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Co-culture for Neuromuscular Junctions is Just Better

Adam Tozer
Cells don’t act in isolation when in vivo so there’s no reason to culture them that way in vitro when you’re trying to model a complex structure like a neuromuscular junction (NMJ).

So, as part of the PLATFORMA Project (an EU Horizon 2020-funded program), we developed a microfluidic 2D co-culture system using our human iPSC-derived skeletal muscle cells and human iPSC-derived motor neurons.

iPSC-derived skeletal muscle cells express relevant markers

The initial steps were to confirm that our derived cells express key markers of identity, like MYH4 and 7 (Figure 1). But we also wanted to show that these cells were mature, so we looked at the expression of MyoD1 (a myogenic progenitor marker that decreases as a pure population culture matures) and found that our iPSC-derived muscle cells have a relatively low expression of the MyoD1 marker compared to the primary tissue (Figure1).

Figure 1. Axol iPSC-derived skeletal muscle cells. Cells express skeletal muscle markers when matured for 10 days from myotube progenitor cell. 

We went a little deeper here and ran some TempoSeq gene profiling to look at multiple genes. The profiles genes have been broken down in relevant categories, like NMJ development, axon guidance, or muscle membrane maintenance, for example (Figure2).

Figure 2. TempoSeq gene profiling of iPSC-derived skeletal muscle cells.

iPSC-derived skeletal cells for sarcomere structures

With cellular identity established, we went on to show our iPSC-derived skeletal muscle cells could form functional muscle tissue, i.e. sarcomeres. Using a multi-nuclei assessment of our skeletal muscle, we could look out for muscle strands with three or more multi-nuclei bodies, which is a phenotypically characteristic feature of mature muscle tissues.

The data revealed that our iPSC-derived skeletal muscle cells had, on average, five nuclei per strand (Figure 3, left). You can also clearly make out the typical Sarcomere structure, with the actin-myosin ladder structure, in plated iPSC skeletal muscle (Figure 3, middle). Furthermore, if we stress or chemically stimulate these plated cells, they contract (Figure 3, right).

Figure 3. Multi-nuclei and morphological assessment of Axol iPSC-derived skeletal muscle cells. Left: tissue displays typical Sarcomere structure. The Actin / Myosin ladder structure can be clearly observed in the plated iPSC skeletal muscle. Right: under stress or chemical stimulation, the muscles contract.

Multi-nuclei and morphological assessment of Axol iPSC-derived skeletal muscle cells. Left: Axol iPSC-derived skeletal muscle cells contain 5 nuclei per strand, on average. Middle: tissue displays typical Sarcomere structure

Successful co-culture depends on getting the media right

When it comes to co-culture you have different cell types with varying requirements of media, so getting that right can make or break a co-culture. In the work here we used our multinucleated skeletal cells alongside iPSC-derived motor neurons.

After a lot of work, we developed a complete maturation media that successfully supports the survival and growth of both iPSC cell types. We started this col-culture off in a 10cm dish with the different cell types on opposite sides of the dish – you can see 30 days of culture in the figure below (Figure 4). Compared to standard media, the skeletal muscle in the complete media were also contracting, as seen by detectable action potentials from the motor neurons (Figure 5).

Figure 4. Co-culture of Axol iPSC-derived skeletal muscle cells and motor neurons. We developed the media to enable both skeletal muscle and motor neurons to survive and mature for 30+ days in culture.

Co-culture of Axol iPSC-derived skeletal muscle cells and motor neurons. 
Multi-electrode array data of co-cultured Axol iPSC-derived skeletal muscle cells and 
Figure 5. Multi-electrode array data of co-cultured Axol iPSC-derived skeletal muscle cells and motor neurons in standard vs complete differentiation media. Contraction action potentials are only observed in the cells in the complete differentiation media. 

Putting the co-culture to work in 2D microfluidic model

With proof of the co-culture concept working, it was time to put these cells to work: in a 2D microfluidic neuromuscular junction model. We used a polydimethylsiloxane (PDMS) microfluidic chamber (Xona Microfluidics), with different cell types plated in different compartments, but connected by microgroove channels (Figure 6).

Figure 6. The polydimethylsiloxane (PDMS) microfluidic chamber (Xona Microfluidics) setup.

The polydimethylsiloxane (PDMS) microfluidic chamber (Xon

The co-culture of motor neurons and skeletal muscle cells in the microfluidic chamber were allowed to mature and form synapses.

We stained with NeuN (yellow), labeled alpha-bungarotoxin as an indicator of synapse formation (green), and titin (red) to visualize muscle cells. You can see that the motor neuron nerve completely overlaps the postsynaptic acetylcholine receptors) and muscle (Figure 7).

Figure 7. Axol 2D microfluidic neuromuscular junction model. Images are 15 days post plating. Top: the co-culture showing the development of the neuromuscular junction. Bottom: close up of a single neuron. 

2D co-culture is an excellent method to study neuronal development and differentiation

As you can see from the data, by using co-culture of human iPSC-derived skeletal muscle cells and motor neurons in a 2D microfluidic chamber, you can develop a powerful in vitro model to study NMJ development and behavior. Being able to separate the cells in the chamber means you can make a clear distinction between cell types and markers, making it easier than ever to look deeper into cellular innervation and synapse development.

If you’d like to try a similar co-culture in your own lab, have a look at our Human iPSC-derived Motor Neurons.

Skeletal cells coming very soon – register your interest to be the first to know.

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