Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells

Central nervous system (CNS) disorders remain a formidable challenge for the development of efficient therapies. Cell and gene therapy approaches are promising alternatives that can have a tremendous impact by treating the causes of the disease rather than the symptoms, providing specific targeting and prolonged duration of action. Hampering translation of gene-based therapeutic treatments of neurodegenerative diseases from experimental to clinical gene therapy is the lack of valid and reliable pre-clinical models that can contribute to evaluate feasibility and safety. Herein we describe a robust and reproducible methodology for the generation of 3D in vitro models of the human CNS following a systematic technological approach based on stirred culture systems. We took advantage of human midbrain-derived neural progenitor cells (hmNPCs) capability to differentiate into the various neural phenotypes and of their commitment to the dopaminergic lineage to generate differentiated neurospheres enriched in dopaminergic neurons. Furthermore, we describe a protocol for efficient gene transfer into differentiated neurospheres using CAV-2 viral vectors and stable expression of the transgene for at least 10 days. CAV-2 vectors, derived from canine adenovirus type 2, are promising tools to understand and treat neurodegenerative diseases, in particular Parkinson's disease. CAV-2 vectors preferentially transduce neurons and have an impressive level of axonal retrograde transport in vivo. Our model provides a practical and versatile in vitro approach to study the CNS in a 3D cellular context. With the successful differentiation and subsequent genetic modification of neurospheres we are increasing the collection of tools available for neuroscience research and contributing for the implementation and widespread utilization of 3D cellular CNS models. These can be applied to study neurodegenerative diseases such as Parkinson's disease; to study the interaction of viral vectors of therapeutic potential within human neural cell populations, thus enabling the introduction of specific therapeutic genes for treatment of CNS pathologies; to study the fate and effect of delivered therapeutic genes; to study toxicological effects. Furthermore these methodologies may be extended to other sources of human neural stem cells, such as human pluripotent stem cells, including patient-derived induced pluripotent stem cells.     

Gene expression profiling of embryonic human neural stem cells and dopaminergic neurons from adult human substantia nigra

Neural stem cells (NSC) with self-renewal and multipotent properties serve as an ideal cell source for transplantation to treat neurodegenerative insults such as Parkinson's disease. We used Agilent's and Illumina Whole Human Genome Oligonucleotide Microarray to compare the genomic profiles of human embryonic NSC at a single time point in culture, and a multicellular tissue from postmortem adult substantia nigra (SN) which are rich in dopaminergic (DA) neurons. We identified 13525 up-regulated genes in both cell types of which 3737 (27.6%) genes were up-regulated in the hENSC, 4116 (30.4%) genes were up-regulated in the human substantia nigra dopaminergic cells, and 5672 (41.93%) were significantly up-regulated in both cell population. Careful analysis of the data that emerged using DAVID has permitted us to distinguish several genes and pathways that are involved in dopaminergic (DA) differentiation, and to identify the crucial signaling pathways that direct the process of differentiation. The set of genes expressed more highly at hENSC is enriched in molecules known or predicted to be involved in the M phase of the mitotic cell cycle. On the other hand, the genes enriched in SN cells include a different set of functional categories, namely synaptic transmission, central nervous system development, structural constituents of the myelin sheath, the internode region of axons, myelination, cell projection, cell somata, ion transport, and the voltage-gated ion channel complex. Our results were also compared with data from various databases, and between different types of arrays, Agilent versus Illumina. This approach has allowed us to confirm the consistency of our obtained results for a large number of genes that delineate the phenotypical differences of embryonic NSCs, and SN cells.     

Gene expression profile of quinacrine-cured prion-infected mouse neuronal cells

Prion diseases are transmissible fatal neurodegenerative diseases of humans and animals, characterised by the presence of an abnormal isoform (scrapie prion protein; PrP^Sc) of the endogenous cellular prion protein (PrP^C). The pathological mechanisms at the basis of prion diseases remain elusive, although the accumulation of PrP^Sc has been linked to neurodegeneration. Different genomic approaches have been applied to carry out large-scale expression analysis in prion-infected brains and cell lines, in order to define factors potentially involved in pathogenesis. However, the general lack of overlap between the genes found in these studies prompted us to carry an analysis of gene expression using an alternative approach. Specifically, in order to avoid the complexities of shifting gene expression in a heterogeneous cell population, we used a single clone of GT1 cells that was de novo infected with mouse prion-infected brain homogenate and then treated with quinacrine to clear PrP^Sc. By comparing the gene expression profiles of about 15 000 genes in quinacrine-cured and not cured prion-infected GT1 cells, we investigated the influence of the presence or the absence of PrP^Sc. By real-time PCR, we confirmed that the gene encoding for laminin was down-regulated as a consequence of the elimination of PrP^Sc by the quinacrine treatment. Thus, we speculate that this protein could be a specific candidate for further analysis of its role in prion infection and pathogenesis.     

Differentiation of single cell derived human mesenchymal stem cells into cells with a neuronal phenotype: RNA and microRNA expression profile

The adult bone marrow contains a subset of non-haematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi-lineage differentiation. The molecular mechanisms governing the self-renewal and differentiation of MSCs remain largely unknown. In a previous paper we demonstrated the ability to induce human clonal MSCs to differentiate into cells with a neuronal phenotype (DMSCs). In the present study we evaluated gene expression profiles by Sequential Analysis of Gene Expression (SAGE) and microRNA expression profiles before and after the neuronal differentiation process. Various tissue-specific genes were weakly expressed in MSCs, including those of non-mesodermal origin, suggesting multiple potential tissue-specific differentiation, as well as stemness markers. Expression of OCT4, KLF4 and c-Myc cell reprogramming factors, which are modulated during the differentiation process, was also observed. Many peculiar nervous tissue genes were expressed at a high level in DMSCs, along with genes related to apoptosis. MicroRNA profiling and correlation with mRNA expression profiles allowed us to identify putative important genes and microRNAs involved in the differentiation of MSCs into neuronal-like cells. The profound difference in gene and microRNA expression patterns between MSCs and DMSCs indicates a real functional change during differentiation from MSCs to DMSCs.     

Maternal gametic transmission of translocations or inversions of human chromosome 11p15.5 results in regional DNA hypermethylation and downregulation of CDKN1C expression

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33 Mb of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in "chromatin context".     

Paternal imprinting of the SLC22A1LS gene located in the human chromosome segment 11p15.5

Prostate cancer is the most commonly diagnosed cancer in men and one of the leading causes of cancer deaths. There is strong genetic evidence indicating that a large proportion of prostate cancers are caused by heritable factors but the search for prostate cancer susceptibility genes has thus far remained elusive. TGFBR1*6A, a common hypomorphic variant of the type I Transforming Growth Factor Beta receptor, is emerging as a tumor susceptibility allele that predisposes to the development of breast, colon and ovarian cancer. The association with prostate cancer has not yet been explored. A total of 907 cases and controls from New York City were genotyped to test the hypothesis that TGFBR1*6A may contribute to the development of prostate cancer. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67). Our data suggest that TGFBR1*6A does not contribute to the development of prostate cancer.     

Gene expression profile of immunoregulatory cytokines secreted from bone marrow and adipose derived human mesenchymal stem cells in early and late passages

Molecular Biology Reports - Mesenchymal Stem Cells (MSCs) have therapeutic potential in a variety of diseases; however, the safety and efficacy of their use remain ambiguous. Clinical applications...     

Gene expression profile of human lymphoid CEM cells sensitive and resistant to glucocorticoid-evoked apoptosis

Three closely related clones of leukemic lymphoid CEM cells were compared for their gene expression responses to the glucocorticoid dexamethasone (Dex). All three contained receptors for Dex, but only two responded by undergoing apoptosis. After a time of exposure to Dex that ended late in the interval preceding onset of apoptosis, gene microarray analyses were carried out. The results indicate that the expression of a limited, distinctive set of genes was altered in the two apoptosis-prone clones, not in the resistant clone. That clone showed altered expression of different sets of genes, suggesting that a molecular switch converted patterns of gene expression between the two phenotypes: apoptosis-prone and apoptosis-resistant. The results are consistent with the hypothesis that altered expression of a distinctive network of genes after glucocorticoid administration ultimately triggers apoptosis of leukemic lymphoid cells. The altered genes identified provide new foci for study of their role in cell death.     

In vitro differentiation of human amniotic fluid‐derived cells: Augmentation towards a neuronal dopaminergic phenotype

Amniotic fluid is known to yield a number of cell types which are multipotent, ethically derived, genetically stable, easily grown, expanded and possess favourable immunogenicity, which has resulted in an increasing interest for use in various neuronal disorders such as Parkinson's disease. The neuronal potential of cells derived from the adherent fraction of amniotic fluid, routinely taken by amniocentesis, are least explored. The aim of the present study was to investigate the capacity of these cells for neuronal and dopaminergic differentiation using in vitro differentiation protocols with canonical inductive factors not previously tested. To do this, samples derived from multiple donors were grown under four conditions: standard serum‐containing media, NB (neurobasal) media designed specifically for propagation and maintenance of neuronal cells, NB media with addition of retinoic acid and BDNF (brain‐derived neurotrophic factor) for NI (neuronal induction), and NB media with addition of FGF8 (fibroblast growth factor‐8) and Shh (sonic hedgehog) after NI. Our results showed the presence of multiple neuronal markers after growth in serum‐containing medium [TUJ1, MAP2, NF‐M, TH (tyrosine hydroxylase)], which was significantly up‐regulated after serum withdrawal in NB medium alone with induction of NeuN (neuronal nuclei) and NSE (neuron‐specific enolase). NI and DA.I (dopaminergic induction) was accompanied by further increases in expression and a distinct transition to a sustained neuronal morphology. Western blot analysis confirmed increasing TH expression and NURR1, expressed in base serum‐containing media, found to be down‐regulated after induction. In conclusion, these cells possess a highly favourable base neuronal and dopaminergic prepotential, which may easily be accentuated by standard induction protocols.     

Neuropharmacological properties of neurons derived from human stem cells

Human pluripotent stem cells have enormous potential value in neuropharmacology and drug discovery yet there is little data on the major classes and properties of receptors and ion channels expressed by neurons derived from these stem cells. Recent studies in this lab have therefore used conventional patch-clamp electrophysiology to investigate the pharmacological properties of the ligand and voltage-gated ion channels in neurons derived and maintained in vitro from the human stem cell (hSC) line, TERA2.cl.SP12.TERA2.cl.SP12 stem cells were differentiated with retinoic acid and used in electrophysiological experiments 28-50 days after beginning differentiation. HSC-derived neurons generated large whole cell currents with depolarizing voltage steps (−80 to 30 mV) comprised of an inward, rapidly inactivating component and a delayed, slowly deactivating outward component. The fast inward current was blocked by the sodium channel blocker tetrodotoxin (0.1 μM) and the outward currents were significantly reduced by tetraethylammonium ions (TEA, 5 mM) consistent with the presence of functional Na and K ion channels. Application of the inhibitory neurotransmitters, GABA (0.1-1000 μM) or glycine (0.1-1000 μM) evoked concentration dependent currents. The GABA currents were inhibited by the convulsants, picrotoxin (10 μM) and bicuculline (3 μM), potentiated by the NSAID mefenamic acid (10-100 μM), the general anaesthetic pentobarbital (100 μM), the neurosteroid allopregnanolone and the anxiolytics chlordiazepoxide (10 μM) and diazepam (10 μM) all consistent with the expression of GABA_A receptors. Responses to glycine were reversibly blocked by strychnine (10 μM) consistent with glycine-gated chloride channels. The excitatory agonists, glutamate (1-1000 μM) and NMDA (1-1000 μM) activated concentration-dependent responses from hSC-derived neurons. Glutamate currents were inhibited by kynurenic acid (1 mM) and NMDA responses were blocked by MgCl₂ (2 mM) in a highly voltage-dependent manner.Together, these findings show that neurons derived from human stem cells develop an array of functional receptors and ion channels with a pharmacological profile in keeping with that described for native neurons. This study therefore provides support for the hypothesis that stem cells may provide a powerful source of human neurons for future neuropharmacological studies.     

In vitro maturation of dopaminergic neurons derived from mouse embryonic stem cells: implications for transplantation

The obvious motor symptoms of Parkinson's disease result from a loss of dopaminergic neurons from the substantia nigra. Embryonic stem cell-derived neural progenitor or precursor cells, adult neurons and fetal midbrain tissue have all been used to replace dying dopaminergic neurons. Transplanted cell survival is compromised by factors relating to the new environment, for example; hypoxia, mechanical trauma and excitatory amino acid toxicity. In this study we investigate, using live-cell fluorescence Ca(2+) and Cl(-) imaging, the functional properties of catecholaminergic neurons as they mature. We also investigate whether GABA has the capacity to act as a neurotoxin early in the development of these neurons. From day 13 to day 21 of differentiation [Cl(-)](i) progressively dropped in tyrosine hydroxylase positive (TH(+)) neurons from 56.0 (95% confidence interval, 55.1, 56.9) mM to 6.9 (6.8, 7.1) mM. At days 13 and 15 TH(+) neurons responded to GABA (30 μM) with reductions in intracellular Cl(-) ([Cl(-)](i)); from day 21 the majority of neurons responded to GABA (30 μM) with elevations of [Cl(-)](i). As [Cl(-)](i) reduced, the ability of GABA (30 μM) to elevate intracellular Ca(2+) ([Ca(2+)](i)) did also. At day 13 of differentiation a three hour exposure to GABA (30 μM) or L-glutamate (30 μM) increased the number of midbrain dopaminergic (TH(+) and Pitx3(+)) neurons labeled with the membrane-impermeable nuclear dye TOPRO-3. By day 23 cultures were resistant to the effects of both GABA and L-glutamate. We believe that neuronal susceptibility to amino acid excitotoxicity is dependent upon neuronal maturity, and this should be considered when isolating cells for transplantation studies.     

Proliferative characteristics of progeny derived from normal human hemopoietic progenitor cells

Cell cycle times of cells derived from human hemopoietic progenitor cells were measured directly by using 5-bromodeoxyuridine (BrdU) incorporation and sister chromatid differential staining. Umbilical cord cells were cultured in the mixed-colony assay for 96 h prior to the addition of BrdU and then harvested at 6-12 h intervals. Individual cell cycle times ranging from 8.5-23.4 h were observed while population mean cell cycle times ranged from 17.5-24 h. These results show that some early progeny of hemopoietic progenitor cells have short cell cycle times and that there is considerable heterogeneity within the proliferating cell population.     

Neurite formation by neurons derived from adult rat hippocampal progenitor cells is susceptible to myelin inhibition

Myelin-associated inhibitors expressed following injury to the adult central nervous system (CNS) induce growth cone collapse and retraction of the axonal cytoskeleton. Myelin-associated glycoprotein (MAG) is a bi-functional molecule that promotes neuritogenesis in some immature neurons during development then becomes inhibitory to neurite outgrowth as neurons mature. Progress is being made towards the elucidation of the downstream events that regulate myelin inhibition of regeneration in neuronal populations. However it is not known how adult-derived neural stem cells or progenitors respond to myelin during neuronal differentiation and neuritogenesis. Here we examine the effect of MAG on neurons derived from an adult rat hippocampal progenitor cell line (AHPCs). We show that, unlike their developmental counterparts, AHPC-derived neurons are susceptible to MAG inhibition of neuritogenesis during differentiation and display a 57% reduction in neurite outgrowth when compared with controls. We demonstrate that this effect can be overcome (by up to 69%) by activation of the neurotrophin, cyclic AMP and protein kinase A pathways or by Rho-kinase suppression. We also demonstrate that combination of these factors enhanced neurite outgrowth from differentiating neurons in the presence of MAG. This work provides important information for the successful generation of new neurons from adult neural stem cell populations within compromised adult circuitry and is thus directly relevant to endogenous repair and regeneration of the adult CNS.