Generation of a human induced pluripotent stem cell line (INEUi001-A) from an amyotrophic lateral sclerosis/frontotemporal dementia patient with a C9ORF72 G4C2 genotype of <2 (GGGGCCG) and 10 repeats.

Human induced pluripotent stem cell (hiPSC) line INEUi001-A was reprogrammed from peripheral blood mononuclear cells (PBMC) using the lentiviral-hSTEMCCA-loxP vector. PBMCs were obtained from a 75- year-old female ALS/FTD disease patient carrying a heterozygous deletion within the C9ORF72 hexanucleotide repeat region resulting in a GGGGCCG sequence (∼1.16 repeats). C9ORF72 genotype was maintained and stemness and pluripotency confirmed in INEUi001-A hiPSC line.

Galectins as Emerging Glyco-Checkpoints and Therapeutic Targets in Glioblastoma.

Despite recent advances in diagnosis and treatment, glioblastoma (GBM) represents the most common and aggressive brain tumor in the adult population, urging identification of new rational therapeutic targets. Galectins, a family of glycan-binding proteins, are highly expressed in the tumor microenvironment (TME) and delineate prognosis and clinical outcome in patients with GBM. These endogenous lectins play key roles in different hallmarks of cancer by modulating tumor cell proliferation, oncogenic signaling, migration, vascularization and immunity. Additionally, they have emerged as mediators of resistance to different anticancer treatments, including chemotherapy, radiotherapy, immunotherapy, and antiangiogenic therapy. Particularly in GBM, galectins control tumor cell transformation and proliferation, reprogram tumor cell migration and invasion, promote vascularization, modulate cell death pathways, and shape the tumor-immune landscape by targeting myeloid, natural killer (NK), and CD8+ T cell compartments. Here, we discuss the role of galectins, particularly galectin-1, -3, -8, and -9, as emerging glyco-checkpoints that control different mechanisms associated with GBM progression, and discuss possible therapeutic opportunities based on inhibition of galectin-driven circuits, either alone or in combination with other treatment modalities.

White matter fiber density abnormalities in cognitively normal adults at risk for late-onset Alzheimer's disease.

Tau accumulation affecting white matter tracts is an early neuropathological feature of late-onset Alzheimer's disease (LOAD). There is a need to ascertain methods for the detection of early LOAD features to help with disease prevention efforts. The microstructure of these tracts and anatomical brain connectivity can be assessed by analyzing diffusion MRI (dMRI) data. Considering that family history increases the risk of developing LOAD, we explored the microstructure of white matter through dMRI in 23 cognitively normal adults who are offspring of patients with Late-Onset Alzheimer's Disease (O-LOAD) and 22 control subjects (CS) without family history of AD. We also evaluated the relation of white matter microstructure metrics with cortical thickness, volumetry, in vivo amyloid deposition (with the help of PiB positron emission tomography -PiB-PET) and regional brain metabolism (as FDG-PET) measures. Finally we studied the association between cognitive performance and white matter microstructure metrics. O-LOAD exhibited lower fiber density and fractional anisotropy in the posterior portion of the corpus callosum and right fornix when compared to CS. Among O-LOAD, reduced fiber density was associated with lower amyloid deposition in the right hippocampus, and greater cortical thickness in the left precuneus, while higher mean diffusivity was related with greater cortical thickness of the right superior temporal gyrus. Additionally, compromised white matter microstructure was associated with poorer semantic fluency. In conclusion, white matter microstructure metrics may reveal early differences in O-LOAD by virtue of parental history of the disorder, when compared to CS without a family history of LOAD. We demonstrate that these differences are associated with lower fiber density in the posterior portion of the corpus callosum and the right fornix.

Integrin alpha-5 subunit is critical for the early stages of human pluripotent stem cell cardiac differentiation.

The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.

Human embryonic stem cells display a pronounced sensitivity to the cyclin dependent kinase inhibitor Roscovitine.

BACKGROUND: The essentially unlimited expansion potential and the pluripotency of human embryonic stem cells (hESCs) make them attractive for cell-based therapeutic purposes. Although hESCs can indefinitely proliferate in culture, unlike transformed cancer cells, they are endowed with a cell-intrinsic property termed mitochondrial priming that renders them highly sensitive to apoptotic stimuli. Thus, all attempts to broaden the insights into hESCs apoptosis may be helpful for establishing pro-survival strategies valuable for its in vitro culture and further use in clinical applications. Cyclin-dependent kinases (CDKs), a family of serine/threonine protein kinases originally identified as regulators of the eukaryotic cell cycle, can also regulate transcription and differentiation. Moreover, there are compelling data suggesting that its activities are involved in certain apoptotic programs in different cell types. Currently, it is not completely determined whether CDKs regulate apoptotic processes in rapidly proliferating and apoptosis-prone hESCs. In this study, to elucidate the effect of CDKs inhibition in hESCs we used Roscovitine (ROSC), a purine analogue that selectively inhibits the activities of these kinases. RESULTS: Inhibition of CDKs by ROSC triggers programmed cell death in hESCs but not in proliferating somatic cells (human fibroblasts). The apoptotic process encompasses caspase-9 and -3 activation followed by PARP cleavage. ROSC treatment also leads to p53 stabilization, which coincides with site-specific phosphorylation at serine 46 and decreased levels of Mdm2. Additionally, we observed a transcriptional induction of p53AIP1, a repression of pro-survival factor Mcl-1 and an up-regulation of pro-apoptotic BH3-only proteins NOXA and PUMA. Importantly, we found that the role of CDK2 inhibition appears to be at best accessory as an active CDK2 is not required to ensure hESCs survival. CONCLUSION: Our experimental data reveal that hESCs, contrary to fibroblasts, exhibit a pronounced sensitivity to ROSC.

Deep Learning Neural Networks Highly Predict Very Early Onset of Pluripotent Stem Cell Differentiation.

Deep learning is a significant step forward for developing autonomous tasks. One of its branches, computer vision, allows image recognition with high accuracy thanks to the use of convolutional neural networks (CNNs). Our goal was to train a CNN with transmitted light microscopy images to distinguish pluripotent stem cells from early differentiating cells. We induced differentiation of mouse embryonic stem cells to epiblast-like cells and took images at several time points from the initial stimulus. We found that the networks can be trained to recognize undifferentiated cells from differentiating cells with an accuracy higher than 99%. Successful prediction started just 20 min after the onset of differentiation. Furthermore, CNNs displayed great performance in several similar pluripotent stem cell (PSC) settings, including mesoderm differentiation in human induced PSCs. Accurate cellular morphology recognition in a simple microscopic set up may have a significant impact on how cell assays are performed in the near future.

MicroRNA characterization in equine induced pluripotent stem cells.

Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. We aim to generate horse iPSCs, characterize them and evaluate the expression of different microRNAs (miR-302a,b,c,d, miR-205, miR-145, miR-9, miR-96, miR-125b and miR-296). Two equine iPSC lines (L2 and L3) were characterized after the reprogramming of equine fibroblasts with the four human Yamanaka's factors (OCT-4/SOX-2/c-MYC/KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2 and c-MYC. In vitro differentiation to embryo bodies (EBs) showed the capacity of the iPSCs to differentiate into ectodermal, endodermal and mesodermal phenotypes. MicroRNA analyses resulted in higher expression of the miR-302 family, miR-9 and miR-96 in L2 and L3 vs. fibroblasts (p<0.05), as previously shown in human pluripotent cells. Moreover, downregulation of miR-145 and miR-205 was observed. After differentiation to EBs, higher expression of miR-96 was observed in the EBs respect to the iPSCs, and also the expression of miR-205 was induced but only in the EB-L2. In addition, in silico alignments of the equine microRNAs with mRNA targets suggested the ability of miR-302 family to regulate cell cycle and epithelial mesenchymal transition genes, miR-9 and miR-96 to regulate neural determinant genes and miR-145 to regulate pluripotent genes, similarly as in humans. In conclusion, we could obtain equine iPSCs, characterize them and determine for the first time the expression level of microRNAs in equine pluripotent cells.

Activation of apoptotic signalling events in human embryonic stem cells upon Coxsackievirus B3 infection.

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate to a wide range of specialized cells and hold great promise as models for human development and disease, as well as for drug discovery and cell-replacement therapies. Group B Coxsackie viruses (CVBs) produce acute myocarditis, pancreatitis, non-septic meningitis and encephalitis in neonates, children and young adults. Moreover, CVBs can produce spontaneous miscarriage after early embryo infection. It was reported that hESCs express CVBs receptors and are susceptible to CVB3 infection. Apoptosis is one of the hallmarks of CVBs infection although details regarding CVB3 involvement in the apoptotic processes remain elusive. In order to evaluate the mechanisms of cell death induced by CVB3 in these pluripotent cells, we infected HUES-5 (H5) and WA01 (H1) hESC lines with CVB3. After validating the maintenance of stemness in these hESC lines when grown as confluent monolayers in feeder-free conditions, we analysed several aspects of programmed cell death triggered by CVB3. In all cases, we detected chromatin condensation, DNA fragmentation and caspase-9 and 3 cleavages. Moreover, we observed the presence of cleaved PARP product which was preceded by the appearance of p17, the catalytically active fragment of caspase-3. Mitochondrial function assays revealed a MOI dependent decrease in cell viability at 24 h post-infection (pi). No appreciable modifications in Bcl-2, Bcl-X(L) and Bax protein levels were observed upon CVB3 infection during 5-24 h observation period. However, a marked decrease in pro-apoptotic Bad abundance was detected without changes in its mRNA levels. In this study we found that the hESCs are highly susceptible to CVB3 infection and display elevated apoptosis rates, thus emerging as suitable human non-transformed in vitro models to study CVB3-induced apoptosis and resulting relevant to understand CVBs pathogenesis.

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iß treatment.

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48 h post-infection ranged from 105-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iß significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.

Heme oxygenase-1 is expressed in carotid atherosclerotic plaques infected by Helicobacter pylori and is more prevalent in asymptomatic subjects.

BACKGROUND AND PURPOSE: It is not well established what are the features, if any, that distinguish symptomatic from asymptomatic carotid atherosclerotic plaques. Inducible heme oxygenase-1 (HO-1) is a component of cellular defense mechanisms against oxidative stress. We aimed to assess the presence of Helicobacter pylori (H pylori) and the expression of HO-1 in carotid atherosclerotic plaques of patients with and without prior neurologic symptoms attributable to the operated artery. METHODS: We examined 25 symptomatic and 23 asymptomatic carotid atherosclerotic plaques removed during endarterectomy and 7 normal carotid arteries obtained at autopsy. We investigated the presence of H pylori DNA in the vessel wall and performed immunohistochemical detection of HO-1. RESULTS: H pylori DNA was present in 28 plaques and HO-1 was expressed in 30 plaques. HO-1 was found in 27 H pylori-positive specimens but in only 3 H pylori-negative specimens (P<0.001). All 7 normal carotid arteries were negative for both H pylori and HO-1. Although 82% of asymptomatic specimens were positive for H pylori and 87% for HO-1, only 36% of symptomatic specimens were positive for both H pylori and HO-1 (P<0.01). CONCLUSIONS: This study suggests a strong association between H pylori infection and expression of HO-1 in carotid atherosclerotic plaques. There was a substantial prevalence of these features in specimens obtained from asymptomatic subjects.