Project

NEUROMUS: toward multi-organ in vitro systems

In the NEUROMUS project, researchers work toward multi-organ in vitro systems, for instance, brain-muscle and brain-gut connections. These systems will be used for human health and livestock research. The focus of NEUROMUS will be on nerve cells and innervation.

The central nervous system (CNS) integrates sensory information to indirectly coordinate the functions of tissues and organs. The primary mediators of these nerve–organ connections are axons, fibrous projections from nerve cells called neurons. Neuronal connections mediate precise junctions (innervation) with end target cells and play roles in the development, maturation, function, regulatory control, and regeneration and pathology of tissues and organs. Unfortunately, such innervation is lost during in vitro cell, tissue or organoid cultures that do contain different cell types but not neurons. Therefore, innervation through neural tissue engineering and/or axon guidance strategies should be considered key to in vitro organogenesis across multiple tissue and organ systems.

In the NEUROMUS project, three cell types stand out in relevance to achieve functional innervation: (1) neuronal cells, (2) muscle (skeletal, cardiac and smooth) cells and (3) intestinal cells. Moreover, co-cultures of neuronal- and Schwann and glial cells, yielding complex cell systems, will allow to obtain biologically correct myelination, and co-cultures of neurons with astrocytes and microglia can be used to study inflammation of the brain, for example induced by infection or by proinflammatory cytokine signals. The complex cell systems the researchers propose to generate in vitro can be used to study the effects of food and feed-based components, (microbial) metabolites and medicine on health, disease, and pathology. Such in vitro models could potentially further reduce laboratory animal use.

Main research questions

  1. What is the role of mitochondrial metabolism and signalling on the formation and functionality of neuromuscular junctions (NMJ) in health and disease?
  2. How is the chemical signalling between the brain and the gut regulated?

Progress (September 2022)

To address the research questions, the researchers have reached several milestones. They have obtained human stem cells and materials to create neuronal stem cell stocks and have obtained the corresponding approvals via MTAs. Stem cells have been stably transfected using the MYOD gene to induce muscle differentiation from iPSCs (induced pluripotent stem cells). The researchers are currently validating transformation by karyotyping and functional characterisation of iPSCs prior to stimulating differentiation of iPSCs into muscle and neurons, and investigating neuromuscular junctions (NMJs).

Starting with lab mice, they have isolated and genomically characterised skeletal muscles and neurons from the spinal cord, generated MyoD construct to induce muscle differentiation from iPSCs, and isolated mouse embryonic fibroblasts (MEFs). The team is currently working on reprogramming MEFs into iPSCs using a nonintegrating viral vector system and stably transfecting reprogrammed iPSCs with MyoD constructs. As with human cells, the researchers will validate transformation by karyotyping and functional characterisation of iPSCs prior to stimulating differentiation of iPSCs into muscle, neurons, and NMJs.

Results

In the NEUROMUS project, the researchers successfully created a differentiation protocol to create muscle cells from human iPSCs. They also successfully reprogrammed mouse embryonic fibroblasts, with a mutated Nicotinamide Nucleotide Transhydrogenase gene, into iPSCs.