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Development of a single-cell Ampli-seq based method for determining the
stages in cellular differentiation of embryonic stem cell to dopaminergic neuron

Jooyeon Lee , Seong-Ho Park , Woojeung Song ,Hee-chang Moon,Yohan Oh & Dokyoung Kim & Junho K. Hur *
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1. Department of biomedical science, Graduate School of Biomedical Science & Engineering, Hanyang University, Seoul, Korea
2. Department of medicine, in medical genetics, Graduate School, Hanyang University, Seoul, Korea
3. Department of Genetics, College of Medicine, Hanyang University, Seoul, Korea
4. Department of Anatomy and Neurobiology, College of Medicine, Kyung Hee University
(A) (B)
Introduction
Parkinson's disease (PD) is a chronic progressive degenerative
disease of the nervous system caused by the loss of dopaminergic
neurons. 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 (slow movement), tremors at
rest, muscle stiffness, and postural instability. While there is no cure (C) (D)
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 (iPSc) of a patient [1].
In this study, the differentiation of the stem cells into dopaminergic Figure 2. Gel electrophoresis of 8 markers at the bulk level
neurons were conducted in distinct stages that were distinguished In bulk, only 8 of the 47 markers we selected are selected and used as
using a fluorescent probe. However, the cellular information from a control. Pick 2 markers that are up in stages 1, 2, 3, and 4. (A) Stage
fluorescence-based labeling was insufficient for precise distinction 1 (D1) gel electrophoresis for 8 markers. (B) Stage 2 (D2) gel
between the differentiation stages. To address the challenge, we electrophoresis for 8 markers. (C) Stage 3 (D3) gel electrophoresis for
8 markers. (D) Stage 4 (D4) gel electrophoresis for 8 markers.
conducted transcriptome analyses of four major stages, and selected (A)
45 gene markers that showed distinct expression patterns between (B)
the stages of cellular differentiation. Single-cell level multiplexed
sequencing (Ampli-seq) that enabled us to analyze the select key
genes at lower cost compared to single-cell mRNA sequencing. We
conducted single-cell Ampli-seq to differentiation cells and compared
the results with commercially available Ampli-seq panel provided by
Illumina. In summary, we found that single-cell Ampli-seq enabled
distinguishing the stages of stem cell differentiation to dopaminergic
neuron at the single-cell level. Figure 3. Single cell sorting
(A) Using the mFP marker CORIN and the ATP target probe AAP-1
Result probe, single cells were sorted into 96 wells through FACS. (P5:
CORIN+, AAP-1+++, P6: CORIN +, AAP-1+, P8: CORIN -,AAP-1+++,
P9: CORIN -, AAP-1+). (B) Check the presence of single cells with
(A) (B) GAPDH. As a result of checking with GAPDH, it was confirmed that
single cells were present.
Log2 CPM heat map (Custom1)
(A) (B) (C) (D)

(C) C 1 C 2 C 3 C 4
Log2 CPM heat map (Custom2)

C C C C
1 2 3 4
(D)
Figure 4. Gel electrophoresis of 8 markers at the single level
Check 8 markers in a single cell. (A) P5 gel electrophoresis for 8
Log2 CPM heat map (Custom3)
Log2 CPM heat map (Custom3)
markers. (B) P6 gel electrophoresis for 8 markers. (C) P8 gel
Figure 1. Ampli-seq custom panel heat map electrophoresis for 8 markers. (D) P9 gel electrophoresis for 8
(A) RNA-seq of stage 1,2,3,4. According to markers. The target size is 150bp, but a lot of non-specific bands
RNA seq. we select markers 47 expressed in appeared. Because it is a single cell level, the amount of cDNA is
each stage. (B)-(E) NGS was performed with C 1 C 2 C 3 C 4 small, so the primer can be attached to other places, so the result
custom panels ordered by Illumina. Heat map of (E) may be like this. However, it seems that there is no problem in
markers expressed in Stage1 (B), Stage2 (C), Log2 CPM heat map (Custom4) distinguishing the stages as the target band is the lightest.
Stage3 (D) and Stage4 (E). In the heat map,
down-regulated for each stage is shown in blue, Reference
and up-regulated is shown in red. It seems that
stages 1, 2, 3, and 4 can be distinguished 1. Schweitzer, J. S., et al. (2020). "Personalized iPSC-Derived Dopamine Progenitor
through each marker. C 1 C 2 C 3 C 4 Cells for Parkinson’s Disease." New England Journal of Medicine 382(20): 1926-
1932.