Dataset Information

In Vitro Differentiation of Human Neural Progenitor Cells Into Striatal GABAergic Neurons.

ABSTRACT:

Unlabelled

: Huntington's disease (HD) results from a CAG repeat expansion in the gene encoding the huntingtin protein. This inherited disorder is characterized by progressive neurodegeneration. In particular, HD progression involves the loss of striatal projection neurons. The limited availability of reliable sources of human striatal projection neurons currently hampers our understanding of HD mechanisms and hinders the development of novel HD treatments. In this paper, we described two- and three-step methods for differentiating human neural progenitor cells toward striatal projection neurons. In the two-step differentiation protocol, 90%, 54%, and 6% of MAP2-positive cells were immunopositive for GABA, calbindin (CALB1), and DARPP-32/PPP1R1B, respectively. In the three-step differentiation protocol, 96%, 84%, and 21% of MAP2-positive cells were immunopositive for GABA, calbindin, and DARPP-32/PPP1R1B, respectively. In line with a striatal projection neuron phenotype, cells differentiated with our protocols displayed significantly increased expression of MAP2, CALB1, DARPP-32/PPP1R1B, ARPP21, and CTIP2. Application of glutamate receptor agonists induced calcium influx; accordingly, the cells also expressed various ionotropic glutamate receptor subunits. Differentiated cells also released GABA on stimulation. We suggest that our three-step differentiation protocol presents a reliable and simplified method for the generation of striatal projection neurons, yielding a critical resource for neuronal physiology and neurodegenerative disorder studies.

Significance

The earliest changes in the neurodegenerative disorder Huntington's disease affect a specific type of brain neurons, the so-called medium spiny neurons of the striatum. In this study, two protocols were developed for the differentiation of neural progenitor cells into striatal medium spiny neurons, and the differentiated neurons were extensively characterized. The data indicate that the three-step differentiation protocol presents a reliable and simplified method for the generation of striatal medium spiny neurons. The generated striatal medium spiny neurons could represent a critical resource for the study of neurodegenerative disorders, a model system for drug discovery, and a step toward cell-based regeneration therapies.

SUBMITTER: Lin L

PROVIDER: S-EPMC4479615 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Publications

In Vitro Differentiation of Human Neural Progenitor Cells Into Striatal GABAergic Neurons.

Lin Lin L Yuan Juan J Sander Bjoern B Golas Monika M MM

Stem cells translational medicine 20150513 7

<h4>Unlabelled</h4>: Huntington's disease (HD) results from a CAG repeat expansion in the gene encoding the huntingtin protein. This inherited disorder is characterized by progressive neurodegeneration. In particular, HD progression involves the loss of striatal projection neurons. The limited availability of reliable sources of human striatal projection neurons currently hampers our understanding of HD mechanisms and hinders the development of novel HD treatments. In this paper, we described tw ...[more]

PMID: 25972145

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Project description:This experiment was designed to characterize the temporal gene expression dynamics of differentiating human neural progenitor cells through time in culture. In vitro models of neuronal differentiation are emerging as an important tool for high-throughput and high-content screening in neurodevelopmental toxicology. However, little has been done to characterize normal temporal pathway dynamics of differentiation in vitro or to anchor processes captured in vitro to developmental processes in vivo that are vulnerable to toxicant perturbation. We cultured human neural progenitor cell (hNPCs) up to 21 days in differentiation conditions, examining changes in morphology, protein expression and global gene expression. Over time, hNPCs acquired morphological characteristics of mature neuronal networks and increased protein expression of neuronal markers, including beta tubulin III, MAP2, and alpha-synuclein. Significantly changed genes were organized according to temporal expression patterns using K-means clustering, revealing 3 phases of gene expression. Quantitative pathway analysis identified gene ontology (GO) terms enriched among genes expressed in each of these phases and created a quantitative summary of temporal pathway trends in vitro. These observations of morphology, protein and gene expression provide a timeline of progression through differentiation, facilitating identification of key phases of sensitivity. We compared gene expression in vitro with publicly available gene expression data from developing human brain tissue in vivo and found substantial concordance in relative gene expression intensity. Genes highly expressed in both samples were enriched for key processes of brain development, including proliferation, migration, differentiation, synapse formation, and neurotransmission. GO terms enriched among genes highly expressed only in vivo or only in vitro reveal important differences between systems. For example, genes highly expressed in vitro are enriched for more stress and apoptosis pathways. This analysis provides a temporal roadmap of in vitro neuronal differentiation and anchors gene expression patterns in vitro to gene expression during sensitive windows of in vivo development. By anchoring in vitro dynamics to in vivo reference points, this work clarifies the extent to which fundamental processes of brain development are captured in our model.

Project description:The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC) culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP) of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia), and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs). These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments.

Dataset Information

In Vitro Differentiation of Human Neural Progenitor Cells Into Striatal GABAergic Neurons.

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Significance

Publications

In Vitro Differentiation of Human Neural Progenitor Cells Into Striatal GABAergic Neurons.

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