Human Midbrain-derived Neural Progenitor Cells Research Articles

Modeling human neural functionality in vitro: three-dimensional culture for dopaminergic differentiation.

Advances in mechanistic knowledge of human neurological disorders have been hindered by the lack of adequate human in vitro models. Three-dimensional (3D) cellular models displaying higher biological relevance are gaining momentum; however, their lack of robustness and scarcity of analytical tools adapted to three dimensions hampers their widespread implementation. Herein we show that human midbrain-derived neural progenitor cells, cultured as 3D neurospheres in stirred culture systems, reproducibly differentiate into complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Moreover, an extensive toolbox of analytical methodologies has been adapted to 3D neural cell models, allowing molecular and phenotypic profiling and interrogation. The generated neurons underwent synaptogenesis and elicit spontaneous Ca(2+) transients. Synaptic vesicle trafficking and release of dopamine in response to depolarizing stimuli was also observed. Under whole-cell current-and-voltage clamp, recordings showed polarized neurons (Vm=-70 mV) and voltage-dependent potassium currents, which included A-type-like currents. Glutamate-induced currents sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate antagonists revealed the existence of functional glutamate receptors. Molecular and phenotypic profiling showed recapitulation of midbrain patterning events, and remodeling toward increased similarity to human brain features, such as extracellular matrix composition and metabolic signature. We have developed a robust and reproducible human 3D neural cell model, which may be extended to patient-derived induced pluripotent stem cells, broadening the applicability of this model.

1H-NMR spectroscopy for human 3D neural stem cell cultures metabolic profiling

Background The current lack of predictable central nervous system (CNS) models in pharmaceutical industry early stage development strongly contributes for the high attrition rates registered for new therapeutics [1]. Thus, there is an increasing need for a paradigm shift towards more human relevant cell models, which can closely recapitulate the in vivo cell-cell interactions, presenting higher physiological relevance by bridging the gap between animal models and human clinical trials. In this context, human 3D in vitro models are promising tools with great potential for preclinical research, as they can mimic some of the main features of tissues, such as cell-cell and cell-extracellular matrix (ECM) interactions [2,3]. Moreover these complex cell models are suitable for high-throughput screening (HTS) platforms, essential in drug discovery pipelines by reducing both costs and time in clinical trials [2,4]. However, despite important advances in the last years and the increasing clinical and biological relevance, the full establishment of human 3D in vitro models in pre-clinical research requires a significant increase in the power of the available analytical methodologies towards more robust and comprehensive readouts [4]. With the emergence of systems biology field and several “-omics” technologies, such as metabolomics, it became possible to have a more mechanistic approach in the understanding of cellular programs. H-nuclear magnetic resonance (H-NMR) spectroscopy is a powerful and widely accepted high resolution methodology for a number of applications, including metabolic profiling [5]. Despite the low sensitivity when compared with mass spectrometry (MS), H-NMR profiling presents several advantages, enabling a non-invasive and non-destructive quantitative analysis requiring only minimal sample preparation [5]. In this work we present the development of a robust and optimized workflow for the exometabolome profiling of 3D in vitro cultures of human midbrain-derived neural progenitor cells (hmNPC).

Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

BackgroundHuman fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson’s disease. While glutamate and GABAA receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown.Methodology/Principal FindingsHere we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na+-K+-Cl− co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. Conclusions/SignificanceThese data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

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 10days. 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.

P119 Functional and molecular analysis of glycine receptors in human midbrain-derived neural progenitor cells

P119 Functional and molecular analysis of glycine receptors in human midbrain-derived neural progenitor cells

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain-derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson's disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole-cell patch-clamp recordings, l-glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration-response plots of differentiated NPCs yielded an EC(50) of 2.2 microM for glutamate and an EC(50) of 36 microM for NMDA. Glutamate-induced currents were markedly inhibited by memantine in contrast to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) suggesting a higher density of functional NMDA than alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. NMDA-evoked currents and calcium signals were blocked by the NR2B-subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage-gated calcium channels by verapamil abolished AMPA-induced calcium responses but only partially reduced NMDA-evoked transients suggesting the expression of calcium-impermeable, GluR2-containing AMPA receptors. Quantitative real-time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 microM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase-immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.