Download Hlutverk transforming Growth factor beta (TGFβ) í stofnfrumum úr

Hlutverk transforming Growth factor beta (TGFβ) í
stofnfrumum úr fósturvísum músa og manna
Útdráttur á íslensku
Einangrun stofnfruma úr fósturvísum manna hefur opnað möguleika fyrir stofnfrumuígræðslu
til lækninga á sjúkdómum, svo sem sykursýki, Parkinson og hjartabilum. Þau ræktunarskilyrði
sem stuðla að endurnýjun eða sérhæfingu stofnfruma úr fóstuvísum músa (mES) og manna
(hES) eru flókin og illa skilgreind. Til þess að hægt verði að nota hES frumur til lækninga á
sjúkdómum er nauðsynlegt að skilja samspil þeirra þátta sem ákvarða endurnýjun og
sérhæfingu. Flestar rannsóknir á þessu viðfangsefni hingað til, hafa verið gerðar á mES
frumum en hES frumur staðið á hakanum. Samt sem áður er ljóst að vaxtarstjórnun hES
fruma er ólík því sem gerist í mES frumum. Valið á milli þess hvort stofnfruma endurnýi sig
eða sérhæfi er ákvarðað af ýmsum vaxtarþáttum, þekktum og óþekktum. Það er því brýnt að
greina þessa vaxtarþætti og ákvarða hvernig innanfrumuboðleiðir þeirra eru virkjaðar. Fram til
þessa hefur verið sýnt fram á að þrjár innanfrumuboðleiðir í stofnfrumum úr fósturvísum músa
gegna hlutverki til að viðhalda sjálfendurnýjun. Þessar boðleiðir leiða til virkjunar
umritunarþátta sem finnast bæði í mES- og hES frumum en hversu mikið boðleiðirnar skarast
er ekki vitað.
Tilgangur þessa verkefnis er að kanna hlutverk TGFβ stórfjölskyldunnar á umritunarþætti sem
stjórna viðhaldi og sérhæfingu stofnfruma úr fósturvísum. Við athugun á vefjasérhæfðum ES
frumum munu augu okkar beinast að æðaþelsfrumum (endothelial cells) og
hjartavöðvafrumum (cardiomyocytes). Niðurstöður úr “knockout” músarannsóknum gefa til
kynna að TGFβ vaxtarþátturinn hefur bein áhrif á fósturþroskun. Með þessum
stofnfrumutilraunum er stefnt að því að kryfja til mergjar hvaða hlutverki TGFβ stórfjölskyldan
gegnir í örlögum stofnfruma úr fósturvísum músa og manna. Markmið stofnfrumurannsókna er
að nýta stofnfrumurnar sem ótakmarkaða uppsprettu til að sérhæfa þær í frumulínur sem nota
mætti til að græða í skaddaðan vef. Niðurstöðurnar munu bæta við þekkingu okkar í
stofnfrumulíffræði auk þess sem þær eru skref í framþróun stofnfrumulækninga á mönnum.
Við munum bæði nota mES og hES frumulínur (D3 mES frumulínan og viðurkenndar hES
frumulínur sem fást frá Dr. D. Melton). Samkvæmt lögum um tæknifrjóvgun (#55, maí, 1996)
er notkun stofnfrumulína úr fósturvísum sem hafa verið útbúnar erlendis leyfð og hefur
rannsakandi ennfremur fengið leyfi hjá Vísindasiðanefnd til að rækta þessar frumur.
Origin of human embryonic stem cells and their potential
Human embryonic stem cells (hES cells) are derived from embryos that are left over from in
vitro fertilization treatments. 4-5 days after fertilization, the embryo has formed a blastocyst
(fig. 1). The outer layer, trophoectoderm, gives rise to the extraembryonic tissues, such as the
placenta (Smith, 2001). The inner cells give rise to all of the cells of the new individual.
day 5
tr
ICM
Early blastocyst
Figure 1. Human blastocyst 3 days after thawing at the 8-cell stage. The trophectoderm (tr) is removed
to isolate the inner cell mass (ICM) which will grow out into ES-like colonies.
If the inner cells are carefully separated from the trophoectoderm and cultured on feeder cells,
stem cells will grow that retain their potency to form all cells of the body (Thomson et al.,
1998). ES cells have the unique ability to propagate indefinitely in the primitive
undifferentiated state while remaining pluripotent (Passier and Mummery, 2003). This unique
ability has opened possibilities for using embryonic stem cells to treat diseases such as
diabetes, Parkisons, Alzheimer and heart failure.
Upscaling technology needs to be applied to both undifferentiated and differentiated hES cells
before transplantation of differentiated hES cells comes to clinical reality.
Undifferentiated/pluripotent state of embryonic stem cells
In order to retain an undifferentiated pluripotent state in culture, ES cells depend on culture on
feeder cell layers; in mouse embryonic stem cells (MESC), these feeder cells can be replaced
by leukemia inhibitory factor (LIF), which acting through gp130, activates the transcription
factor STAT3 (Smith, 2001). Importantly, LIF is effective only in serum containing conditions
but LIF in combination with BMP4 alone is sufficient to maintain the MESC in a pluripotent
state and allow their de novo derivation in the absence of serum (Ying et al., 2003). In
contrast to MESC, LIF has no effect on human embryonic stem cells (hES cells) renewal
(Sato et al, 2004). It is essential that hES cells that will be used in human therapy will be free
of animal culture systems, in which exposure to mouse retroviruses can be avoided. The
factors produced by feeder cells, which are responsible for maintaining the hES cells in a
pluripotent state are unknown although some candidates have been proposed. Amit and
coworkers presented a novel feeder layer-free culture system for hES maintenance. This
culture is based on a medium supplemented with 15 % serum replacement, a combination of
growth factors including Transforming growth factor β1, LIF, basic fibroblast growth factor
(bFGF) and fibronectin matrix (Amit et al., 2004). hES cells are known to express growth
factor receptors including bFGF, stem cells factor (SCF) and fetal liver tyrosine kinase-3
ligand (Flt3L). Thus, Carpenter and colleges asked if these growth factors could maintain hES
cells in an undifferentiated state in the absence of conditioned medium (CM). They concluded
that bFGF supports undifferentiated hES cell growth without CM (Xu et al., 2005). Thomson et
al. extended these findings and reported that bFGF and suppression of BMP signaling sustain
undifferentiated proliferation in hES cells (Xu et al., 2005). Hayek et al. showed that ActivinA,
a member of the TGFβ superfamily, is secreted by mouse embryonic feeders (MEFs) and that
cultured medium enriched with ActivinA maintains HES cells undifferentiated without feeder
layer conditioned medium from MEFs or STAT activation (Beattie et al., 2005). This data is
supported by Hemmati-Brivanlou and colleges who reported that the TGFβ / Activin / nodal
branch is activated through the transcription factors Smad2/3 in undifferentiated cells while
the BMP branch is only activated in isolated mitotic cells (through the transcription factors
Smad 1/5) (James et al., 2005; Besser, 2004). They extended their findings by showing that
Smad 2/3 activation is required downstream of Wnt signaling, which they previously showed
to be sufficient to maintain the undifferentiation state of hES cells. Taken together, bFGF,
TGFβ branch activation Smad2/3 and Wnt signaling are all promising candidates in
maintaining undifferentiation and pluripotency in hES cells.
Recently, Nanog was identified as a transcription factor that maintains self-renewal in both
mouse and human ES cells (Chambers et al., 2003; Mitsui et al. 2003; Sato et al., 2004).
Even in the absence of LIF, ES cells overexpressing Nanog remain undifferentiated and they
do not require addition of BMP. Overexpression of Nanog was shown to sustain Id3 at high
levels in MESC, hence there may be a strong connection between the BMP signalling
pathway and Nanog activity (Ying et al., 2003). The N-terminus of Nanog is rich in Ser/Thr
residues, suggesting that it may be regulated by phosphorylation (Pan and Pei, 2003).
Furthermore, it has recently been reported that human and mouse ES cells can maintain their
pluripotency via activation of the Wnt signalling pathway and might be mediated by
transcriptional regulation of Nanog (Sato et al., 2004).
Transforming growth factor signal transduction
Transforming growth factor beta (TGFβ) is a multipotent growth factor, acting
throughout the body, where it affects most cell types. TGFβ has been implicated in regulating
cell growth, differentiation, migration, extracellular matrix, deposition and apoptosis (Shi and
Massague, 2003). TGFβ transduces signals from the membrane to the nucleus by binding to
a heteromeric complex of serine/threonine kinase receptors known as TGFβ type I (TβRI) and
type II (TβRII) receptors (figure 2). The type I receptor, also known as activin receptor-like
kinase (ALK), acts downstream of the type II receptor and propagates the phosphorylation
signal through specific downstream mediators of the Smad family, known as Smads receptorregulated R-Smads (Heldin et al, 1997; Shi og Massague, 2003). Phosphorylated R-Smads
form complexes with the common partner (Co)- Smad, and accumulate in the nucleus where
they regulate transcriptional activity of their target genes (Derynck et al., 1997). The Inhibitory
Smads (I-Smads) prevent activation of R- and Co-Smads by occupying the phosphorylation
site of the TβRI. Other members of the TGF-β superfamily are BMP that lead to activation of
Smad 1/5/8 through BMP type I and type II receptors and activin leading to Smad 2/3
activation through activin type I and type II receptors.
TGFβ
Cytoplasm
Type II
TypeI
P
Smad4
P P
R-Smad
P P
R-Smad
Smad4
P P
Nucleus
R-Smad
Smad4
TF
P
4
ad
Sm
ad
Sm
R
p300/CBP
Figure 2. The TGFβ signal transduction pathway
Bone Morphogenetic Proteins (BMPs) are members of the TGFβ superfamily. The BMP
ligands and their downstream effectors, named Smads, play an essential role in determining
ES cell fate not only by facilitating self-renewal but, in the absence of JAK/STAT activation,
causing them to adopt a mesodermal fate instead of differentiating into the ectodermal
lineage (Johansson and Wiles, 1995; Monteiro et al., 2004). BMPs have been shown to
phosphorylate Smad1/5 in both human and mouse ES cells (Pera et al., 2004; Ying et al.,
2003) and, at least in MESC, to upregulate Id proteins (Ying et al., 2003). The effect on
MESC cells appears to be specific for BMPs/Smad1/5 since TGFβ is apparently without effect
(Ying et al., 2003) although a trivial point may be that they lack specific TGFβ type II binding
receptors (Goumans et al., 1998). BMPs have been shown to upregulate Id proteins
(Inhibitors of differentiation), which in turn inhibit bHLH transcription factors, thereby blocking
differentiation of embryonic stem cells (Hollnagel et al., 1999) and exogenous expression of Id
mimicked effects of BMP on MESC. This effect has not been shown in hES cells and in fact
Thomson and colleges have shown that repression of BMP sustains undifferentiated hES
cells. Additionally, they have shown that BMP stimulation in hES cells in conditioned medium
containing bFGF promotes trophoblast differentiation. Pera and colleges reported that
blocking BMP activity in serum does not maintain hES cell self-renewal, but instead enhances
primitive endoderm differentiation (Pera et al., 2004).
Transcriptional regulation in embryonic stem cells
The choice that a stem cell makes to undergo either differentiation or self-renewal is decided
by cues of growth factors or inhibitors present in the stem cell niche. It is an emerging concept
that the response of a cell not only relies on a particular signalling pathway but rather on the
integration of signals from multiple pathways. It is therefore essential to understand how their
signalling transduction machinery is activated by means of their downstream transcription
factors that regulate target gene expression in order to maintain the ES cells' pluripotency or
to manipulate them to differentiate into the required cell type for the development of cell
based transplantation therapy. In particular, mechanism of cross-talk between the different
signal transduction pathways has been poorly investigated although its importance is clearly
illustrated by the striking result that simultaneous activation of the JAK/STAT and the Smad
pathways by exogenous LIF and BMP, respectively, is sufficient to maintain pluripotency, at
least of MESC, as described above.
For mouse embryonic stem (ES) cells, self-renewal is dependent on signals from the cytokine
leukaemia inhibitory factor (LIF) and from either serum or bone morphogenetic proteins
(BMPs). In addition to the extrinsic regulation of gene expression, intrinsic transcriptional
determinants are also required for maintenance of the undifferentiated state. These include
Oct4, a member of the POU family of homeodomain proteins (Tomilin et al., 1998) and a
second recently identified homeodomain protein, Nanog (Chambers et al., 2003)(figure 3). A
number of other genes have been implicated as markers for pluripotency, including Cripto (Xu
et al., 1999) and UTF-1 (Okuda et al., 1998). However, these genes are not expressed
exclusively by the inner cell mass of the blastocyst and their exact roles in ES cells still
remains to be determined. Interestingly, it has been suggested that UTF-1 is a target of a
TGFβ superfamily member, which might connect the puzzling data of the role of the TGFβ
family in pluripotency and differentiation as well as the discrepancy between mouse and
human ES cells together.
TGFβ/Activin/Nodal
BMP
Wnt
LIF
P-Smad1/5
Figure 3. Signalling pathways
that maintain pluripotency
in ES cells. LIF and BMP enhance
self-renewal in mouse ES cells,
Wnt and TGFβ/Activin enhance
self-renewal in human ES cells.
STAT3
P-Smad2/3
β-catenin
Id1/3
Nanog
Oct3/4
UTF-1
Differentiation of embryonic stem cells
The cascade of events involving sequential gene activation taking place during human
embryonic development is hindered by the unavailability of postimplantation embryos at
different stages of development. Fortunately, spontaneous differentiation of hES cells can
occur by means of the formation of embryoid bodies (EBs), which resemble early embryos in
some aspects. ES cells can be induced to differentiate by replacing the conditioned medium
(conditioned on fibroblasts overnight with addition of bFGF) to an unconditioned medium
(including knockout serum replacement) and allowing them to grow on a monolayer (Xu et al.,
2005) or as aggregates in suspension and form EBs, which contain a heterogenous mixture
of cell types (Feraud and Vittet, 2003); this provides an easy in vitro model for studying the
molecular mechanisms controlling differentiation and early stages of development normally
inaccessible in mammals, particularly humans.
Since Thomson and colleges succeeded in deriving hES cells, it has been shown that they
can terminally differentiate into many cell types in vitro, including insulin producing cells, heart
cells, bone cells, blood cells and liver cells.
Differentiation of ES cells into endothelial cells and cardiomyocytes
In the embryo, vasculogenesis is the process in which blood vessel formation occurs by
differentiation of vascular endothelial cells (ECs) from angioblastic precursors, which in turn
expand and coalesce to give rise to the primitive vascular plexus (Risau, 1997). In contrast,
angiogenesis refers to the process in which sprouting occurs from preexisting vessels or split
to form new vessels (Carmeliet, 2000). During vasculogenesis, embryonic vessels are
thought to be formed by endothelial cells that arise from Flk1-expressing (Flk1+) mesoderm
cells, surrounded by mural cells derived from mesoderm, neural crest, or epicardial cells. It
was believed that endothelial and vascular smooth muscle cells (VSMC) arise from separate
precursors. However, recent findings have shown that embryonic vascular progenitor cells are
capable of differentiating into both mural and endothelial cells. (Yamashita et al., 2000;
Gerecht-Nir et al., 2004). Furthermore, bone marrow derived vascular progenitors circulating
in adult peripheral blood have been shown to include progenitors that can give rise to both
types of cells and contribute to tumour angiogenesis. Tumour-derived angiogenic factors
attract these circulating endothelial precursors (CEPs) to the growing new blood vessels,
where they become incorporated into the growing vasculature (Asahara et al., 1997).
Additionally, mature vascular endothelium has been shown to give rise to SMC via
transdifferentiation, co-expressing both endothelial and SMC-specific markers (DeRuiter et
al., 1997). Endothelial embryonic cells have also been shown to transdifferentiate into cardiac
muscle cells under specific conditions (Condorelli et al., 2001). However the molecular
mechanisms that regulate their differentiation and proliferation remains to be elucidated. It
has been shown that members (Watabe et al., 2003) of the TGFβ superfamily play important
roles during differentiation of vascular progenitor cells derived from the mES cells as well as
in mouse embryonic endothelial cells (Goumans et al, 2002, 2003).
Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle
cells) is essential for development of vascular tissues and maintenance of their homeostasis
in both embryonic and adult tissues (Folkman and D’Amore, 1996). TGFβ superfamily
members and Platelet derived growth factor (PDGF) have been implicated among the
cytokines that mediate such interaction (Carmeliet, 2000).
Since Thomson and colleges succeeded in deriving hES cells, it has been shown that they
can terminally differentiate into many cell types, including ECs (Levenberg et al., 2002).
Spontanously differentiated ECs were isolated from EBs using an endothelial specific marker.
These sorted cells had purity of 80 % and showed endothelial characteristics, such as vessellike formation both in vitro and in vivo. The expression kinetics of specific endothelial markers
during spontaneous hEB formation were analysed (Levenberg et al., 2002). The levels of
endothelial markers increased during the first two weeks of hEB differentiation, reaching a
maximum on days 13-15 and indicating a differentiation process toward ECs.
Gerecht-Nir and colleagues explored the genes that were upregulated during 4 weeks of
development of EBs focusing on vascular development. Among the upregulated genes were
the members of the TGFβ superfamily, TGFβ3, TβRII, Endoglin as well as PDGFB and
PDGFRβ known to mediate the binding of SMC and ECs (Gerecht-Nir et al., 2005). In
contrast to mES cells, Flk-1 was shown to be expressed in undifferentiated hES cells
(Levenberg et al., 2002). Hence, different markers seem to play a different role in human and
mouse vasculogenesis, meaning that murine systems are not necessarily predictive of human
systems.
Our previous findings show that in endothelial cells (ECs), TGFβ can activate two distinct type
I receptor/Smad signalling pathways with opposite cellular responses. In most cell types,
TGFβ signals via the TGFβ type I receptor, ALK5. However, ECs express a predominant
endothelial type I receptor, named ALK1. Whereas the TGFβ/ALK1 signalling leads to
activation, the TGFβ/ALK5 pathway results in an inhibition of the activation state. This
suggests that TGFβ regulates the activation state of the endothelium via a fine balance
between these two pathways (Goumans et al., 2002, 2003).
We identified genes that are specifically induced by TGFβ mediated ALK1 or ALK5 activation.
Id1 was found to be the target gene of the ALK1/Smad1/5 pathway while induction of
plasminogen activator inhibitor-1 was activated only by ALK5/Smad2 pathway. Bone
morphogenetic protein (BMP) is a member of the TGFβ superfamily and signals through
Smad1/5. The BMP/Smad1/5 pathway was found to potently activate the endothelium. Id1
was identified as an important BMP target gene in ECs and was sufficient and necessary for
BMP-induced EC migration (Valdimarsdottir et al., 2002).
The isolation of endothelial cells from hES cells could prove to have potential therapeutic
implications, including cell transplantation for repair of ischemic tissues and tissue
engineering of vascular grafts. In transplantations, the newly grafted tissue needs to be
surrounded by capillaries to survive. It is therefore critical to isolate human embryonic
endothelial cells (ECs) or their precursors for such applications.
Our focus
The TGFβ superfamily members have enormous influence on ES fate decision, not only by
enhancing self-renewal but also to direct their differentiation into mesodermal lineage.
This study will be undertaken to analyze hES cells as an in vitro model for human vascular
development and further to evolve the techniques used to simplify vascular cell derivation for
future clinical applications. Because of their importance, we will use TGFβ superfamily
members to study their role on the regulation of downstream target genes and their function in
self-renewal and differentiation of hES cells into mesodermal-derived endothelial cells.
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