Abstract:
Investigations into diseases of the human central nervous system are restricted by limited access to human brain tissue and to clinically relevant animal models. The reprogramming of adult human somatic cells into pluripotent or multipotent intermediates holds promise for the generation of live disease-affected human cell models. Two-dimensional culture of reprogrammed cells has demonstrated the ability for these cells to generate clinically relevant human models of neurological diseases, but produces cells with different morphology, adhesions, and behaviour to cells in a 3-dimensional (3D) in vivo setting. To overcome these limitations, a range of 3D culture systems are being designed for use with reprogrammed human cells. This portfolio established an optimised protocol for 3D culture of cortical spheroids generated from human induced pluripotent stem cells (hiPSCs). These spheroids are self-organising, long-term cultures which generate mature neuronal and astrocytic cell populations, as well as regions of ongoing proliferation. The imaging of 3D models while maintaining the 3D architecture creates an additional challenge. This portfolio also optimised a protocol for less-toxic optical clearing, allowing us to image spheroids in toto and capture fine structural details. This was demonstrated to be effective for use with cortical spheroids using a range of immunofluorescence antibodies. To provide an initial validation of our cortical spheroid protocol to model Alzheimer’s disease (AD), we generated 40-day old cortical spheroids from patients with familial AD. These cortical spheroids exhibited amyloid-β pathologies in agreement with AD patient brains. In summary, the protocols developed in this portfolio allows for the generation of complex 3D cell models from healthy and patient-derived cells which are capable of exhibiting disease phenotypes and can be imaged effectively in toto.