Poster Title:
Protocol Development for the Mass Production of hiPSC-Derived SOX6+ A9 Dopaminergic Neurons Using a High-Dimensional Design-of-Experiments Approach
Poster Presentation
Presenter:
Nooshin Amini, PhD
Scientific Director, Trailhead Biosystems
Objective:
Developing an unbiased and data-driven method to systematically approach generation of A9 dopaminergic neurons using HD-DoE® technology in a reasonable timeframe and budget.
Why A9 Dopaminergic Neurons?
Parkinson’s Disease (PD) affected over 8.5 million people in 2019 alone and has the fastest-growing population of affected individuals of any neurological disorder (WHO). PD is characterized by the loss of the A9 subset of Dopaminergic Neurons (DN). While efforts have been made using mouse models to identify drugs and test cell therapies involving brain tissue grafts, one major barrier to this has been the lack of available DNs for drug discovery screening.
Video Transcript
Hi, my name is Nooshin, I’m a scientific director at Trailhead Biosystem.
In this poster, I’m going to explain Trailhead’s approach to generate iPSC-derived dopaminergic neurons that are SOX6+.
This population is important because this is the main population that is lost in Parkinson’s disease patients.
Here at Trailhead, we use directed differentiation protocols to differentiate human induced pluripotent stem cells to different cell types.
To develop these protocols, we use High-Dimensional Design of Experiments (HD-DoE®) technology.
This technology allows us to design unbiased and mathematically driven experiments, screening a large number of inputs including small molecules and proteins, and studied their individual and combinatorial effects on cell behavior.
Here, to generate A9 dopaminergic neurons, we first focus on committing the cells to the midbrain region.
Therefore, we optimize the expression of genes of the region, such as FOXA2, LMX1A and OTX2.
Next step, we focus on optimizing expression of SOX6 transcription factor to make sure that we have the correct progenitors.
Later, the cells were terminally differentiated to dopaminergic neurons. Mature neurons express markers such as tyrosine hydroxylase, dopamine transporter, KCNJ6 and neurofilament marker.
We also use flow cytometry to look at the purity of the culture, and also performed bulk RNA sequencing to understand the gene profile of cells at different time points during the differentiation.
We also looked at the dopamine release from the cells using ELISA assay, and when compared to other commercially available cells, we learned that our cells can produce dopamine at a significantly higher concentration compared to these other cells that are available right now.
It was very important to us to ensure we don’t have contamination of other cell populations in this region, specifically A10 dopaminergic neurons that express Calbindin-1 (CALB1).
Therefore, we stained the cells with this marker and confirmed that our mature dopaminergic neurons don’t express Calbindin-1.
At this point, we wanted to check the robustness of the protocol.
Therefore, we tested the protocol on another iPSC line and confirmed that the generated cells, similar to the original line, are more than 70% TH positive, and we have less than 10% expression of CD44, which is a glial marker.
Lastly, we wanted to transfer our protocol to the bioreactors so that we can produce these cells at a larger scale.
We were able to translate our protocol to the suspension culture successfully and generated cells that were validated using immunocytochemistry, flow cytometry and RT-PCR.
Similar to the adherent culture, we don’t have expression of Calbindin-1 in these cells, and more than 70% of the cells are TH positive.
Next, we are going to look at the function of the cells using electrophysiology and make an inventory of the cells so they can be available to researchers that want to do drug screening, disease modelling or cell therapy related to Parkinson’s disease.