Glial and neuronal dysfunction in streptozotocin-induced diabetic rats

Neuronal dysfunction has been noted very soon after the induction of diabetes by streptozotocin injection in rats. It is not clear from anatomical evidence whether glial cell dysfunction accompanies the well-documented neuronal deficit. Here, we isolate the Müller cell driven slow-P3 component of the full-field electroretinogram and show that it is attenuated at 4 weeks following the onset of streptozotocin-hyperglycaemia. We also found a concurrent reduction in the sensitivity of the phototransduction cascade, as well as in the components of the electroretinogram known to indicate retinal ganglion cell and amacrine cell integrity. Our data support the idea that neuronal and Müller cell dysfunction occurs at the same time in streptozotocin-induced hyperglycaemia.

The ventral habenulae of zebrafish develop in prosomere 2 dependent on Tcf7l2 function

The conserved habenular neural circuit relays cognitive information from the forebrain into the ventral mid- and hindbrain. In zebrafish, the bilaterally formed habenulae in the dorsal diencephalon are made up of the asymmetric dorsal and symmetric ventral habenular nuclei, which are homologous to the medial and lateral nuclei respectively, in mammals. These structures have been implicated in various behaviors related to the serotonergic/dopaminergic neurotransmitter system. The dorsal habenulae develop adjacent to the medially positioned pineal complex. Their precursors differentiate into two main neuronal subpopulations which differ in size across brain hemispheres as signals from left-sided parapineal cells influence their differentiation program. Unlike the dorsal habenulae and despite their importance, the ventral habenulae have been poorly studied. It is not known which genetic programs underlie their development and why they are formed symmetrically, unlike the dorsal habenulae. A main reason for this lack of knowledge is that the vHb origin has remained elusive to date.

Endothelial lineage differentiation from induced pluripotent stem cells is regulated by microRNA-21 and transforming growth factor beta2 (TGF-β2) pathways

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In the paper we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in pre-differentiated iPSCs induced EC marker upregulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-β2 mRNA and secreted protein level, consistent with the strong upregulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21 and we showed that PTEN knockdown is required for miR-21 mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.

Regulation of TGF-β1-Driven Differentiation of Human Lung Fibroblasts: Emerging Roles of Cathepsin B and Cystatin C

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-β1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF) and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation and led to an accumulation of intracellular 50-kDa TGF-β1. Moreover addition of Cat B generated 25-kDa mature form of TGF-β1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation, but had no effect on p38 MAPK and JNK phosphorylation indicating that Cat B mostly disturbs TGF-β1-driven canonical Smad signaling pathway. While mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-β1-induced differentiation of IPF and CCD-19Lu fibroblasts. In addition cystatin C participated in the control of extracellular Cats, since its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-β1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.

Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony–forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel–forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >10⁸ ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF₁₆₅ as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.

MicroRNA-199b modulates vascular cell fate during iPS cell differentiation by targeting the notch ligand jagged1 and enhancing VEGF signaling

Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalised medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation towards ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration.

Methods and Results: In this study we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in VEGF transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation towards SMCs. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis.

Conclusions: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses.

The anti-atherogenic effects of eicosapentaenoic and docosahexaenoic acid are dependent on the stage of THP-1 macrophage differentiation.

In this study LC n-3 PUFA-specific effects on the degree of monocyte differentiation and macrophage foam cell formation were investigated by treating PMA-induced immature and mature macrophage models with LC n-3/n-6 PUFA during and post-differentiation. During immature macrophage differentiation LC n-3 PUFA alone decreased TNFα mRNA levels. EPA, and the n-6 PUFAs, linoleic acid and arachidonic acid, decreased CD36 mRNA levels, and EPA also downregulated CD49d cell-surface expression. Both LC n-3 PUFA reduced LDLr mRNA levels in immature macrophages, while DHA alone reduced levels in mature macrophages. Post-differentiation, n-3 and -6 PUFA reduced basal, but not oxidised LDL dependent cholesterol levels in immature macrophages. LC n-3 PUFA-specific reductions in LDLr and LOX-1 mRNA expression were also observed.

This study found LC n-3 PUFA specific, anti-atherogenic effects were more significant in immature macrophages. LC n-3 PUFA effects may be modulated by the extent of monocyte to macrophage differentiation.

Comparison Method for Differentiation of IEC 61850 based Substation Configuration Files

The increased construction and reconstruction of smart substations has exposed a problem with version management of substation configuration description language (SCL) files due to frequent changes. This paper proposes a comparative approach for differentiation of smart substation SCL configuration files. A comparison model for SCL configuration files is built in this method, which is based on the SCL structure and abstract model defined by IEC 61850. The proposed approach adopts the algorithms of depth-first traversal, sorting, and cross comparison in order to rapidly identify differences of changed SCL configuration files. This approach can also be utilized to detect malicious tampering or illegal manipulation tailoring for SCL files. SCL comparison software is developed using the Qt platform to validate the feasibility and effectiveness of the proposed approach.

Phosphorylated DegU Manipulates Cell Fate Differentiation in the Bacillus subtilis Biofilm

Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation.

Spinal cord injury reveals multilineage differentiation of ependymal cells

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.