Microfluidic models to study the nervous system and its connection to the periphery

Abstract

Amyotrophic lateral sclerosis (ALS) is an incompletely understood disease in which motor neurons and skeletal muscle cells are affected. One of the major hurdles that the field had to overcome, involved obtaining sufficient cells to study the disease in a controlled environment in the laboratory. The advent of induced pluripotent stem cell (iPSC) technology has allowed this. Importantly, iPSC-derived cells are increasingly being combined with culture methods that are more relevant than 2-dimensional approaches. In chapter 1, we highlight recent advances the ALS field has made, which in vitro models would be relevant to study the disease, and how microfluidic technology can be used to generate such models. Chapter 2 describes the differentiation of iPSCs from healthy and ALS donors into motor neurons and skeletal muscle cells. Importantly, motor neurons progenitors could be cryopreserved and subsequently demonstrated good viability upon thawing. Finally, we highlight how motor neurospheres and skeletal muscle cells can be co-cultured to form a neuromuscular junction (NMJ) model, and how the motor neurons can be integrated into the OrganoPlate. We subsequently developed a model that separates axons from a somatodendritic compartment in chapter 3. Diseases such as ALS are characterized by various axonal defects. Being able to separate the axons allows their specific interrogation. We demonstrate that the outgrowth is robust and can be targeted using a specific tubulin inhibitor, while axonal guidance cues deter outgrowing axons. We therefore argue that the neurite outgrowth model can be used in the fields of toxicology and regenerative medicine. We then utilized the developed protocols to compare iPSC-derived motor neurons from healthy and ALS donors in the OrganoPlate platform in chapter 4. For these comparisons, we focused on ALS risk gene ATXN2. Using different read-outs, donor-dependent differences could be elucidated. While these were not ALS-specific, it demonstrates the sensitivity of the assays. Additional read-outs and ALS genes should be considered, while model complexity can be increased to study additional ALS pathways. In chapter 5, we developed an approach to study calcium bursts in skeletal muscle and neuronal cells. Both iPSC-derived and primary skeletal muscle cells form multinucleated myotubes that contract upon an electrical stimulus. We demonstrate that contraction could be quantified through the assessment of calcium concentrations, and that this process can be inhibited. We finally demonstrate that calcium bursts in neuronal cells can be induced and inhibited as well. In chapter 6, the implications of this thesis are highlighted. We discuss how the developed models and read-outs can be used for specific applications. First, the models can be used to assess toxicological effects on neuronal or muscle cells, which holds potential in the field of drug development. In addition, increasing culture complexity may pave the way for the development of novel, relevant read-outs. The addition of glia and skeletal muscle cells are specifically highlighted. The high throughput of the OrganoPlate may ultimately be leveraged to screen a large number of therapeutics in the fields of toxicology and disease modeling.