Method for inducing human dorsal cortical neurons from pluripotent stem cells using tilapia collagen gel

Technology for inducing neuronal differentiation using tilapia collagen gel that mimics brain tissue stiffness

This technology involves chemically cross-linking collagen derived from tilapia skin using NHS and EDC to create a gel with a flat surface and brain-like stiffness of 1,700 Pa or less, which is then used to differentiate human pluripotent stem cells into neurons.

While it is known that substrate stiffness dictates stem cell fate, it has been difficult to consistently reproduce the extremely soft stiffness of the brain. Conventional polyacrylamide hydrogels are hard to tune to brain-level stiffness and pose toxicity risks to neurons due to acrylamide.

This technology uses fish collagen, which has a low risk of zoonotic infection, and adjusts the degree of cross-linking to replicate the stiffness of living brain tissue, inducing differentiation into dorsal cortical neurons on a ~1,500 Pa gel. It can be applied to stem cell culture substrates, neuron production for brain disease modeling, and nerve regeneration research, allowing for the selective, non-toxic acquisition of desired neuronal subtypes.

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Key Features:

• Step of seeding human pluripotent stem cells on a tilapia skin collagen gel composition and culturing them in neuronal differentiation medium to induce differentiation
• Tilapia collagen gel chemically cross-linked with an NHS to EDC ratio of 0.1:1 to 1:1, featuring a stiffness of 1,700 Pa or less and uniform flat surface morphology
• Step of obtaining collagen extract from tilapia skin and chemically cross-linking it by adding EDC in the 20–200 mM range along with NHS
• Stem cell culture composition containing cross-linked tilapia skin collagen gel to induce differentiation into human dorsal cortical neurons

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This invention was developed with support from the Ministry of Science and ICT for research into the causes, diagnosis, and control of brain development and brain diseases.

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