National Institute for Basic Biology

Cell Biology

LABORATORY OF STEM CELL BIOLOGY

Associate Professor

TSUBOUCHI, Tomomi

Specially Appointed Assistant Professor:
KURASHIMA, Kiminori
SOKENDAI Graduate Student: KUMAZAKI, Taisei
MATSUMOTO, Akino
Technical Assistant:
ASAI, Yuriko
NAGANUMA, Maipresence of replication obstacles. However, the direct cause
and the underlying mechanism remains to be uncovered. To
date, studies on cell cycle regulation in ES cells have notbeen as straightforward compared to that of other cell types,
as many commonly used cell-synchronization protocols areineffective for ES cells. We have now established several
protocols to synchronize ES cells (Tsubouchi et al., Cell,2013; unpublished), which has allowed us to investigate
specific stages of the ES cell cycle. So far, we have foundthat DNA replication is regulated differently in ES cells,
to the extent that DNA replication of the whole genome ismore accurate in ES cells. We are currently aiming to address
how such differences are interlinked with pluripotency bycarrying out side-by-side analyses between ES cells and differentiated cells.

II. Induction of Pluripotency through Nuclear
Reprogramming and Role of Cell Cycle
Regulation
Pluripotent stem cells (PSCs) are defined as stem cells
capable of producing all cell types that compose our body.
In vivo, PSCs appear only transiently during the early stageof development and are lost as development proceeds.
Molecular features that underlie pluripotency have been
extensively studied, and accumulating evidences suggest thatthe unique cell cycle regulation of PSC is tightly linked tothe maintenance of pluripotency. Curiously, they proliferatewith truncated gap phases while lengths of the S (DNA replication) and M (mitosis) phases remain similar to non-pluripotent cell types. In particular, the G1 phase of PSCs needs tobe kept short to maintain pluripotency: artificial elongationof the G1 phase makes them susceptible to differentiation.
On the other hand, active proliferation with truncated gap
phases can cause cellular stress as in some cancer cells,
leading to genome instability. Our laboratory is interested in
the role of cell cycle regulations in the maintenance of pluripotency and genome integrity of PSCs.

In mammals, PSCs are not maintained in a fully-developedorganism. However, differentiated cells can regain pluripotency upon experimental trigger, albeit at a low efficiency.
Factors that limit active reprogramming, and conditions
that potentiate reprogramming, are the subjects under activeinvestigation.
One of the main obstacles when investigating molecularmechanism underlying reprogramming is the time it takes tostart seeing any sign of reprogramming. In order to overcome
this problem, we are taking advantage of the cell-to-cellfusion approach, in which a target cell is fused to a pluripotent stem cell to induce pluripotency within a target nucleus,
in a short time frame. The cell fusion system is a simple, versatile way to induce reprogramming towards another lineage,
and is not limited to pluripotency. Using this system, the firstsign of reprogramming can be detected within one day afterfusion, thus allowing us to monitor the initial events of reprogramming after induction.
Using this system, we previously found that DNA synthesis is an important event for successful reprogramming(Tsubouchi et al., Cell, 2013). Recent reports indicate that
reprogramming may cause genetic instability, some of which
are thought to arise as DNA replication errors. To investigate
the nature of such errors and how they are linked to repro-

Visual overview of this lab’s work.

I. Self-renewal of Embryonic Stem Cells andTheir Genome-Maintenance Mechanisms

Embryonic stem (ES) cells are derived from the blastocyst
stage of embryonic development, and are capable of differentiating into all of the cell types that compose our body(i.e., ES cells are “pluripotent”). Recent reports suggest that,compared to other cell types, DNA replication machineryof ES cells proceed at lower speed, a feature generally infer24

Figure 1. Cellular fusion to study reprogramming: a human lymphoblastoid nucleus can be induced to undergo nuclear reprogramming towards
pluripotency upon fusion with mouse ES cells (green). Lamin B1 isendogenously tagged with GFP in ES cells, allowing us to distinguish ES
vs lymphoblastoid nucleus (unpublished).

gramming-specific events, we have set up a system to isolateand track a single fused cell (Figure 1) through live-imaging.

III. Future Perspectives

While the fundamental mechanisms that maintain genome
integrity have been widely studied using various models, thedanger a cell might face when its identity is being altered(through differentiation, reprogramming etc.) are largely
unknown. Recent studies of cancer genome sequencing
repeatedly identified mutations in the factors that govern
cellular identities, leading us to hypothesize that cells may
experience genome instability when their identity is unstable.
Our goal is to gain a comprehensive understanding of how
genome integrity is maintained in ES cells and cells undergoing reprogramming.

Publication List:〔Review article〕

T., and Pereira, C.-F. (2021). Reprogramming Stars #1:• Tsubouchi,
Genome Programming Through the Cell Cycle-An Interview with Dr.
Tomomi Tsubouchi. Cell. Reprogram. 23, 153–157. DOI: 10.1089/
cell.2021.29039.tt

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