopodes – very long prolongations that establish intercellular contacts with various types of cells. Telocytes have been found in many organs and various species and have been characterized ultrastructurally, immunophenotypically and electrophysiologically ( www.telocytes.com ). Telocytes are distributed through organ stroma forming a three‐dimensional network in close contacts with blood vessels, nerve bundles and cells of the local immune system. Moreover, it has been shown that TCs express a broad range of microRNAs, such as pro‐angiogenic and stromal‐specific miRs. In this study, the gene expression profile of murine lung TCs is compared with other differentiated interstitial cells (fibroblasts) and with stromal stem/progenitor cells. More than 2000 and 4000 genes were found up‐ or down‐regulated, respectively, in TCs as compared with either MSCs or fibroblasts. Several components or regulators of the vascular basement membrane are highly expressed in TCs, such as Nidogen, Collagen type IV and Tissue Inhibitor of Metalloproteinase 3 (TIMP3). Given that TCs locate in close vicinity of small vessels and capillaries, the data suggest the implication of TCs in vascular branching. Telocytes express also matrix metalloproteases Mmp3 and Mmp10, and thus could regulate extracellular matrix during vascular branching and de novo vessel formation. In conclusion, our data show that TCs are not fibroblasts, as the ultrastructure, immunocytochemistry and microRNA assay previously indicated. Gene expression profile demonstrates that TCs are functionally distinct interstitial cells with specific roles in cell signalling, tissue remodelling and angiogenesis.     

Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells,fibroblasts, alveolar type II cells,airway epithelial cells and lymphocytes

Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC‐specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8⁺ T cells from bronchial lymph nodes (T‐BL) and CD8⁺ T cells from lungs (T‐LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up‐regulated and 70% down‐regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over‐expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types.