miR-7 Modulates hESC Differentiation into Insulin-Producing Beta-like Cells and Contributes to Cell Maturation.

Human pluripotent stem cells retain the extraordinary capacity to differentiate into pancreatic beta cells. For this particular lineage, more effort is still required to stress the importance of developing an efficient, reproducible, easy, and cost-effective differentiation protocol to obtain more mature, homogeneous, and functional insulin-secreting cells. In addition, microRNAs (miRNAs) have emerged as a class of small non-coding RNAs that regulate many cellular processes, including pancreatic differentiation. Some miRNAs are known to be preferentially expressed in islets. Of note, miR-375 and miR-7 are two of the most abundant pancreatic miRNAs, and they are necessary for proper pancreatic islet development. Here we provide new insight into specific miRNAs involved in pancreatic differentiation. We found that miR-7 is differentially expressed during the differentiation of human embryonic stem cells (hESCs) into a beta cell-like phenotype and that its modulation plays an important role in generating mature pancreatic beta cells. This strategy may be exploited to optimize the potential for in vitro differentiation of hESCs into insulin-producing beta-like cells for use in preclinical studies and future clinical applications as well as the prospective uses of miRNAs to improve this process.

Resveratrol ameliorates the maturation process of ß-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 ß-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 ß-cell-like cells and demonstrates that RSV improves the maturation process.

Mesothelial cells: a cellular surrogate for tissue engineering of corneal endothelium.

PURPOSE: To evaluate whether mouse adipose tissue mesothelial cells (ATMCs) share morphologic and biochemical characteristics with mouse corneal endothelial cells (CECs) and to evaluate their capacity to adhere to the decellularized basal membrane of human anterior lens capsules (HALCs) as a potential tissue-engineered surrogate for corneal endothelium replacement. METHODS: Adipose tissue mesothelial cells were isolated from the visceral adipose tissue of adult mice, and their expression of several corneal endothelium markers was determined with quantitative RT-PCR, immunofluorescence, and Western blotting. Adipose tissue mesothelial cells were cultured in a mesothelial retaining phenotype medium (MRPM) and further seeded and cultured on top of the decellularized basal membrane of HALCs. ATMC-HALC composites were evaluated by optical microscopy, immunofluorescence, and transmission electron microscopy. RESULTS: Mesothelial retaining phenotype medium-cultured ATMCs express the corneal endothelium markers COL4A2, COL8A2, SLC4A4, CAR2, sodium- and potassium-dependent adenosine triphosphatase (Na(+)/K(+)-ATPase), ß-catenin, zona occludens-1, and N-cadherin in a pattern similar to that in mouse CECs. Furthermore, ATMCs displayed strong adhesion capacity onto the basal membrane of HALCs and formed a confluent monolayer within 72 hours of culture in MRPM. Ultrastructural morphologic and marker characteristics displayed by ATMC monolayer on HALCs clearly indicated that ATMCs retained their original phenotype of squamous epithelial-like cells. CONCLUSIONS: Corneal endothelial cells and ATMCs share morphologic (structural) and marker (functional) similarities [corrected]. The ATMCs adhered and formed structures mimicking focal adhesion complexes with the HALC basal membrane. Monolayer structure and achieved density of ATMCs support the proposal to use adult human mesothelial cells (MCs) as a possible surrogate for damaged corneal endothelium.

Preconditioning of microglia by α-synuclein strongly affects the response induced by toll-like receptor (TLR) stimulation.

In recent years, it has become accepted that α-synuclein (αSyn) has a key role in the microglia-mediated neuroinflammation, which accompanies the development of Parkinson's disease and other related disorders, such as Dementia with Lewy Bodies and Alzheimer's disease. Nevertheless, the cellular and molecular mechanisms underlying its pathological actions, especially in the sporadic forms of the diseases, are not completely understood. Intriguingly, several epidemiological and animal model studies have revealed a link between certain microbial infections and the onset or progression of sporadic forms of these neurodegenerative disorders. In this work, we have characterized the effect of toll-like receptor (TLR) stimulation on primary murine microglial cultures and analysed the impact of priming cells with extracellular wild-type (Wt) αSyn on the subsequent TLR stimulation of cells with a set of TLR ligands. By assaying key interleukins and chemokines we report that specific stimuli, in particular Pam3Csk4 (Pam3) and single-stranded RNA40 (ssRNA), can differentially affect the TLR2/1- and TLR7-mediated responses of microglia when pre-conditioned with αSyn by augmenting IL-6, MCP-1/CCL2 or IP-10/CXCL10 secretion levels. Furthermore, we report a skewing of αSyn-primed microglia stimulated with ssRNA (TLR7) or Pam3 (TLR2/1) towards intermediate but at the same time differential, M1/M2 phenotypes. Finally, we show that the levels and intracellular location of activated caspase-3 protein change significantly in αSyn-primed microglia after stimulation with these particular TLR agonists. Overall, we report a remarkable impact of non-aggregated αSyn pre-sensitization of microglia on TLR-mediated immunity, a phenomenon that could contribute to triggering the onset of sporadic α-synuclein-related neuropathologies.

Glial innate immunity generated by non-aggregated alpha-synuclein in mouse: differences between wild-type and Parkinson's disease-linked mutants.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (α-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular α-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular α-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked α-Syn mutants on this stimulation, are still largely unknown. METHODS AND FINDINGS: In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant α-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with α-Syn, we measured the release of Th1- and Th2- type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1α/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated α-Syn, we found strong differences in the immune response generated by wild-type α-Syn and the familial PD mutants (A30P, E46K and A53T). CONCLUSIONS: These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD.