TRPC3 is required for the survival, pluripotency and neural differentiation of mouse embryonic stem cells (mESCs)
TRPC3 is required for the survival, pluripotency and neural differentiation of mouse embryonic stem cells (mESCs)
Helen Baixia Hao 2
Sarah E. Webb 2
Jianbo Yue 1
Marc Moreau 0
Catherine Leclerc 0
Andrew L. Miller 2
0 Centre de Biologie du Développement (CBD), Centre de Biologie Intégrative (CBI), Université de Toulouse , CNRS, UPS, Toulouse F-31062 , France
1 Department of Biomedical Sciences, City University of Hong Kong , Hong Kong , China
2 Division of Life Science and State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay , Hong Kong , China
•COVER ARTICLE• . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .March 2018 Vol.61 No.3: 253-265 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .https://doi.org/10.1007/s11427-017-9222-9 Transient receptor potential canonical subfamily member 3 (TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mouse embryonic stem cells (mESCs) and during the differentiation of mESCs into neurons. CRISPR/Cas9-mediated knockout (KO) of TRPC3 induced apoptosis and the disruption of mitochondrial membrane potential both in undifferentiated mESCs and in those undergoing neural differentiation. In addition, TRPC3 KO impaired the pluripotency of mESCs. TRPC3 KO also dramatically repressed the neural differentiation of mESCs by inhibiting the expression of markers for neural progenitors, neurons, astrocytes and oligodendrocytes. Taken together, our new data demonstrate an important function of TRPC3 with regards to the survival, pluripotency and neural differentiation of mESCs.
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Citation:
INTRODUCTION
Embryonic stem cells (ESCs) have two distinguishing
properties, they can proliferate in an unlimited manner (i.e.,
have the ability to self-renew) and they can differentiate into
any type of somatic cell (i.e. they are pluripotent)
(Bradley et
al., 1984; Beddington and Robertson, 1989)
. It is because of
these properties that ESCs have the potential for use in
cellbased therapies to treat a range of congenital, developmental,
and degenerative conditions, including neurodegenerative
diseases
(McNeish, 2004; Hipp and Atala, 2008)
. Thus,
elucidating the cellular and molecular mechanisms that
regulate neural fate determination from stem cells is a crucial
first step before they can be successfully applied to
regenerative medicine
(Liu et al., 2013; Engel et al., 2016)
.
The neural differentiation of ESCs is a dynamic and
complex process, which is regulated by combinatorial
signaling pathways, and involves intrinsic transcription factors,
cytokines, and growth factors, as well as epigenetic
modifications
(Dhara and Stice, 2008; Hayashi et al., 2008;
Chuang et al., 2015)
. In addition, Ca2+ is a versatile and
universal intra- and inter-cellular second messenger, which is
known to be vital for embryonic neurogenesis
(Berridge et
al., 2003; Leclerc et al., 2012)
. Ca2+ signaling is also reported
to be key for regulating the differentiation of ESCs into
neuronal progenitor cells. For example, antagonizing the
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activity of RyR2 (an ER-located Ca2+ release channel), CD38
(an enzyme that catalyzes the synthesis of cADPR and
NAADP), neuronatin (a SERCA2 inhibitor), or STIM1 (an
ER Ca2+ sensor), resulted in an inhibition of the neural
differentiation of mouse ESCs (mESCs)
(Yu et al., 2008; Lin et
al., 2010; Hao et al., 2014; Wei et al., 2015; Hao et al., 2016)
.
The role of several of the transient receptor potential
canonical (TRPC) channels in neural differentiation has also been
investigated. For example, the inhibition of TRPC channel
members 1 and 4 (TRPC1, TRPC4; both Ca2+ influx
channels), has been shown to impair the proliferation of
neuroepithelial cells and block neurite extension in post-mitotic
neurons derived from human ESCs (hESCs)
(Weick et al.,
2009)
. The contribution, however, of many other Ca2+
channels or pumps to the regulation of neural differentiation
of ESCs is still largely unknown. However, numerous
studies have shown that TRPC member 3 (TRPC3) is
abundantly expressed in the brain, and various attributed
functions in the central nervous system (CNS) have been
described
(Fusco et al., 2004; Amaral and Pozzo-Miller,
2007)
. For example, using a TRPC3 knockout (KO) mouse
model, it was proposed that TRPC3 mediates
mGluR-dependent synaptic transmission in cerebellar Purkinje cells,
and is crucial for motor coordination
(Hartmann et al., 2008)
.
TRPC3 is a nonselective cation channel, which is
permeable to both Na+ and Ca2+
(Lichtenegger and Groschner,
2014)
. It is proposed to contribute to downstrea (...truncated)