Using iPSC-Derived Neurons

CNS cells derived from human iPSCs have recently emerged as a straightforward, efficient, and replicable in vitro system for assessing aspects of human neuronal function such as neurite outgrowth. These cultures can be derived from healthy or patient-derived iPSCs. They can be studied in monoculture or co-culture to explore single cell mechanisms as well as the influence of neighboring cell types and signaling interplay. This iPSC technology is ideal for mechanistic studies and disease modelling in human cells.

Methodology to asses label-free neurite dynamics

A diagram of the method steps to assess neurite dynamics from human iPSC differentiation to image analysis.

Human iPSCs are terminally differentiated into neurons using a specific neuronal differentiation protocol. The effect of a therapeutic compound or inflammatory stimuli on cell viability and neurite outgrowth dynamic are assessed by NeuroTracker software.

Neurites are critical for communication between neurons. Neurodegeneration seen in neuropathological disorders can be attributed to a loss of this connectivity. Neurite outgrowth, complexity, and dynamics influence network connectivity, number of synapses, cell viability, and response to inflammatory stimuli.

iPSC neurite tracking:

Immunostaining of human iPSC neurons which are terminally differentiated into and live cell imaged.Neuronal differentiation was confirmed by the presence of MAP2 and absence of precursor markers. Neurons are imaged live and the total cell number (yellow) and neurite dynamics (pink) assessed by NeuroTracker software. Live cell imaging was performed using the IncuCyte® S3 platform.

The ability to measure dynamic changes in neurites gives information about the viability of these connections in a healthy or pathological context and the influence of therapeutics on this process.

Studying patient-derived IPSCs can identify drug candidate responders and non-responders to provide a valuable preclinical readout. Overall, they fill an unmet need that bridges the gap between clinical and animal models to support drug development.

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Frequently Asked Questions (FAQs) for Neurite Outgrowth Assays

  • What is neurite outgrowth?

    Neurite outgrowth is a process where developing neurons produce new projections as they grow in response to nerve growth factors or neurotrophins.

  • When does neurite outgrowth become compromised?

    Aging is one of the leading causes of neuron atrophy; however, brain lesions that occur with acute brain injuries or with chronic inflammation from neurodegeneration also result in impaired sprouting/decreased neurite outgrowth.

  • Can non-neuronal cells in the brain influence neurite outgrowth?

    Diminishing glial fibrillary acidic protein (GFAP) levels of old astrocytes or compromised astrocytes due to excess inflammation, can affect neurite outgrowth. Cellular senescence increases with age and several laboratories suggest that both aging and age-related neurodegenerative diseases are accompanied by an increase in senescent cells of non-neuronal origin in the brain.