Page 60 - ebook

[B. Cell Biology/Stem Cell] B-30

Development of a single-cell AmpliSeq based method for

determining the stages in cellular differentiation of

embryonic stem cell to dopaminergic neuron

JooYeon Lee¹, Junho Hur¹*

¹Department of Genetics, College of Medicine, Hanyang University, Seoul 04763, Korea

Parkinson's disease is a neurodegenerative disease by the loss of dopaminergic neurons in substantia nigra. It is the
second-most common neurodegenerative disorder that affects 2–3% of the population over 65 years of age.

Symptoms of Parkinson's disease include kinesia, tremors at rest, muscle stiffness, and postural instability. While
there is no cure for PD, a clinical study demonstrated a successful treatment of Parkinson's disease by delivering

dopaminergic neurons that were differentiated from induced pluripotent stem cell of a patient. In this study, the
differentiation of the stem cells into dopaminergic neurons were conducted in distinct stages that were distinguished

using a fluorescent probe. However, the cellular information from fluorescence-based labeling was insufficient for
precise distinction between the differentiation stages. To address the challenge, we conducted transcriptome

analyses of four major stages, and selected 45 gene markers that showed distinct expression patterns between the
stages of cellular differentiation. Single-cell level multiplexed sequencing that enabled us to analyze the select key
genes at lower cost compared to single-cell mRNA sequencing. We conducted single-cell AmpliSeq to differentiation

cells and compared the results with commercially available AmpliSeq panel provided by Illumina.