7 research outputs found
Study of the molecular mechanisms involved in cell differentiation of embryonic stem cells
Potential
heart
progenitors
from
postnatal
and
adult
heart
have
been
identified
in
rodents
and
humans,
and
were
shown
to
differentiate
in
vitro
into
mature
cardiomyocytes.
However,
in
some
studies
only
immature
cardiac
cells
were
obtained,
leading
to
concerns
about
whether
the
inefficient
cell
electrical
maturation
and
electrical
coupling
of
derivative
cells
will
cause
arrhythmias
upon
cellular
therapies
of
injured
hearts.
Notwithstanding
these
concerns,
recent
clinical
assays
have
indicated
an
improvement
of
heart
function
and
life
quality
of
patients
treated
with
autologous
cardiac
progenitor
cells.
With
the
discovery
of
the
capacity
of
embryonic
stem
cells
(ESC)
to
differentiate
into
all
lineages,
including
the
cardiovascular
lineage,
many
researchers
have
been
focusing
on
developing
strategies
to
efficiently
direct
ESC
differentiation
for
the
production
of
unlimited
numbers
of
cardiomyocytes.
Yet,
the
low
efficiency
of
cardiac
differentiation,
its
teratogenic
nature
and
the
lack
of
reliable
protocols
to
purify
the
desired
cells
from
ESC
cultures
are
holding
back
their
use
for
clinical
application.
Hence,
understanding
the
molecular
mechanisms
that
regulate
commitment
and
differentiation
of
diverse
muscle
and
non-‐muscle
cell
lineages
of
the
heart
is
essential
to
improve
current
therapies
and
to
design
novel
heart
stem
cell-‐
based
therapeutic
strategies
for
cardiac
regenerative
medicine.
Many
transcription
factors,
including
Nkx2.5,
Gata4
and
Isl1,
have
been
described
as
master
regulators
of
cardiac
morphogenesis.
Indeed,
they
are
required
for
the
commitment
and
differentiation
of
cardiac
progenitors
in
early
embryonic
development,
as
well
as
for
the
differentiation
of
ESC
into
cardiovascular
lineages.
Furthermore,
mutations
in
these
genes
have
been
associated
with
congenital
heart
defects
in
humans.
This
finding
underscores
the
clinical
relevance
of
studying
the
role
of
cardiac
transcription
factors
during
heart
development.
Cited2
is
a
transcriptional
regulator
essential
for
mouse
embryonic
development.
Amongst
a
wide
spectrum
of
developmental
defects
presented
by
Cited2
knockout
mouse
embryos,
the
cardiovascular
and
left-‐right
patterning
defects
are
the
most
prominent.
Importantly,
heterozygous
mutations
of
human
CITED2
in
association
with
congenital
heart
disease
have
been
reported
by
several
research
groups.
Moreover,
it
has
been
suggested
that
Cited2
is
important
for
ESC-‐derived
cardiovascular development
and
essential
in
the
epiblast
or
its
derivatives
for
normal
cardiac
left-‐right
patterning.
Although
Cited2
was
shown
to
be
required
for
the
maintenance
of
pluripotency
and
self-‐renewal
of
ESC,
its
role
in
early
embryonic
development
and
its
mechanism
of
action
are
still
not
well
explored.
In
the
present
study,
we
investigated
the
role
of
Cited2
in
mouse
ESC
differentiation
towards
cardiac
cells.
By
performing
increasing
and
absence
of
Cited2
expression
in
ESC,
we
have
established
that
Cited2
is
essential
for
cell
commitment
and
differentiation
towards
the
mesoderm,
ectoderm
and
endoderm
lineages.
Indeed,
Cited2
depletion
impaired
ESC
differentiation
to
several
cell
lineages.
More
importantly,
Cited2
was
required
for
the
normal
emergence
of
mesoderm
cells
from
early
ESC
specification
to
cardiac
lineages.
Our
results
suggest
that
this
response
is
primarily
associated
with
the
direct
or
indirect
modulation
of
Nodal
and
Brachyury
gene
expression
during
mesodermal
cell
commitment,
thus
enabling
a
signaling
cascade
promoting
cardiogenesis.
Additionally,
our
results
indicated
that
loss
of
Cited2
generated
comparatively
less
cardiomyocytes
in
differentiated
ESC
cultures,
whereas
gain
of
Cited2
led
to
higher
numbers
of
cardiomyocytes.
These
findings
suggest
Cited2
is
an
important
protein
for
cardiac
function.
Furthermore,
our
data
suggest
that
Cited2
can
modulate
the
gene
expression
(Islet1,
Gata4,
Nkx2.5
and
Tbx5)
in
cardiac
progenitors,
thus
playing
an
important
role
during
cardiac
cell
commitment.
We
have
also
found
that
Cited2
plays
a
role
in
cardiomyocyte
proliferation,
as
indicated
by
the
downregulation
of
Tbx5
and
its
targets
genes
upon
Cited2
depletion.
Furthermore,
by
overexpressing
human
CITED2
and
Gata4
in
murine
ESC,
we
found
that
Cited2
could
not
prevent
endoderm
commitment
induced
by
Gata4
overexpression.
The
same
overexpression
approach
was
used
to
test
the
role
of
CITED2-‐ISLET1
interaction
in
cardiac
differentiation,
and
the
results
indicate
that
the
cardiogenic
response
of
Cited2
might
be
additive
in
association
with
that
induced
by
Islet1
(Isl1)
overexpression.
We
have
also
found
Cited2
expression
in
multipotent
cardiac
progenitor
population,
specifically
in
secondary
heart
field
(SHF)
progenitors,
which
are
marked
by
the
expression
of
Isl1
protein.
Our
data
generated
by
increasing
and
absence
of
Cited2
Cited2
expression
in
SHF
progenitor,
suggest
that
finely
regulated
Cited2
gene
expression
levels
could
allow
or
disrupt
the
SHF-‐differentiation
program.
Moreover,
by
performing
GST-‐pull
down
and
by
bi-‐molecular
fluorescence
complementation
assays,
we
gathered
evidence
indicating
that
CITED2
physically
interacts
with
ISL1.
By performing
chromatin
immunoprecipitation
assays
we
have
also
found
that
CITED2
binds
the
Isl1
promoter
region.
Taken
together,
our
results
indicate
that
Cited2
is
important
for
proper
cardiovascular
differentiation
by
acting
at
different
levels
during
cardiac
stem
cell
commitment:
firstly,
by
inducing
ESC
towards
mesoderm
at
early
events
during
ESC
commitment;
and
secondly,
by
inducing
and
maintaining
the
expression
of
cardiac
transcription
factors
important
for
cardiogenesis.
Although
many
Cited2
functions
remain
uncovered,
our
data
indicate
that
Cited2
is
an
important
cardiac
differentiation
regulator
that
can
participate
in
heart
regeneration,
and
therefore
be
useful
for
the
development
of
alternative
approaches
to
treat
or
prevent
heart
failure,Potenciais
progenitores
cardíacos
de
coração
pós-‐natal
e
adulto
foram
identificados
em
roedores
e
seres
humanos,
e
verificou-‐se
serem
capazes
de
se
diferenciar
in
vitro
em
cardiomiócitos
maduros.
No
entanto,
em
alguns
estudos
foram
obtidos
apenas
células
cardíacas
imaturas,
assim
suscitando
questões
sobre
se
ineficiente
maturação
elétrica
celular
e
acoplamento
eléctrico
das
células
derivadas
não
causará
arritmias
no
contexto
da
aplicação
de
terapias
celulares
em
lesões
cardíacas.
Apesar
destas
questões,
ensaios
clínicos
recentes
indicaram
uma
melhoria
da
função
cardíaca
e
da
qualidade
de
vida
dos
pacientes
tratados
com
células
progenitoras
cardíacas
autólogas.
Com
a
descoberta
da
capacidade
de
células
estaminais
embrionárias
(ESC)
se
diferenciarem
em
todas
as
linhagens,
incluindo
a
linhagem
cardiovascular,
muitos
investigadores
se
têm
concentrado
no
desenvolvimento
de
estratégias
para
dirigir
eficientemente
a
diferenciação
de
ESC
para
a
produção
de
um
número
ilimitado
de
cardiomiócitos.
No
entanto,
a
baixa
eficiência
da
diferenciação
cardíaca,
a
sua
natureza
teratogénica
e
a
falta
de
protocolos
bem
validados
para
purificar
as
células
desejadas
das
culturas
de
ESC
estão
a
atrasar
o
seu
uso
para
aplicações
clínicas.
Assim,
a
compreensão
dos
mecanismos
moleculares
que
regulam
o
comprometimento
e
diferenciação
de
diversas
linhagens
de
células
musculares
e
não-‐
musculares
do
coração
é
essencial
para
melhorar
as
terapias
atuais
e
para
conceber
novas
estratégias
terapêuticas
baseadas
em
células
estaminais
para
a
medicina
regenerativa
cardíaca.
Muitos
factores
de
transcrição,
incluindo
Nkx2.5,
Gata4
e
ISL1,
têm
sido
descritos
como
reguladores
importantes
da
morfogénese
cardíaca.
Com
efeito,
eles
são
necessários
para
o
comprometimento
e
diferenciação
de
células
progenitoras
cardíacas
no
desenvolvimento
embrionário
inicial,
assim
como
para
a
diferenciação
das
ESC
em
linhagens
cardiovasculares.
Além
disso,
mutações
nestes
genes
foram
associadas
a
doenças
cardíacas
congénitas
em
pacientes
humanos.
Esta
observação
reforça
a
relevância
clínica
do
estudo
do
papel
dos
fatores
de
transcrição
cardíacos
no
desenvolvimento
do
coração.
O
Cited2
é
um
regulador
de
transcrição
essencial
para
o
desenvolvimento
embrionário
murino.
Entre
um
amplo
espetro
de
defeitos
de
desenvolvimento apresentados
por
embriões
de
ratinho
Cited2
knockout,
defeitos
cardiovasculares
e
de
padronização
esquerda-‐direita
são
os
mais
proeminentes.
É
importante
ressaltar
que
mutações
heterozigóticas
de
CITED2
humano
em
associação
com
cardiopatias
congénitas
têm
sido
relatados
por
vários
grupos
de
investigação.
Além
disso,
tem
sido
sugerido
que
Cited2
é
importante
para
o
desenvolvimento
cardiovascular
derivado
de
ESC
e
é
essencial
no
epiblasto
ou
seus
derivados
para
a
padronização
esquerda-‐direita
cardíaca.
Embora
o
Cited2
seja
necessário
para
a
manutenção
da
pluripotência
e
auto-‐
renovação
de
ESC,
o
seu
papel
no
desenvolvimento
embrionário
precoce
e
o
seu
mecanismo
de
ação
ainda
não
foram
bem
explorados.
No
presente
estudo,
investigamos
o
papel
de
Cited2
em
diferenciação
de
ESC
de
ratinho
em
células
cardíacas.
Através
da
realização
de
experiências
de
ganho
e
de
perda
de
função
de
Cited2
em
ESC,
nós
verificámos
que
Cited2
é
essencial
para
o
comprometimento
e
diferenciação
em
células
de
linhagens
de
mesoderme,
ectoderme
e
endoderme.
De
facto,
a
depleção
de
Cited2
perturbou
a
diferenciação
de
ESC
para
diversas
linhagens
de
células.
Mais
importante
ainda,
Cited2
foi
necessário
para
o
aparecimento
normal
de
células
da
mesoderme
a
partir
da
especificação
precoce
de
ESC
para
linhagens
cardíacas.
Os
nossos
resultados
sugerem
que
esta
resposta
está
principalmente
associada
com
a
modulação
direta
ou
indireta
da
expressão
dos
genes
Nodal
e
Brachyury
durante
o
comprometimento
em
célula
mesodermal,
permitindo
assim
uma
cascata
de
sinalização
que
promove
a
cardiogénese.
Além
disso,
os
nossos
resultados
indicam
que
a
perda
de
Cited2
levou
à
geração
de
menos
cardiomiócitos
em
culturas
de
ESC
diferenciadas,
enquanto
que
o
ganho
de
Cited2
levou
a
um
maior
número
de
cardiomiócitos.
Estes
resultados
sugerem
portanto
que
Cited2
é
uma
proteína
importante
para
a
função
cardíaca.
Além
disso,
nossos
dados
sugerem
que
Cited2
pode
modular
a
expressão
génica
(Islet1,
Gata4,
Nkx2.5
e
Tbx5)
em
progenitores
cardíacos,
desempenhando
assim
um
papel
importante
durante
o
comprometimento
de
células
cardíacas.
Nós
também
descobrimos
que
Cited2
desempenha
um
papel
na
proliferação
de
cardiomiócitos,
como
indicado
pela
regulação
negativa
de
Tbx5
e
seus
genes-‐alvo
na
situação
de
depleção
de
Cited2.
Além
disso,
através
de
sobreexpressão
de
CITED2
humano
e
Gata4
em
ESC,
descobrimos
que
Cited2
não
impede
o
comprometimento
em
endoderme
induzido
pela
sobreexpressão
de
Gata4.
A
mesma
abordagem
de
sobre-‐expressão
foi
usada
para
testar
o
papel
da
interação
CITED2-‐
ISLET1
na
diferenciação
cardíaca,
e
os
resultados
indicam
que
a
resposta
cardiogénica de
Cited2
será
aditiva
em
associação
com
aquela
induzida
pela
sobreexpressão
de
Islet1
(ISL1).
Também
detetámos
expressão
de
Cited2
em
populações
de
progenitores
cardíacos
multipotentes,
especificamente
em
progenitores
do
campo
cardíaco
secundário
(SHF),
que
são
marcados
pela
expressão
da
proteína
ISL1.
Os
nossos
dados
obtidos
em
experiências
de
ganho
e
perda
da
função
de
Cited2
em
progenitores
SHF
sugerem
que
níveis
de
expressão
do
gene
Cited2
finamente
regulados
podem
permitir
ou
interromper
o
programa
de
diferenciação
SHF.
Além
disso,
através
da
realização
de
ensaios
de
GST-‐pull
down
e
ensaios
de
fluorescência
de
complementação
bi-‐molecular,
obtivemos
dados
indicando
que
CITED2
interage
fisicamente
com
ISL1.
Através
da
realização
de
ensaios
de
imunoprecipitação
de
cromatina
também
descobrimos
que
CITED2
se
liga
à
região
do
promotor
de
ISL1.
Em
conjunto,
os
nossos
resultados
indicam
que
Cited2
é
importante
para
uma
diferenciação
cardiovascular
adequada,
agindo
em
diferentes
níveis
durante
o
comprometimento
de
células
estaminais
cardíacas:
em
primeiro
lugar
através
da
indução
de
ESC
para
mesoderme
em
estadios
precoces
durante
o
comprometimento
de
ESC;
e
em
segundo
lugar
através
da
indução
e
manutenção
da
expressão
de
fatores
de
transcrição
cardíacos
importantes
para
a
cardiogénese.
Embora
muitas
funções
de
Cited2
permaneçam
por
descobrir,
os
nossos
dados
indicam
que
Cited2
é
um
importante
regulador
da
diferenciação
cardíaca,
que
pode
participar
na
regeneração
do
coração
e,
portanto,
ser
útil
para
o
desenvolvimento
de
abordagens
alternativas
para
o
tratamento
ou
prevenção
da
insuficiência
cardíaca.Fundação para a Ciência e Tecnologia - bolsa
de doutoramento FCT (SFRH/BD/62054/2009)Universidade do Algarve, Departamento de Ciências Biomédicas e MedicinaMinistério
Ciência,
Tecnologia
e
Ensino
Superior - Programa
de
Investigação
em
Medicina
Regenerativa
da
Universidade
do
Algarv
Cdkn2a inactivation promotes malignant transformation of mouse immature thymocytes before the β-selection checkpoint
CDKN2A deletion is the most frequent genetic alteration in T-cell
acute lymphoblastic leukemia (T-ALL), occurring across all molecular and immunophenotypic subtypes. CDKN2A encodes two functionally unrelated tumor suppressor proteins, ARF and INK4a,
which are critical regulators of cell cycle and proliferation. Arf has
been reported to suppress T-ALL development in post−b-selection
thymocytes, but whether CDKN2A acts as a tumor suppressor gene
in immature, pre−b-selection thymocytes remains to be elucidated.
Resorting to a Rag2-deficient model of T-ALL, driven by the ETV6::
JAK2 fusion, we report that Cdkn2a haploinsufficiency at early
stages of T-cell development facilitates leukemia developmentPPBI-POCI-01-0145-FEDER-022122; POCI-01-0145-FEDER-007274; NORTE01-0145-FEDER-000029info:eu-repo/semantics/publishedVersio
StemChecker: a web-based tool to discover and explore stemness signatures in gene sets
Stem cells present unique regenerative abilities, offering great potential for treatment of prevalent pathologies such as diabetes, neurodegenerative and heart diseases. Various research groups dedicated significant effort to identify sets of genes-so-called stemness signatures-considered essential to define stem cells. However, their usage has been hindered by the lack of comprehensive resources and easy-to-use tools. For this we developed StemChecker, a novel stemness analysis tool, based on the curation of nearly fifty published stemness signatures defined by gene expression, RNAi screens, Transcription Factor (TF) binding sites, literature reviews and computational approaches. StemChecker allows researchers to explore the presence of stemness signatures in user-defined gene sets, without carrying-out lengthy literature curation or data processing. To assist in exploring underlying regulatory mechanisms, we collected over 80 target gene sets of TFs associated with pluri- or multipotency. StemChecker presents an intuitive graphical display, as well as detailed statistical results in table format, which helps revealing transcriptionally regulatory programs, indicating the putative involvement of stemness-associated processes in diseases like cancer. Overall, StemChecker substantially expands the available repertoire of online tools, designed to assist the stem cell biology, developmental biology, regenerative medicine and human disease research community. StemChecker is freely accessible at http://stemchecker.sysbiolab.eu.Portuguese Fundacao para a Ciencia e Tecnologia (FCT) [RH/BPD/96890/2013, SFRH/BPD/70718/2010, PTDC/BIA/GEN/116519/2010, IF/00881/2013, PTDC/BIA-GEN/116519/2010]; Programa Doutoral ProRegeM-Mecanismos de Doenca e Medicina Regenerativa [PD/00117/2012]info:eu-repo/semantics/publishedVersio
Acute Loss of Cited2 Impairs Nanog Expression and Decreases Self-Renewal of Mouse Embryonic Stem Cells
Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.National Portuguese through FCT-Fundacao para a Ciencia e a Tecnologia [PEst-OE/EQB/LA0023/2013, PTDC/SAU-ENB/111702/2009]; Camara Municipal de Oeiras; Merck Sharp & Dhome Foundation-Portugal; CRUK; Leukaemia & Lymphoma Research; Kay Kendall Leukaemia Fund; Wellcome Trust; Medical Research Council; Cancer Research UK [12796, 14633]; Great Ormond Street Hospital Childrens Charity [W1062]; Medical Research Council [MC_U137973817, G1000801g, MC_qA137913
Estudio del efecto de los principales componentes del medio de cultivo agotado de Azotobacter vinelandii sobre la produccion de alginato y su peso molecular
Alginate Biosynthesis in Azotobacter vinelandii
The Gram-negative bacterium Azotobacter vinelandii can synthetize the biopolymer alginate that has material properties appropriate for plenty of applications in industry as well as in medicine. In order to settle the foundation for improving alginate production without compromising its quality, a better understanding of the polymer biosynthesis and the mechanism of regulation during fermentation processes is necessary. This knowledge is crucial for the development of novel production strategies. Here, we highlight the key aspects of alginate biosynthesis that can lead to producing an alginate with specific material properties with particular focus on the role of oxygen availability linked with the molecular mechanisms involved in the alginate production
NF-kappa B-dependent RANKL expression in a mouse model of immature T-cell leukemia
Activation of the receptor activator of nuclear factor-kappa B (RANK) by its ligand (RANKL) is involved in both solid and hematological malignancies, including multiple myeloma, acute myeloid leukemia and B-cell leukemia. Although RANKL expression has been described in normal T cells, a potential role in T-cell leukemia remains undefined. Here, we used a model of immature T-cell leukemia/lymphoma, the TEL-JAK2 transgenic mice, to assess RANKL expression in leukemic cells and its regulatory mechanisms. We found that Rankl mRNA was significantly overexpressed in leukemic T cells when compared to wild-type thymocytes, their nonmalignant counterparts. Moreover, Rankl mRNA and RANKL surface expression in leukemic cells was induced by T-cell receptor (TCR) signaling activation, dependently on the NFKB signaling pathway. These results indicate that TCR-activated leukemic T cells express high levels of RANKL and are potential inducers of RANK signaling in microenvironmental cells. (C) 2019 Elsevier Inc. All rights reserved.Fundacao para a Ciencia e a Tecnologia (Portugal)Portuguese Foundation for Science and Technology [PTDC/SAU-OBD/103336/2008, UID/BIM/04773/2013, POCI-01-0145-FEDER-007274, SFRH/BD/75137/2010, IF/00056/2012]European Regional Development Fund through COMPETE 2020 programEuropean Union (EU) [NORTE-01-0145-FEDER-000029