Chemically defined substrates in a ready to use format that promote differentiation, accelerated maturation and neurite extension of multiple neuronal subtypes for screening applications

Abstract: Human neural cells manufactured from induced pluripotent stem cells (iPSCs) hold great promise for modeling neurodevelopmental disorders, screening for potential risks from environmental toxins and as cell replacement therapies.  However, most protocols require the use of complex, animal derived substrates that need to be prepared prior to cell plating and introduce variability to culture and assay workflows.  We have developed chemically defined substrates employing norbornene-functionalized polyethylene glycol and synthetic peptides that can be applied to cell culture surfaces, dehydrated, packaged, sterilized, and stored ready for re-hydration and use.  We employed Design of Experiment (DOE) methodology utilizing Box-Behnken response surface modeling to screen for formulations that promoted viability, cell adhesion, desired morphology, and accelerated maturation of cortical glutamatergic and motor neurons or differentiation to glutamatergic neurons.  These formulations were tested for compatibility with other commercially available iPSC-derived neurons including GABAergic neurons and transcriptionally induced excitatory neurons. Transcriptional profiling utilizing bulk RNASeq analysis was performed comparing neural cultures on the substrates to those cultured on standard substrates including charged polymers (poly-lysines) or animal-derived substrates (PLO-Laminin or Geltrex).  Cells cultured on Stem Pharm’s hydrogel substrates demonstrated an increase in transcription of gene ontology sets expressing neuronal maturation, neuron projection guidance and cell-substrate adhesion in day seven samples and enhanced cell differentiation, migration, signaling at the cell periphery and vesicles at day fourteen.  The pre-plated substrates reduce time for experimental preparation, reduce the substrate complexity, eliminate ethical considerations of animal-derived substrates, and improve assay metrics when applied to screening campaigns.