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Human iPSC-Derived Motor Neurons: Expert tips on best cell-culture practices to enhance your research

Axol Bioscience Ltd

Despite intensive research, there is still no known cure or standard treatment for Amyotrophic Lateral Sclerosis (ALS), a Motor Neuron Disease (MND) subtype. Researchers have traditionally used animal models (usually mice) to screen candidate compounds, but these models are now known to lack physiological relevance to the human pathology, which could limit translational drug development .

As such, it is crucial to develop physiologically relevant models that represent human neurological pathways involved in MND/ALS. Achieving this will help improve our understanding of the disease pathology as well as boost success rates in drug development. This is one of our most important research aims here at Axol, where we have leveraged advances in stem cell biology to develop motor neurons derived from human induced pluripotent stem cells (hiPSCs) .

Our ready-to-use hiPSC-derived motor neuron progenitors, developed from both healthy and patient donors, can be cultured in vitro to provide a physiologically relevant alternative to conventional animal models. Implementing this innovative technology could help you make novel discoveries about early disease mechanisms and pathogenetic pathways, as well as improve the accuracy of your proof-of-concept candidate drug screening tests to enhance success rates in drug development.

Transitioning to hiPSC-derived motor neuron progenitors takes just a few simple steps, which we outline below. For each step, we provide our top tips on best in vitro culture practices (based on our own research insights) to enable you to harness the enormous potential of our hiPSC-derived motor neuron progenitors as a research and drug screening tool.

Our top tips on culturing hiPSC-derived Motor Neuron Progenitors

Axol’s hiPSC-derived motor neuron progenitors arrive at the bench ready to use, so you don’t need to waste any time getting started. Along with the cells, you will receive a comprehensive protocol for culturing the cells , together with an optimal cell culture system. This includes motor neuron maintenance and recovery media, SureBond+ReadySet optimized surface coating for neural cultures, and Unlock cell detachment solution (all Axol Bioscience). Other recommended reagents to support motor neuron progenitor culture include retinoic acid, Brain-derived Neurotrophic Factor, Ciliary Neurotrophic Factor and ROCK inhibitor (Y-27632 2HCl).

Culturing hiPSC-derived motor neuron progenitors is typically a straightforward process. But in the unlikely event that you run into any problems, Axol’s expert customer support can ensure your experiments run as smoothly as possible. To help get you started, below we have laid out our tips on best practices at each stage of the culturing process.

Step 1: Initial culture

On Day 0, you can plate the cells in a T-25 flask or in 60mm dishes using motor neuron recovery medium with SureBond+ReadySet coating solution to optimize cell expansion over an initial one-week period. The recovery medium is mixed with lyophilized retinoic acid (RA) to a final concentration of 0.1 µM immediately before use. For the first 24 hours, the recovery medium is also supplemented with ROCK inhibitor to a final concentration of 10 µM.

Tips for Step 1

  • We recommend using T-25 flasks for plating the cells for the initial one-week expansion period, because this provides them with more space to grow compared to 60mm dishes. The motor neurons will proliferate from two million seeded cells after five days, you should expect to see a five-fold increase in cell number.
  • Do not over seed the cells, because if the confluency is too high then this can result in cell stress and potentially cell death.
  • The day after plating, and then every two days subsequently, all of the recovery medium should be replaced with fresh pre-warmed (37°C) recovery medium (without ROCK inhibitor) using a fresh aliquot of RA with each media change.
  • Once you have opened the RA ampule, it is advisable to use all the powder immediately because the product is extremely sensitive to UV light, air, and oxidizing agents. Any unused product should be protected by an atmosphere of inert gas and protected from light.
  • Before adding RA to the recovery medium, the RA solution should be prepared in 1 mM dimethyl sulfoxide (DMSO) solvent and stored in light-protected vials at -20°C. This solution cannot be stored for more than 2-3 weeks.
  • Expect the hiPSC-Derived motor neuron progenitors to grow as aggregated clumps, similarly to that shown in Figure 1.
Figure 1: Left to right: Day 1 & 3 of initial cell expansion period. It is normal to see the hiPSC-derived motor neuron progenitors growing as aggregated clumps. Day 5 provides a visual representation of when the cells should be passaged. Images provided by Lucia Marani and Prof Mark Lewis, Loughborough University.

Step 2: Cell counts and passaging

After a 5-7 day initial cell expansion period (depending on confluency), the hiPSC-derived motor neuron progenitors will be ready to passage. It is very important to count the cells at this time.

Tips for Step 2

  • After passaging, the seeding density should be higher than it is for the initial expansion period.
  • Always remember to count cells post-expansion before passaging, as this will help you to determine the correct density of cells in the receiving plate, which is 150,000-200,00 cells/cm 2 .

Step 3: Post-passaging culture

On Day 2 post-passaging, the cells are seeded onto any plate size at the correct seeding density, as determined by the cell counts. At this stage, the hiPSC-derived motor neuron progenitors should be matured for a minimum of 19-35 days in order to observe key motor neuron markers.

Tips for Step 3

  • It is important to ensure you do not grow the cells to over confluency during this stage, so that the optimum proportion of the cell culture flask/dish surface is covered by the adherent cells to achieve optimal and consistent results.
  • During this stage, it is normal for the cells to develop long axons and multiple dendrons and dendrites to carry signal transmissions to and afferent signals away, from the cell body. It is also normal to see the cell bodies aggregating with each other, as shown in Figure 2.
Figure 2: Left to right: Day 1 & 3 of initial cell expansion period. It is normal to see the hiPSC-derived motor neuron progenitors growing as aggregated clumps. Day 5 provides a visual representation of when the cells should be passaged. Images provided by Lucia Marani and Prof Mark Lewis, Loughborough University.

Step 4: Immunocytochemistry

In the post-passaging culture, immunocytochemistry (ICC) can be performed to identify hiPSC-derived motor neurons a combination of key markers can help to confirm the presence of motor neurons e.g. using antibodies to stain for choline acetyltransferase (ChAT), neurofilament protein (SMI-32), motor neuron and pancreas homeobox 1 (MNX1) (also known as HB9). The expression of other markers, including Oligodendrocyte transcription factor (OLIG2), LIM homobox 3(LIM3), Insulin gene enhancer protein (ISL1), can be used to reveal whether the maturation stages of hiPSC-Derived motor neuron progenitors are motor neuron-specific (see Table 1 for marker specificity).

Table 1 : Specificity of ICC markers that can be used to reveal the morphological characteristics and maturation stage of cultured hiPSC-derived motor neurons.
Molecular Markers   Description
LIM3   Transcription factor that is present in motor neuron progenitors and developing motor neurons
OLIG2   Transcription factor that is present in motor neuron progenitors: spinal cord motor neuron precursor marker
ISL1   Islet-1 is present in postmitotic somatic and visceral motor neurons making it an early marker for motor neurons
SMI-32   Marker for non-phosphorylated neurofilaments, of which motor neurons are rich
HB9   Transcription factor that is present in motor neurons
ChAT   Mature motor neuron marker, stains cholinergic neurons
Beta-III Tubulin (TUJ1)   Neuron-specific cytoskeleton marker

Tips for Step 4

After Day 35 post-passage, the ICC will usually reveal ISL1 and ChAT expression, as well as TUJ1, NeuN and HB9 expression, to indicate that cultured hiPSC-derived motor neuron progenitors have the typical maturation and morphology of motor neurons (see Figure 3).

Figure 3: ICC at Day 35 post-passaging showing expression of NeuN, Tau, HB9, ISL1, TUJ1 and DAPI counterstain.

Traditional animal models of MND/ALS are now recognized as a poor representation of the pathological pathways underlying this debilitating group of neurodegenerative diseases, and so may be limiting research translation and drug development.

Now that Axol has developed hiPSC-derived motor neurons, you now have access to biologically relevant in vitro models that display human neurological characteristics. Implementing these in your research could enable you to obtain novel pathogenetic insights and enhance your proof-of-principle drug screening assays to boost the success and translation of your research.

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