TY  - THES
AB  - We used an in vitro model of Pluripotent Stem Cell (PSC) development
in mice to analyze dynamic changes in transcriptomes of hundreds
of individual cells which were undergoing an induced transition from
naïve mouse Embryonic Stem Cells (mESC) towards primed pluripotent
Epiblast Stem Cells (EpiSC). <br />
The differentiation of mESCs to EpiSC-like cells takes about five
days after induction. We collected cell samples in 24-hour intervals
for four days after induction as well as untreated mESCs and primed
state EpiSCs. Single-cell isolation and scRNA-seq library preparation for
each time point were done on the commercial Fluidigm C1 platform. In
addition, we sampled C1-Cap Analysis of Gene Expression (C1-CAGE)
libraries for the same set of time points to enable detection of non-
coding RNAs (ncRNAs) such as anti-sense RNAs or enhancer RNAs. <br />
This C1-CAGE protocol was new and still undergoing optimization
at the beginning of our experiments. C1-CAGE was first published
by Kouno *et al.* (2019) and the author of this thesis contributed as a
co-author. Throughout the work on this project a data management
platform called SCPortalen was developed to share all data among
project collaborators. SCPortalen’s publication was also co-authored
by the author of this thesis (Abugessaisa et al., 2018). <br />
The combination of transcriptome datasets from two different
protocols allowed the elucidation of expression dynamics of the naïve-
to-primed stem cell conversion. We independently identified two
subpopulations of cells during the transition process with both the
Fluidigm scRNA-seq and C1-CAGE dataset. Pseudotime analysis
revealed the developmental trajectory of cells and is a powerful tool to
reliably identify developmental stages of cells without prior knowledge
of their actual stage. Among these two transition phase subpopu-
lations, one showed wide-spread repression of gene expression. The
small nuclear RNA (snRNA) *Rn7sk* was identified as one potential
regulator of this population specific phenomenon. The second subpop-
ulation shared some characteristics with primed EpiSCs such as cell
morphology and the expression of known primed state marker genes, but
it could be shown that cells from this population were still undergoing
Epithelial-Mesenchymal Transition (EMT). That is a clear sign that
these cells have not yet fully transitioned to primed pluripotent stem
cells. Interestingly, the characteristics of this subpopulation largely
match a predicted third pluripotency state called “formative” (Smith,
2017). Therefore, we believe that our dataset not only contains naïve
and primed pluripotent stem cells, but also formative pluripotent stem
cells. Thus, our dataset represents a unique resource to compare and
study this proposed formative pluripotency state. Last but not least,
we found several marker gene candidates for all developmental stages
of the naïve-to-primed transition, which will facilitate classification of
cells in future experiments. For example, we propose *Cd59a* as a highly
specific marker gene for primed EpiSCs. <br />
The results of this thesis project have also been compiled into
a manuscript for publication in a peer reviewed journal and will be
submitted soon after the submission of this thesis.
DA  - 2020
DO  - 10.4119/unibi/2945588
LA  - eng
PY  - 2020
TI  - Single-cell transcriptome analyses on developmental transitions in mouse pluripotent stem cells
UR  - https://nbn-resolving.org/urn:nbn:de:0070-pub-29455884
Y2  - 2024-11-22T02:24:51
ER  -