These hiPSC lines can be produced from any individual and can be used to engineer replacement tissue for diseased or damaged organs 5. In 2007, Takahashi and Yamanaka showed that it was possible to reprogram human fibroblasts into a pluripotent state by upregulating the expression of four transcription factors, producing human induced pluripotent stem cells (hiPSCs) 2. Pluripotent stem cells detect and respond to chemical and mechanical cues from their micro-environment, which direct them to become specialized for their desired role in the body 4. These two properties make them an attractive option for treating degenerative diseases and traumatic injuries that occur in the central nervous system 3. Human pluripotent stem cells can divide indefinitely and adopt the identity of any cell type in the body, giving them the potential to regenerate specific tissue types 1, 2. These mechanically stable genipin-crosslinked fibrin scaffolds support hiPSC-derived neural aggregates and induce neurite outgrowth while remaining intact for 2 weeks as opposed to 5 days for unmodified fibrin scaffolds. Here we show that genipin enhances neuronal differentiation of neural progenitors derived from human induced pluripotent stem cells (hiPSCs) in 2D culture and genipin concentration influences the morphological and mechanical properties of 3D fibrin scaffolds. It also possesses neuritogenic and neuroprotective properties, making it particularly attractive for engineering neural tissue from pluripotent stem cells. Genipin crosslinking alters the physical characteristics of the fibrin scaffolds, which influences the behaviour of the differentiating cells seeded inside. The addition of crosslinking agents, such as genipin, during the polymerization process increases scaffold stability while decreasing the degradation rate of fibrin. However, the rapid degradation rate of fibrin remains a major limitation when differentiating human pluripotent stem cells for tissue engineering applications. Our plugin is an attractive alternative to existing tracing tools because it is fully automated and ready for use within a freely accessible imaging program.Recent work demonstrated that 3D fibrin scaffolds function as an effective substrate for engineering tissues from pluripotent stem cells. We reveal the utility of NeuriteTracer by demonstrating its ability to detect the neurite outgrowth promoting capacity of the rho kinase inhibitor Y-27632. Values obtained by NeuriteTracer correlate strongly with those obtained by semi-manual tracing with NeuronJ and by using a sophisticated analysis package, MetaXpress. We find that NeuriteTracer accurately measures neurite outgrowth from cerebellar, DRG and hippocampal neurons. Given user-defined thresholds, the plugin counts neuronal nuclei, and traces and measures neurite length.
The plugin analyzes fluorescence microscopy images of neurites and nuclei of dissociated cultured neurons. To automate these measurements, we have written NeuriteTracer, a neurite tracing plugin for the freely available image-processing program ImageJ.
#NEUROTRACKER NEURITE QUANTIFICATION MANUAL#
Generally, these measurements are obtained through labor-intensive manual or semi-manual tracing of images. The quantification of these assays requires accurate measurements of neurite length and neuronal cell numbers in neuronal cultures. In vitro assays to measure neuronal growth are a fundamental tool used by many neurobiologists studying neuronal development and regeneration.