Biomedical Engineering Reference
In-Depth Information
the primitive endoderm lineage (Poon et al., 2006). These data suggest that
TGFb/activin/nodal supports the undifferentiated state of hESCs with bFGF
by preventing differentiation.
The canonical wnt/b-catenin pathway supports self-renewal of both mESCs
and hESCs (Hao et al., 2006; Ogawa et al., 2006; Singla et al., 2006). Wnt
signaling is endogenously activated in ESCs and is downregulated upon differ-
entiation. WNTs are secreted glycoproteins that have widespread roles in tissue
differentiation and organogenesis. The Wnt pathway consists of two distinct
components. In the canonical WNT pathway, binding of WNT to a Frizzled/
LRP-5/6 receptor complex leads to a signaling cascade responsible for the
accumulation and nuclear translocation of b-catenin, which regulates transcrip-
tion in the nucleus. Activation of the Wnt pathway with wnt3a-conditioned
media (but not with purified wnt3a protein) supports the undifferentiated
phenotype in ESCs and sustains expression of the ESCs-specific markers,
such as Oct4, Nanog, REX1 in the absence of LIF (Sato et al., 2004). BIO, a
chemical inhibitor of GSK3 (glycogen synthase kinase 3) has a similar effect on
hESCs. GSK3 phosphorylates b-catenin, which then becomes a target for
ubiqitination and subsequent proteosomal degradation. In mESCs, GSK3
activation/inactivation is closely controlled by the LIF/gp130 pathway. GSK3
is suppressed by PI3K (phosphoinositide-3 kinase)-Akt through the LIF/gp130
pathway; on the other hand, GSK3 is rapidly activated upon withdrawal of
LIF. PI3K also activates Akt, which encodes a serine/threonine protein kinase.
A constitutively active form of Akt (myr-Akt) mutant maintains the undiffer-
entiated phenotypes in mESCs in the absence of LIF (Watanabe et al., 2006).
Wnt signaling upregulates STAT3 expression, suggesting that the Wnt/Frizzle
pathway balances synergistic effects with LIF/STAT3 pathway in mESCs (Hao
et al., 2006). As such, it may constitute the pathway that substitutes for the LIF
pathway in hESCs. Furthermore, the canonical Wnt signaling pathway has
been shown to elevate the level of c-myc, a target gene of STAT3. Interestingly,
GSK3 phosphorylates c-myc (Sato et al., 2004) to promote its degradation.
GSK3 inhibited by BIOmay prevent c-myc degradation by also blocking c-myc
phosphorylation by GSK3. Thus, these Wnt and LIF pathways may converge
on c-myc through STA3 and GSK3. However, recent report suggests a different
probability. The cooperative interactions of the canonical Wnt/b-catenin, Acti-
vin/Nodal, and BMP signaling pathways for hESCs define the differentiation
toward mesoendoderm/endoderm instead of supporting self-renewal and plur-
ipotency (Sumi et al., 2008). Interestingly, in this chapter, the GSK3 inhibitor
BIO supported self-renewal only at low concentrations, whereas BIO promi-
nently induces nuclear translocation of b-catenin and mesoderm differentiation
in hESCs at high concentrations. The authors observed the same effect with
overexpression of catenin/ER (estrogen receptor) fusion protein by various
4OHT (4-hydroxy-tamoxifen) concentrations. At lower concentrations, which
anticipate modest activation of b-catenin, hESCs were seemingly maintained in
a self-renewal state, despite weak induction of mesoderm markers, whereas at
higher concentrations of 4OHT undifferentiated hESCs were abolished. This
