CDK5 Deficiency Does not Impair Neuronal Differentiation of Human Induced Pluripotent Stem Cells but Affects Neurite Outgrowth.

Cyclin-dependent kinase 5 (CDK5) is a protein kinase involved in neuronal homeostasis and development critical for neuronal survival. Besides, its deregulation is linked to neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases. For that reason, we aimed to generate a deficient CDK5 genetic model in neurons derived from human-induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 technology. We obtained a heterozygous CDK5+/- clone for the FN2.1 hiPSC line that retained hiPSC stemness and pluripotent potential. Then, neural stem cells (NSCs) and further neurons were derived from the CDK5+/- KO FN2.1 hiPSCs, and their phenotype was validated by immunofluorescence staining using antibodies that recognize lineage-specific markers (SOX-1, SOX-2, and NESTIN for NSCs and TUJ-1, MAP-5, and MAP-2 for neurons). We found that the proliferation rate increased in CDK5+/- KO hiPSC-derived neurons concomitantly with a reduction in NEUN and P35 expression levels. However, the morphometric analysis revealed that CDK5 deficiency caused an increase in the length of the main, primary, and secondary neurites and the neuronal soma area. As a whole, we found that a deficit in CDK5 does not impair hiPSC neuronal differentiation but deregulates proliferation and neurite outgrowth, favoring elongation. The misregulated activity of specific kinases leads to abnormalities such as impaired axonal connectivity in neurodegenerative diseases. Thus, therapeutic approaches aimed at normalizing the activity of kinases, such as CDK5, may help prevent the degeneration of vulnerable neurons.

Role of hydroxymethylglutharyl-coenzyme A reductase in the induction of stem-like states in breast cancer.

PURPOSE: De novo synthesis of cholesterol and its rate-limiting enzyme, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors and critical for tumor cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stem-like states in tumors remains unclear. METHODS: A compiled public database from breast cancer (BC) patients was analyzed with the web application SurvExpress. Cell Miner was used for the analysis of HMGCR expression and statin sensitivity of the NCI-60 cell lines panel. A CRISPRon system was used to induce HMGCR overexpression in the luminal BC cell line MCF-7 and a lentiviral pLM-OSKM system for the reprogramming of MCF-7 cells. Comparisons were performed by two-tailed unpaired t-test for two groups and one- or two-way ANOVA. RESULTS: Data from BC patients showed that high expression of several members of the cholesterol synthesis pathway were associated with lower recurrence-free survival, particularly in hormone-receptor-positive BC. In silico and in vitro analysis showed that HMGCR is expressed in several BC cancer cell lines, which exhibit a subtype-dependent response to statins in silico and in vitro. A stem-like phenotype was demonstrated upon HMGCR expression in MCF-7 cells, characterized by expression of the pluripotency markers NANOG, SOX2, increased CD44 +/CD24low/ -, CD133 + populations, and increased mammosphere formation ability. Pluripotent and cancer stem cell lines showed high expression of HMGCR, whereas cell reprogramming of MCF-7 cells did not increase HMGCR expression. CONCLUSION: HMGCR induces a stem-like phenotype in BC cells of epithelial nature, thus affecting tumor initiation, progression and statin sensitivity.

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.

Embryoid Bodies-Based Multilineage Differentiation of Human Embryonic Stem Cells Grown on Feeder-Free Conditions.

Human embryonic stem cells (hESCs) can differentiate into any cell lineage (pluripotency potential) derived from the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is usually demonstrated in vitro by spontaneous differentiation of hESCs grown on a monolayer of feeder-cells using an embryoid bodies (EBs)-based method. However, currently hESCs are grown mostly using fully defined media in the absence of a feeder layer. Here we describe a EBs-based protocol that allows multilineage differentiation of hESCs and human induced pluripotent stem cells (hiPSCs) grown on feeder-free conditions.

Protocol for morphometric analysis of neurons derived from human pluripotent stem cells.

The analysis of morphological features of neurons derived from human pluripotent stem cells (hPSCs) is important to describe neuronal phenotypes and changes observed throughout development. Using free and easily accessible tools, we describe a protocol for the morphometric quantification of hPSCs-derived neurons in two- and three-dimensions in vitro cultures. We detail the analysis of soma area and main and secondary dendrites lengths of GFP-transfected neurons and the measurement of area and perimeter of immunostained neurospheres.

Acute severe hypoxia induces apoptosis of human pluripotent stem cells by a HIF-1α and P53 independent mechanism.

Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (hPSCs) that can differentiate into a wide range of specialized cells. Although moderate hypoxia (5% O2) improves hPSC self-renewal, pluripotency, and cell survival, the effect of acute severe hypoxia (1% O2) on hPSC viability is still not fully elucidated. In this sense, we explore the consequences of acute hypoxia on hPSC survival by culturing them under acute (maximum of 24 h) physical severe hypoxia (1% O2). After 24 h of hypoxia, we observed HIF-1α stabilization concomitant with a decrease in cell viability. We also observed an increase in the apoptotic rate (western blot analysis revealed activation of CASPASE-9, CASPASE-3, and PARP cleavage after hypoxia induction). Besides, siRNA-mediated downregulation of HIF-1α and P53 did not significantly alter hPSC apoptosis induced by hypoxia. Finally, the analysis of BCL-2 family protein expression levels disclosed a shift in the balance between pro- and anti-apoptotic proteins (evidenced by an increase in BAX/MCL-1 ratio) caused by hypoxia. We demonstrated that acute physical hypoxia reduced hPSC survival and triggered apoptosis by a HIF-1α and P53 independent mechanism.

miR-302 family, miR-145 and miR-296 temporal expression profile along the cell cycle of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs), like embryonic (hESCs) and induced pluripotent stem cells (hiPSCs), exhibit an unusual cell cycle structure characterized by a short G1 phase and cells being most of time in S phase. hPSCs are receptive to differentiation cues during their transition through G1 phase when lineage determination is decided. Although several MicroRNAs (miRNAs) have been shown to target transcripts that directly or indirectly coordinate the cell cycle of pluripotent cells, its temporal expression profile along hPSCs cell cycle remains poorly characterized. miR-145 and miR-296 are induced during differentiation and silence the self-renewal and pluripotency program. miR-302 family is essential for hPSCs stemness and its expression decreases during differentiation. We aimed to study how the aforementioned miRNAs are regulated along the cell cycle of hPSCs. We demonstrated by pharmacological synchronization and block and release experiments that miR-145, miR-296 and miR-302 family are periodically expressed in hPSCs. Importantly, miR-302 family expression is induced at G1/S boundary and remained high at S phase, presumably to impede differentiation onset. Besides, we confirmed by a gene ontology analysis that many validated miR-302 family target genes are involved in cell cycle regulation.

Generation of a human induced pluripotent stem cell line from a familial Alzheimer's disease PSEN1 T119I patient.

Human induced pluripotent stem cells (hiPSC) line FLENIi001-A was reprogrammed from dermal fibroblasts using the lentiviral-hSTEMCCA-loxP vector. Fibroblasts were obtained from a skin biopsy of a 72-year-old Caucasian male familial Alzheimer's disease patient carrying the T119I mutation in the PSEN1 gene. PSEN1 genotype was maintained and stemness and pluripotency confirmed in the FLENIi001-A hiPSC line.

Chemical hypoxia induces apoptosis of human pluripotent stem cells by a NOXA-mediated HIF-1α and HIF-2α independent mechanism.

Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are self-renewing human pluripotent stem cells (hPSCs) that can differentiate to a wide range of specialized cells. Notably, hPSCs enhance their undifferentiated state and self-renewal properties in hypoxia (5% O2). Although thoroughly analyzed, hypoxia implication in hPSCs death is not fully determined. In order to evaluate the effect of chemically mimicked hypoxia on hPSCs cell survival, we analyzed changes in cell viability and several aspects of apoptosis triggered by CoCl2 and dimethyloxalylglycine (DMOG). Mitochondrial function assays revealed a decrease in cell viability at 24 h post-treatments. Moreover, we detected chromatin condensation, DNA fragmentation and CASPASE-9 and 3 cleavages. In this context, we observed that P53, BNIP-3, and NOXA protein expression levels were significantly up-regulated at different time points upon chemical hypoxia induction. However, only siRNA-mediated downregulation of NOXA but not HIF-1α, HIF-2α, BNIP-3, and P53 did significantly affect the extent of cell death triggered by CoCl2 and DMOG in hPSCs. In conclusion, chemically mimicked hypoxia induces hPSCs cell death by a NOXA-mediated HIF-1α and HIF-2α independent mechanism.

Palbociclib Effectively Halts Proliferation but Fails to Induce Senescence in Patient-Derived Glioma Stem Cells.

Glioblastoma multiforme is the most aggressive primary brain tumor. Current knowledge suggests that the growth and recurrence of these tumors are due in part to the therapy-resistant glioma stem cell subpopulation, which possesses the ability for self-renewal and proliferation, driving tumor progression. In many cancers, the p16INK4a-CDK4/6-pRb pathway is disrupted in favor of cell cycle progression. In particular, the frequent deregulation of CDK4/6 in cancer positions these kinases as promising targets. Palbociclib, a potent and selective CDK4/6 inhibitor, has been approved by the FDA as a first-line treatment of advanced breast cancer and there is currently interest in evaluating its effect on other cancer types. Palbociclib has been reported to be efficient, not only at halting proliferation, but also at inducing senescence in different tumor types. In this study, we evaluated the effect of this inhibitor on four patient-derived glioma stem cell-enriched cell lines. We found that Palbociclib rapidly and effectively inhibits proliferation without affecting cell viability. We also established that in these cell lines CDK6 is the key interphase CDK for controlling cell cycle progression. Prolonged exposure to Palbociclib induced a senescent-like phenotype characterized by flattened morphology, cell cycle arrest, increased ß-galactosidase activity and induction of other senescent-associated markers. However, we found that after Palbociclib removal cell lines resumed normal proliferation, which implies they conserved their replicative potential. As a whole, our results indicate that in patient-derived glioma stem cell-enriched cell lines, Palbociclib induces a senescent-like quiescence rather than true senescence.

The Kinase Fyn As a Novel Intermediate in L-DOPA-Induced Dyskinesia in Parkinson's Disease.

Dopamine replacement therapy with L-DOPA is the treatment of choice for Parkinson's disease; however, its long-term use is frequently associated with L-DOPA-induced dyskinesia (LID). Many molecules have been implicated in the development of LID, and several of these have been proposed as potential therapeutic targets. However, to date, none of these molecules have demonstrated full clinical efficacy, either because they lie downstream of dopaminergic signaling, or due to adverse side effects. Therefore, discovering new strategies to reduce LID in Parkinson's disease remains a major challenge. Here, we have explored the tyrosine kinase Fyn, as a novel intermediate molecule in the development of LID. Fyn, a member of the Src kinase family, is located in the postsynaptic density, where it regulates phosphorylation of the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in response to dopamine D1 receptor stimulation. We have used Fyn knockout and wild-type mice, lesioned with 6-hydroxydopamine and chronically treated with L-DOPA, to investigate the role of Fyn in the induction of LID. We found that mice lacking Fyn displayed reduced LID, ΔFosB accumulation and NR2B phosphorylation compared to wild-type control mice. Pre-administration of saracatinib (AZD0530), an inhibitor of Fyn activity, also significantly reduced LID in dyskinetic wild-type mice. These results support that Fyn has a critical role in the molecular pathways affected during the development of LID and identify Fyn as a novel potential therapeutic target for the management of dyskinesia in Parkinson's disease.