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.

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.

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.

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.

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.

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.

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.

Regulation of cyclin E1 expression in human pluripotent stem cells and derived neural progeny.

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs) show unique cell cycle characteristics, such as a short doubling time due to an abbreviated G1 phase. Whether or not the core cell cycle machinery directly regulates the stemness and/or the differentiation potential of hPSCs remains to be determined. To date, several scenarios describing the atypical cell cycle of hPSCs have been suggested, and therefore there is still controversy over how cyclins, master regulators of the cell cycle, are expressed and regulated. Furthermore, the cell cycle profile and the expression pattern of major cyclins in hESCs-derived neuroprogenitors (NP) have not been studied yet. Therefore, herein we characterized the expression pattern of major cyclins in hPSCs and NP. We determined that all studied cyclins mRNA expression levels fluctuate along cell cycle. Particularly, after a thorough analysis of synchronized cell populations, we observed that cyclin E1 mRNA levels increased sharply in G1/S concomitantly with cyclin E1 protein accumulation in hPSCs and NP. Additionally, we demonstrated that cyclin E1 mRNA expression levels involves the activation of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a mechanism that requires a functional CDK2 but not GSK3ß activity. ABBREVIATIONS: hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III ß-tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium.

The Cell Cycle Inhibitors p21Cip1 and p27Kip1 Control Proliferation but Enhance DNA Damage Resistance of Glioma Stem Cells.

High-grade gliomas are the most prevalent and lethal primary brain tumors. They display a hierarchical arrangement with a population of self-renewing and highly tumorigenic cells called cancer stem cells. These cells are thought to be responsible for tumor recurrence, which make them main candidates for targeted therapies. Unbridled cell cycle progression may explain the selective sensitivity of some cancer cells to treatments. The members of the Cip/Kip family p21Cip1 and p27Kip1 were initially considered as tumor suppressors based on their ability to block proliferation. However, they are currently looked at as proteins with dual roles in cancer: one as tumor suppressor and the other as oncogene. Therefore, the aim of this study was to determine the functions of these cell cycle inhibitors in five patient-derived glioma stem cell-enriched cell lines. We found that these proteins are functional in glioma stem cells. They negatively regulate cell cycle progression both in unstressed conditions and in response to genotoxic stress. In addition, p27Kip1 is upregulated in nutrient-restricted and differentiating cells, suggesting that this Cip/Kip is a mediator of antimitogenic signals in glioma cells. Importantly, the lack of these proteins impairs cell cycle halt in response to genotoxic agents, rendering cells more vulnerable to DNA damage. For these reasons, these proteins may operate both as tumor suppressors, limiting cell proliferation, and as oncogenes, conferring cell resistance to DNA damage. Thus, deepening our knowledge on the biological functions of these Cip/Kips may shed light on how some cancer cells develop drug resistance.