Neural Precursor Cells
Abstract
Australia Huntington’s disease affected human embryonic stem cells are a powerful tool to research pathophysiology and develop therapeutics. Previously, we reported the derivation and characterisation of four HD-affected human embryonic stem cell lines as well as optimised expansion protocols of the undifferentiated cells. In order to be an effective strategy for HD research and drug screening however, stem cells have to be differentiated into relevant cell types in an efficient, reproducible and scalable manner. We developed Sydney IVF NPC Medium for the small-scale generation of neural precursor cells (NPC). In order to scale up NPC production, we have utilized the BioLevitator™, a commercially available three-dimensional culturing platform , which allowed us to generate NPC with far greater efficiencies than traditional flask culture. This neural precursor population is > 80% Sox2+, >70% Nestin+and MAP2+. Sydney IVF NPC can be further in vitro differentiated using Sydney IVF Neural Maturation Medium to yield a population of neural cells positive for GABA (40-60%). This culturing platform combined with Sydney IVF’s four HD-affected human embryonic stem cell lines could be utilized as a reliable cell supply for a high-throughput drug discovery platform.
Introduction
The BioLevitator™ (Hamilton) utilizes protein-coated magnetic microcarrier sas attachment surfaces that are gently rotated to provide suspension-like cell culture. Cells attach to the protein-coated Global Eukaryotic Microcarrier(GEM™). The GEM/cell mixture is then simultaneously moved in the vertical direction by a magnet and horizontally by rotating the culture vessel. This agitation creates a suspension-like culture system that promotes gas exchange and maintains a homogeneous medium composition. |
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Conclusion
Immunocytochemistry Results
Both GEM™ type and passagingmethod had an effect on the cell type produced during neurodifferentiation.
•Passagingmethod 2 produces more MAP2+cells (p=0.017) and Sox2+cells (p=0.0052)
•Collagen & GelatinGEMs™ passagedby Method 1 produce Peripherin+cells
•FibronectinGEMs™ produce less 3CB2+cells than Collagen & GelatinGEMs™ (p=0.00063)
•GelatinGEMs™ produce more FoxG1+cells than Collagen & FibronectinGEMs™ (p=0.011)
NPC can be further in vitro differentiated to express DARPP32, GAD65, TH, VGLUT1, or GFAP
Sydney IVF NPC have the capacity to become mature neurons expressing markers of GABAergicneurons (DARPP32 and GAD65), dopaminergicneurons (TH), glutamatergicneurons (VGLUT1) or astrocytes(GFAP). Cell lines do appear to have intrinsic tendencies to produce certain subsets of neurons. We are currently investigating the use of the BioLevitator™ to offer more control over in vitro differentiation in order to make the process efficient and reproducible. |
Click the "Downloads and Links" tab for more details about the poster and select the file titled A 3D culture system for medium-scale production of HD-affected neural precursor cells.
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