Neurodegenerative diseases, such as Huntington’s disease (HD), cause a progressive loss of neurons with no treatments available. Improved human cell culture models are needed to screen for relevant therapeutics. Induced pluripotent stem cells (iPSCs) can differentiate into all cell types, including neurons affected by HD. Overexpression of neuronal factors by lentiviral gene delivery enables rapid and efficient iPSC differentiation into neurons. These cells are typically cultured on protein-coated stiff substrates, whereas, <i>in vivo,</i> neurons interact with a soft, laminin-rich extracellular matrix. Here, we hypothesized that mimicking the structure and protein content of neural environments may enhance patient-specific iPSC differentiation into neurons. To test this, we identified transcription factors present during neural development. Lentiviral particles encoding these factors were produced, purified, and characterized to optimize viral infectivity for iPSC differentiation. We compared infectivity of lentiviral particles in iPSCs and HEK cells, finding that 20-100 times the amount of lentivirus is required to infect iPSCs. To mimic key aspects of neuronal tissue, bioinert poly(ethylene glycol) hydrogels were selected owing to the precise and independent control afforded of matrix elasticity and biochemical content (e.g., integrin-binding peptides, laminin). Hydrogels were formed by photoinitiated thiol–ene click chemistry and concentrations were varied to achieve a modulus comparable to neural tissue (Young’s modulus, E ~ 0.6 kPa). These hydrogels were modified with bioactive peptides and optimized for attachment of iPSCs and neuroblastoma cells. Ongoing differentiation experiments entail characterization of neuron morphology and markers by immunostaining and RT-PCR. Stem cell differentiation using viral infectivity on biomimetic hydrogels is a viable option for developing neuronal cell culture models.

Controlling differentiation of induced pluripotent stem cells into neurons in well-defined, hydrogel-based microenvironments

251st National Meeting (2016)

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