Differentiation of Human T Cells Alters Their Repertoire of G Protein α-Subunits

Because T cell differentiation leads to an expanded repertoire of chemokine receptors, a subgroup of G protein-coupled receptors, we hypothesized that the repertoire of G proteins might be altered in parallel. We analyzed the abundance of mRNA and/or protein of six G protein α-subunits in human CD4⁺ and CD8⁺ T cell subsets from blood. Although most G protein α-subunits were similarly expressed in all subsets, the abundance of Gα_o, a protein not previously described in hematopoietic cells, was much higher in memory versus naive cells. Consistent with these data, activation of naive CD4⁺ T cells in vitro significantly increased the abundance of Gα_o in cells stimulated under nonpolarizing or T_H17 (but not T_H1 or T_H2)-polarizing conditions. In functional studies, the use of a chimeric G protein α-subunit, Gα_qo5, demonstrated that chemokine receptors could couple to Gα_o-containing G proteins. We also found that Gα_i1, another α-subunit not described previously in leukocytes, was expressed in naive T cells but virtually absent from memory subsets. Corresponding to their patterns of expression, siRNA-mediated knockdown of Gα_o in memory (but not naive) and Gα_i1 in naive (but not memory) CD4⁺ T cells inhibited chemokine-dependent migration. Moreover, although even in Gα_o- and Gα_i1-expressing cells mRNAs of these α-subunits were much less abundant than Gα_i2 or Gα_i3, knockdown of any of these subunits impaired chemokine receptor-mediated migration similarly. Together, our data reveal a change in the repertoire of Gα_i/o subunits during T cell differentiation and suggest functional equivalence among Gα_i/o subunits irrespective of their relative abundance.     

Tra2α Promotes RA Induced Neural Differentiation of P19 Cells

The present study demonstrates a high level of the nuclear Transformer 2 (Tra2) protein in adult mouse brain relative to other tissues, including muscle, heart, liver, lungs, kidney and small intestine, suggesting the potential importance of Tra2 in neural function. The level of Tra2 in mouse cerebrum is developmentally regulated, peaking at neonate stage. In P19 carcinoma cells, Tra2 is transiently up-regulated upon retinoic acid (RA) treatment. Although over-expression of Tra2 protein alone does not elicit P19 differentiation, under these conditions the response of P19 cells to RA is significantly increased. The results suggest that Tra2 proteins may act as a mediator in the signal pathway associated with RA-induced differentiation in P19 cells.     

Interferon-γ Promotes Differentiation of Neural Progenitor Cells via the JNK Pathway

It has been reported that interferon-γ (IFN-γ) facilitates differentiation of PC-12 cells and murine adult neural stem cells. Here we show that IFN-γ promotes the differentiation of C17.2 neural progenitor cells (NPC) into a neuronal phenotype characterized by neurite outgrowth and the expression of the neuronal marker protein β-III tubulin. IFN-γ induced an increase in the activity c-jun N-terminal kinase (JNK) without affecting activities of extracellular signal-regulated kinases (ERKs 1 and 2). An inhibitor of JNK blocked the ability of IFN-γ to promote differentiation of NPC into neurons, whereas an inhibitor of ERKs 1 and 2 did not. Our findings show that the pro-inflammatory cytokine, IFN-γ has the potential to stimulate neurogenesis, suggesting roles for this cytokine in development and repair of the nervous system.     

Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process.     

Calcineurin Inhibitors Suppress Cytokine Production from Memory T Cells and Differentiation of Na?ve T Cells into Cytokine-Producing Mature T Cells

T cells have been classified as belonging to the Th1 or Th2 subsets according to the production of defining cytokines such as IFN-γ and IL-4. The discovery of the Th17 lineage and regulatory T cells shifted the simple concept of the Th1/Th2 balance into a 4-way mechanistic pathway of local and systemic immunological activity. Clinically, the blockage of cytokine signals or non-specific suppression of cytokine predominance by immunosuppressants is the first-line treatment for inflammatory T cell-mediated disorders. Cyclosporine A (CsA) and Tacrolimus (Tac) are commonly used immunosuppressants for the treatment of autoimmune disease, psoriasis, and atopic disorders. Many studies have shown that these compounds suppress the activation of the calcium-dependent phosphatase calcineurin, thereby inhibiting T-cell activation. Although CsA and Tac are frequently utilized, their pharmacological mechanisms have not yet been fully elucidated.In the present study, we focused on the effects of CsA and Tac on cytokine secretion from purified human memory CD4⁺T cells and the differentiation of naïve T cells into cytokine-producing memory T cells. CsA or Tac significantly inhibited IFN-γ, IL-4, and IL-17 production from memory T cells. These compounds also inhibited T cell differentiation into the Th1, Th2, and Th17 subsets, even when used at a low concentration. This study provided critical information regarding the clinical efficacies of CsA and Tac as immunosuppressants.     

A Novel Function of dcf1 During the Differentiation of Neural Stem Cells In vitro

The study of neural dendrite formation is of great significance both in theory and applications. However, the molecular mechanisms of regulation remain unclear. We previously described a novel EST, which has high homology with dentritic cell factors (DCF1), expressed differentially between undifferentiated and differentiated neural stem cells (NSCs). In this study, we cloned, expressed, and silenced the dcf1 gene and offered insight into its function in regulating dendrite formation during the differentiation of NSCs. The results indicated that dcf1 encoded a 42 kD protein and could be successfully expressed both in Escherichia coli and NSCs. In order to silence dcf1 gene, three different kinds of siRNA vectors were constructed and transformed into the NSC line C17.2 and primary NSCs, resulting in down regulation of the dcf1 mRNA. Analysis of immunofluorescence or GFP illuminated that with overexpression of the dcf1 gene, the NSCs were maintained in undifferentiated status. After the dcf1 gene was silenced, cells tended to differentiate into neurons and astrocytes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Activation of Wnt/β-Catenin Signalling Affects Differentiation of Cells Arising from the Cerebellar Ventricular Zone

Development of the cerebellum proceeds under the precise spatio-temporal control of several key developmental signalling pathways, including the Wnt/β-catenin pathway. We recently reported the activity of Wnt/β-catenin signalling in the perinatal cerebellar ventricular zone (VZ), a germinal centre in the developing cerebellum that gives rise to GABAergic and glial cells. In order to investigate the normal function of Wnt/β-catenin signalling in the VZ and the cell lineages it gives rise to, we used a combination of ex vivo cerebellar slice culture and in vivo genetic manipulation to dysregulate its activity during late embryonic development. Activation of the pathway at the cerebellar ventricular zone led to a reduction in the number of cells expressing the glial lineage markers Sox9 and GFAP and the interneuron marker Pax2, but had no consistent effect on either proliferation or apoptosis. Our findings suggest that activation of the Wnt/β-catenin pathway in the cerebellar ventricular zone causes a shift in the cell types produced, most likely due to disruption of normal differentiation. Thus, we propose that regulation of Wnt/β-catenin signalling levels are required for normal development of cells arising from the cerebellar ventricular zone during late embryogenesis.     

Effects of Oxygen Concentration on the Proliferation and Differentiation of Mouse Neural Stem Cells In Vitro

Background and purpose Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro. Methods NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively. Results The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions. Conclusions These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation.     

Differentiation of Human Skeletal Muscle Stem Cells into Odontoblasts Is Dependent on Induction of α1 Integrin Expression

Skeletal muscle stem cells represent an abundant source of autologous cells with potential for regenerative medicine that can be directed to differentiate into multiple lineages including osteoblasts and adipocytes. In the current study, we found that α7 integrin-positive human skeletal muscle stem cells (α7⁺hSMSCs) could differentiate into the odontoblast lineage under specific inductive conditions in response to bone morphogenetic protein-4 (BMP-4). Cell aggregates of FACS-harvested α7⁺hSMSCs were treated in suspension with retinoic acid followed by culture on a gelatin scaffold in the presence of BMP-4. Following this protocol, α7⁺hSMSCs were induced to down-regulate myogenic genes (MYOD and α7 integrin) and up-regulate odontogenic markers including dentin sialophosphoprotein, matrix metalloproteinase-20 (enamelysin), dentin sialoprotein, and alkaline phosphatase but not osteoblastic genes (osteopontin and osteocalcin). Following retinoic acid and gelatin scaffold/BMP-4 treatment, there was a coordinated switch in the integrin expression profile that paralleled odontoblastic differentiation where α1β1 integrin was strongly up-regulated with the attenuation of muscle-specific α7β1 integrin expression. Interestingly, using siRNA knockdown strategies revealed that the differentiation-related expression of the α1 integrin receptor positively regulates the expression of the odontoblastic markers dentin sialophosphoprotein and matrix metalloproteinase-20. These results strongly suggest that the differentiation of α7⁺hSMSCs along the odontogenic lineage is dependent on the concurrent expression of α1 integrin.     

Direct Interaction of CD40 on Tumor Cells with CD40L on T Cells Increases the Proliferation of Tumor Cells by Enhancing TGF-β Production and Th17 Differentiation

It has recently been reported that the CD40-CD40 ligand (CD40L) interaction is important in Th17 development. In addition, transforming growth factor—beta (TGF-β) promotes tumorigenesis as an immunosuppressive cytokine and is crucial in the development of Th17 cells. This study investigated the role of CD40 in breast cancer cells and its role in immunosuppressive function and tumor progression. CD40 was highly expressed in the breast cancer cell line MDA-MB231, and its stimulation with CD40 antibodies caused the up-regulation of TGF-β. Direct CD40-CD40L interaction between MDA-MB231 cells and activated T cells also increased TGF-β production and induced the production of IL-17, which accelerated the proliferation of MDA-MB231 cells through the activation of STAT3. Taken together, the direct CD40-CD40L interaction of breast tumor cells and activated T cells increases TGF-β production and the differentiation of Th17 cells, which promotes the proliferation of breast cancer cells.     

Differentiation,Distribution and γδ T Cell-Driven Regulation of IL-22-Producing T Cells in Tuberculosis

Differentiation, distribution and immune regulation of human IL-22-producing T cells in infections remain unknown. Here, we demonstrated in a nonhuman primate model that M. tuberculosis infection resulted in apparent increases in numbers of T cells capable of producing IL-22 de novo without in vitro Ag stimulation, and drove distribution of these cells more dramatically in lungs than in blood and lymphoid tissues. Consistently, IL-22-producing T cells were visualized in situ in lung tuberculosis (TB) granulomas by confocal microscopy and immunohistochemistry, indicating that mature IL-22-producing T cells were present in TB granuloma. Surprisingly, phosphoantigen HMBPP activation of Vγ2Vδ2 T cells down-regulated the capability of T cells to produce IL-22 de novo in lymphocytes from blood, lung/BAL fluid, spleen and lymph node. Up-regulation of IFNγ-producing Vγ2Vδ2 T effector cells after HMBPP stimulation coincided with the down-regulated capacity of these T cells to produce IL-22 de novo. Importantly, anti-IFNγ neutralizing Ab treatment reversed the HMBPP-mediated down-regulation effect on IL-22-producing T cells, suggesting that Vγ2Vδ2 T-cell-driven IFNγ-networking function was the mechanism underlying the HMBPP-mediated down-regulation of the capability of T cells to produce IL-22. These novel findings raise the possibility to ultimately investigate the function of IL-22 producing T cells and to target Vγ2Vδ2 T cells for balancing potentially hyper-activating IL-22-producing T cells in severe TB.     

PPARα Is Essential for Microparticle-Induced Differentiation of Mouse Bone Marrow-Derived Endothelial Progenitor Cells and Angiogenesis

Background

Bone marrow-derived endothelial progenitor cells (EPCs) are critical for neovascularization. We hypothesized that microparticles (MPs), small fragments generated from the plasma membrane, can activate angiogenic programming of EPCs.

Methodology/Principal Findings

We studied the effects of MPs obtained from wild type (MPs^PPARα+/+) and knock-out (MPs^{PPARα−/−}) mice on EPC differentiation and angiogenesis. Bone marrow-derived cells were isolated from WT or KO mice and were cultured in the presence of MPs^PPARα+/+ or MPs^{PPARα−/−} obtained from blood of mice. Only MPs^PPARα+/+ harboring PPAR^α significantly increased EPC, but not monocytic, differentiation. Bone marrow-derived cells treated with MPs^PPARα+/+ displayed increased expression of pro-angiogenic genes and increased in vivo angiogenesis. MPs^PPARα+/+ increased capillary-like tube formation of endothelial cells that was associated with enhanced expressions of endothelial cell-specific markers. Finally, the effects of MPs^PPARα+/+ were mediated by NF-κB-dependent mechanisms.

Conclusions/Significance

Our results underscore the obligatory role of PPARα carried by MPs for EPC differentiation and angiogenesis. PPARα-NF-κB-Akt pathways may play a pivotal stimulatory role for neovascularization, which may, at least in part, be mediated by bone marrow-derived EPCs. Improvement of EPC differentiation may represent a useful strategy during reparative neovascularization.     

Neural Differentiation of Mesenchymal Stem Cells Influences Their Chemotactic Responses to Stromal Cell-Derived Factor-1α

Mesenchymal stem cells (MSCs) are proposed as a promising source for cell-based therapies in neural disease. Although increasing numbers of studies have been devoted to the delineation of factors involved in the migration of MSCs, the relationship between the chemotactic response and the differentiation status of these cells is still unclear. In the present study, we demonstrated that MSCs in varying neural differentiation states display various chemotactic responses to stromal cell-derived factor-1α (SDF-1α). The chemotactic responses of MSCs under different differentiation stages in response to SDF-1α were analyzed by Boyden chamber, and the results showed that cells of undifferentiation, 24-h preinduction, 5-h induction, and 18-h maintenance states displayed a stronger chemotactic response to SDF-1α, while 48-h maintenance did not. Further, we found that the phosphorylation levels of PI3K/Akt, ERK1/2, SAPK/JNK, and p38MAPK are closely related to the differentiation states of MSCs subjected to SDF-1α, and finally, inhibition of SAPK/JNK signaling significantly attenuates SDF-1α-stimulated transfilter migration of MSCs of undifferentiation, 24-h preinduction, 18-h maintenance, and 48-h maintenance, but not MSCs of 5-h induction. Meanwhile, interference with PI3K/Akt, p38MAPK, or ERK1/2 signaling prevents only cells at certain differentiation state from migrating in response to SDF-1α. Collectively, these results demonstrate that MSCs in varying neural differentiation states have different migratory capacities, thereby illuminating optimization of the therapeutic potential of MSCs to be used for neural regeneration after injury.     

Retinoic Acid Promotes the Generation of Pancreatic Endocrine Progenitor Cells and Their Further Differentiation into β-Cells

The identification of secreted factors that can selectively stimulate the generation of insulin producing β-cells from stem and/or progenitor cells represent a significant step in the development of stem cell-based β-cell replacement therapy. By elucidating the molecular mechanisms that regulate the generation of β-cells during normal pancreatic development such putative factors may be identified. In the mouse, β-cells increase markedly in numbers from embryonic day (e) 14.5 and onwards, but the extra-cellular signal(s) that promotes the selective generation of β-cells at these stages remains to be identified. Here we show that the retinoic acid (RA) synthesizing enzyme Raldh1 is expressed in developing mouse and human pancreas at stages when β-cells are generated. We also provide evidence that RA induces the generation of Ngn3⁺ endocrine progenitor cells and stimulates their further differentiation into β-cells by activating a program of cell differentiation that recapitulates the normal temporal program of β-cell differentiation.     

Bilobalide Induces Neuronal Differentiation of P19 Embryonic Carcinoma Cells via Activating Wnt/β-Catenin Pathway

Bilobalide, a natural product extracted from Ginkgo biloba leaf, is known to exhibit a number of pharmacological activities. So far, whether it could affect embryonic stem cell differentiation is still unknown. The main aim of this study was to investigate the effect of bilobalide on P19 embryonic carcinoma cells differentiation and the underlying mechanisms. Our results showed that bilobalide induced P19 cells differentiation into neurons in a concentration- and time-dependent manner. We also found that bilobalide promoted neuronal differentiation through activation of Wnt/β-catenin signaling pathway. Exposure to bilobalide increased inactive GSK-3β phosphorylation, further induced the nuclear accumulation of β-catenin, and also up-regulated the expression of Wnt ligands Wnt1 and Wnt7a. Neuronal differentiation induced by bilobalide was totally abolished by XAV939, an inhibitor of Wnt/β-catenin pathway. These results revealed a novel role of bilobalide in neuronal differentiation from P19 embryonic cells acting through Wnt/β-catenin signaling pathway, which would provide a better insight into the beneficial effects of bilobalide in brain diseases.