Generation and Neuronal Differentiation of Patient-Specific Induced Pluripotent Stem Cells Derived from Niemann-Pick Type C1 Fibroblasts.

Patient-specific induced pluripotent stem cells (iPSCs) are discussed to provide a powerful tool to investigate pathological mechanisms of diseases. Moreover, such cells might be a future platform for individualized personal treatment of diseases with a broad spectrum of mutations and thus resulting in phenotypical specificities.Here, we present a protocol for the induction of induced pluripotent stem cells from patient fibroblasts with Niemann-Pick type C1 disease (NPC1). The induction is based on a retroviral system, using the "classical" transcription factors, which were described by Takahashi and colleagues in 2007. To obtain a neuronal in vitro model system of NPC1, human iPSCs were differentiated to neural progenitor cells (NPCs) and subsequently to cells of the neural lineage, namely, neurons and glial cells. iPSCs, NPCs, and terminal neuronal differentiated cells (NDCs) were characterized by means of immunocytochemistry as well as patch clamp recordings and calcium imaging to prove the functional maturation.

HES5 is a key mediator of Wnt-3a-induced neuronal differentiation.

Human neural stem/progenitor cell (hNPC)-derived neuronal progeny has been suggested as a promising cell source in a variety of neurodegenerative diseases. Understanding the underlying mechanisms that regulate neuronal differentiation is essential for efficient cell-based therapies. Wnt and Notch signaling has been shown to be crucial in this process. However, their interactions in the process of neuronal differentiation remain elusive. By using human fetal (ReNcell VM) and iPS-derived hNPCs we demonstrate that Wnt-3a immediately induced a transient HES1 upregulation and a sustained HES5 repression that was accompanied by upregulation of the proneural gene MASH1. Conversely, overexpression of HES5 resulted in reduced MASH1 expression. Remarkably, HES5 overexpression efficiently blocked Wnt-3a as well as γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT)-induced neuronal differentiation that was accompanied by a strong MASH1 downregulation thus directly linking HES5 repression/MASH1 induction to the proneurogenic effect of Wnt-3a. Stabilized ß-catenin or treatment with the specific glycogen synthase kinase 3 beta (GSK3ß) inhibitor SB-216763 failed to or only partially mimicked these effects, suggesting a GSK3ß- and ß-catenin-independent mechanism. Further, inhibition of Wnt-3a-LDL-receptor-related protein 5/6 (LRP5/6) interactions using Dickkopf-1 (Dkk-1) failed to inhibit the modulatory effect of Wnt-3a on HES1/5 and neuronal differentiation. Taken together, these data identify HES5 as a key mediator of the Wnt-3a proneurogenic effect occurring independently of the classical Wnt/ß-catenin signaling cascade thus further deciphering crosstalk mechanisms of Wnt and Notch signaling pathways regulating cell fate of hNPCs.

Glucosylsphingosine is a highly sensitive and specific biomarker for primary diagnostic and follow-up monitoring in Gaucher disease in a non-Jewish, Caucasian cohort of Gaucher disease patients.

BACKGROUND: Gaucher disease (GD) is the most common lysosomal storage disorder (LSD). Based on a deficient ß-glucocerebrosidase it leads to an accumulation of glucosylceramide. Standard diagnostic procedures include measurement of enzyme activity, genetic testing as well as analysis of chitotriosidase and CCL18/PARC as biomarkers. Even though chitotriosidase is the most well-established biomarker in GD, it is not specific for GD. Furthermore, it may be false negative in a significant percentage of GD patients due to mutation. Additionally, chitotriosidase reflects the changes in the course of the disease belatedly. This further enhances the need for a reliable biomarker, especially for the monitoring of the disease and the impact of potential treatments. METHODOLOGY: Here, we evaluated the sensitivity and specificity of the previously reported biomarker Glucosylsphingosine with regard to different control groups (healthy control vs. GD carriers vs. other LSDs). FINDINGS: Only GD patients displayed elevated levels of Glucosylsphingosine higher than 12 ng/ml whereas the comparison controls groups revealed concentrations below the pathological cut-off, verifying the specificity of Glucosylsphingosine as a biomarker for GD. In addition, we evaluated the biomarker before and during enzyme replacement therapy (ERT) in 19 patients, demonstrating a decrease in Glucosylsphingosine over time with the most pronounced reduction within the first 6 months of ERT. Furthermore, our data reveals a correlation between the medical consequence of specific mutations and Glucosylsphingosine. INTERPRETATION: In summary, Glucosylsphingosine is a very promising, reliable and specific biomarker for GD.

Niemann-Pick type C1 patient-specific induced pluripotent stem cells display disease specific hallmarks.

BACKGROUND: Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired, resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However, the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore, a human model using patient-specific induced pluripotent stem cells provides a promising approach. METHODS: We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation. RESULTS: The hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81 and SSEA4. Using the embryoid body assay, the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice, demonstrating their pluripotency. In addition, neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels, spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient, derived hiPSCs, and neural progenitor cells, but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay, demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient. CONCLUSIONS: We generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts, providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease.

Spontaneous calcium transients in human neural progenitor cells mediated by transient receptor potential channels.

Calcium signals affect many developmental processes, including proliferation, migration, survival, and apoptosis, processes that are of particular importance in stem cells intended for cell replacement therapies. The mechanisms underlying Ca(2+) signals, therefore, have a role in determining how stem cells respond to their environment, and how these responses might be controlled in vitro. In this study, we examined the spontaneous Ca(2+) activity in human neural progenitor cells during proliferation and differentiation. Pharmacological characterization indicates that in proliferating cells, most activity is the result of transient receptor potential (TRP) channels that are sensitive to Gd(3+) and La(3+), with the more subtype selective antagonist Ruthenium red also reducing activity, suggesting the involvement of transient receptor potential vanilloid (TRPV) channels. In differentiating cells, Gd(3+) and La(3+)-sensitive TRP channels also appear to underlie the spontaneous activity; however, no sub-type-specific antagonists had any effect. Protein levels of TRPV2 and TRPV3 decreased in differentiated cells, which is demonstrated by western blot. Thus, it appears that TRP channels represent the main route of Ca(2+) entry in human neural progenitor cells (hNPCs), but the responsible channel types are subject to substitution under differentiating conditions. The level of spontaneous activity could be increased and decreased by lowering and raising the extracellular K(+) concentration. Proliferating cells in low K(+) slowed the cell cycle, with a disproportionate increased percentage of cells in G1 phase and a reduction in S phase. Taken together, these results suggest a link between external K(+) concentration, spontaneous Ca(2+) transients, and cell cycle distribution, which is able to influence the fate of stem and progenitor cells.

Interference of a novel indolylmaleimide with microtubules induces mitotic arrest and apoptosis in human progenitor and cancer cells.

Indolylmaleimides display a broad spectrum of biological activity and offer great opportunity to influence several aspects of cell fate, as proliferation and differentiation. In this study we describe the effect of PDA-66, a newly synthesised indolylmaleimide, showing a strong dose dependent anti-proliferative effect on immortalised human progenitor and cancer cells. We demonstrated a highly depolymerizing effect on in vitro tubulin assembly and conclude that PDA-66 acts as microtubule destabilising agent. In addition we found that PDA-66 induces mitotic arrest of cells in the G2/M phase of the cell cycle. Subsequently cells undergo apoptosis, indicating the major mechanism of the anti-proliferative effect. To prove a potential anti-cancer activity of PDA-66 we examined the effect of PDA-66 on human SH-SY5Y neuroblastoma and A-459 lung cancer cells, showing a significant reduction in cancer cell proliferation in a dose dependent manner. Thus PDA-66 is a new anti-mitotic compound with an indole-core with the potential to be used for cancer therapy.

Small molecules in stem cell research.

Stem cells possess great promise as therapeutic tools for neurological disorders such as neurodegenerative diseases (Parkinson's disease and Huntington's disease), cerebrovascular diseases (stroke), neurotraumata (spinal cord injury) and demyelinating diseases (multiple sclerosis). This aspiration is based on the cells` ability to maintain a status of self-renewal and to differentiate into the various cell types of an organism. The use of the cells ranges from in vitro to in vivo studies in animal models, ending with clinical applications in humans. The self-renewal and commitment of stem/progenitor cells to differentiate and mature involves complex events leading to the generation of different phenotypes via distinctive developmental programs. Small molecules provide a tool with which to influence these regulatory changes in a controlled manner and to help understand the underlying mechanisms. Furthermore, substantial progress in generating induced pluripotent stem cells has been made using small molecules to replace reprogramming factors and enhance the reprogramming efficiency and kinetics, thus generating cells more compatible with the requirements for cell replacement therapies. In this review we will present the recent progress on the use of small molecules in embryonic and induced pluripotent stem cell research. In the final section we will give a short summary of the clinical approaches using these cells.

Human neural progenitor cells show functional neuronal differentiation and regional preference after engraftment onto hippocampal slice cultures.

The transplantation of stem cells offers potential therapies for many neurodegenerative disorders that currently have limited or no treatment options. However, relatively little is known about how the host environment affects the development and integration of these cells. In this study we have engrafted immortalized human midbrain neural progenitor cells (NPCs) onto rat hippocampal brain slice cultures to examine the influence of a neural environment on differentiation. Patch clamp recordings revealed that the transplanted progenitor cells could express neuronal-type voltage-gated currents and rapidly receive synaptic input from the hippocampal brain slice. The distribution of progenitor cells across the hippocampal slices was strongly influenced by the neural architecture, with most cells located in the fissural regions and sending processes parallel to the laminar structure, while in contrast, cells located in the dentate gyrus showed no organized pattern. Almost no cells were found in the stratum radiatum or pyramidal cell layers. Together, these results demonstrate the potential for the architecture of the host environment to regulate the integration of transplanted cells, and highlight the utility of coculture systems for studying the mechanisms underlying the migration, integration, and differentiation of human NPCs in structured neural environments.

Decreased expression of myelin gene regulatory factor in Niemann-Pick type C 1 mouse.

Niemann-Pick type C 1 (NPC1) disease is an autosomal recessive cholesterol transport defect resulting in a neurodegenerative process in patients mainly at an early age, although some patients may start with manifestation in adult. Since loss of myelin is considered as a main pathogenetic factor, the precise mechanism inducing dysmylination in NPC1 disease is still unclear. In the present study, a quantitative evaluation on the myelin protein and its regulatory factors of oligodendrocytes, such as SRY-related HMG-box 10 (Sox10), Yin Yang 1 factor (YY1) and myelin gene regulatory factor (MRF), in different parts of the brain and spinal cord was performed in NPC1-mutant mice. The results showed that NPC1 protein was expressed in oligodendrocytes and the amount of myelin protein was generally decreased in all parts of the brain and spinal cord in NPC1-mutant mice. Compared to wild type, the amount of Sox10 and YY1 was not different in NPC1-mutant mice, but MRF was significantly decreased, suggesting a possible mechanism perturbing differentiation of oligodendrocytes and the myelination process in the NPC1-mutant mouse.

Quantitative and kinetic profile of Wnt/ß-catenin signaling components during human neural progenitor cell differentiation.

ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/ß-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/ß-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized ß-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and ß-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/ß-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/ß-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.

Small molecule GSK-3 inhibitors increase neurogenesis of human neural progenitor cells.

Human neural progenitor cells provide a source for cell replacement therapy to treat neurodegenerative diseases. Therefore, there is great interest in mechanisms and tools to direct the fate of multipotent progenitor cells during their differentiation to increase the yield of a desired cell type. We tested small molecule inhibitors of glycogen synthase kinase-3 (GSK-3) for their functionality and their influence on neurogenesis using the human neural progenitor cell line ReNcell VM. Here we report the enhancement of neurogenesis of human neural progenitor cells by treatment with GSK-3 inhibitors. We tested different small molecule inhibitors of GSK-3 i.e. LiCl, sodium-valproate, kenpaullone, indirubin-3-monoxime and SB-216763 for their ability to inhibit GSK-3 in human neural progenitor cells. The highest in situ GSK-3 inhibitory effect of the drugs was found for kenpaullone and SB-216763. Accordingly, kenpaullone and SB-216763 were the only drugs tested in this study to stimulate the Wnt/ß-catenin pathway that is antagonized by GSK-3. Analysis of human neural progenitor differentiation revealed an augmentation of neurogenesis by SB-216763 and kenpaullone, without changing cell cycle exit or cell survival. Small molecule inhibitors of GSK-3 enhance neurogenesis of human neural progenitor cells and may be used to direct the differentiation of neural stem and progenitor cells in therapeutic applications.

Erythropoietin and the effect of oxygen during proliferation and differentiation of human neural progenitor cells.

BACKGROUND: Hypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process. RESULTS: In this study we demonstrate that differentiation of human fetal neural progenitor cells under hypoxic conditions results in an increased neurogenesis. In addition, expansion and proliferation under lowered oxygen conditions also increased neuronal differentiation, although proliferation rates were not altered compared to normoxic conditions. Erythropoietin partially mimicked these hypoxic effects, as shown by an increase of the metabolic activity during differentiation and protection of differentiated cells from apoptosis. CONCLUSION: These results provide evidence that hypoxia promotes the differentiation of human fetal neural progenitor cells, and identifies the involvement of erythropoietin during differentiation as well as different cellular mechanisms underlying the induction of differentiation mediated by lowered oxygen levels.

Novel indolylmaleimide acts as GSK-3beta inhibitor in human neural progenitor cells.

The Wnt pathway is involved in cellular processes linked to either proliferation or differentiation. Therefore small molecules offer an attractive opportunity to modulate this pathway, whereas the key enzyme GSK-3beta is of special interest. In this study, non-symmetrically substituted indolylmaleimides have been synthesized and their ability to function as GSK-3beta inhibitors has been investigated in a human neural progenitor cell line. Among the newly synthesized compounds, the substance IM-12 showed a significant activity in several biological tests which was comparable or even outplayed the effects of the known GSK-3beta inhibitor SB-216763. Furthermore the treatment of human neural progenitor cells with IM-12 resulted in an increase of neuronal cells. Therefore we conclude that indolylmaleimides act via the canonical Wnt signalling pathway by inhibition of the key enzyme GSK-3beta.

Differentiation of human neural progenitor cells regulated by Wnt-3a.

Wnt ligands play pivotal roles in the control of cell growth and differentiation during central nervous system development via the Wnt signaling pathway. In this study, we investigated the effects of Wnt-3a and ß-catenin on the differentiation of ReNcell VM human neural progenitor cells. After overexpression of Wnt-3a or mutant-stabilized ß-catenin in ReNcell VM cells, their effects on TCF-mediated transcription, Wnt target gene expression and differentiation into neuronal and glial cells were investigated. Our results show that activation of Wnt/ß-catenin signaling increases TCF-mediated transcription and the expression of the Wnt target genes Axin2, LEF1 and CyclinD1 in ReNcell VM cells. In contrast to mutant-stabilized ß-catenin, Wnt-3a increases neurogenesis during the differentiation of ReNcell VM cells. Thus, our data suggest that neurogenesis induced by Wnt-3a is independent of the transcriptional activity of Wnt/ß-catenin pathway in ReNcell VM cells.

2-DE profiling of GDNF overexpression-related proteome changes in differentiating ST14A rat progenitor cells.

Targeted differentiation of neural progenitor cells (NPCs) is a challenge for treatment of neurodegenerative diseases by cell replacement therapy and cell signalling manipulation. Here, we applied a proteome profiling approach to the rat striatal progenitor model cell line ST14A in order to elucidate cellular differentiation processes. Native cells and cells transfected with the glial cell line-derived neurotrophic factor (GDNF) gene were investigated at the proliferative state and at seven time points up to 72 h after induction of differentiation. 2-DE combined with MALDI-MS was used to create a reference 2-DE-map of 652 spots of which 164 were identified and assigned to 155 unique proteins. For identification of protein expression changes during cell differentiation, spot patterns of triplicate gels were matched to the 2-DE-map. Besides proteins that display expression changes in native cells, we also noted 43 protein-spots that were differentially regulated by GDNF overexpression in more than four time points of the experiment. The expression patterns of putative differentiation markers such as annexin 5 (ANXA5), glucosidase II beta subunit (GLU2B), phosphatidylethanolamine-binding protein 1 (PEBP1), myosin regulatory light chain 2-A (MLRA), NASCENT polypeptide-associated complex alpha (NACA), elongation factor 2 (EF2), peroxiredoxin-1 (PRDX1) and proliferating cell nuclear antigen (PCNA) were verified by Western blotting. The results reflect the large rearrangements of the proteome during the differentiation process of NPCs and their strong modification by neurotrophic factors like GDNF.