Wnt-YAP interactions in the neural fate of human pluripotent stem cells and the implications for neural organoid formation

Figures & data

Figure 1. Illustration of the interactions of extracellular microenvironment (soluble factors, matrices etc.) with Wnt/β-catenin signaling through biochemical regulation. The effects of Wnt activation (e.g., Wnt 3A or CHIR99021) and inhibition (e.g., DKK1 or IWP4) on pluripotent stem cell (PSC) lineage commitment are shown. GSK-3β: glycogen synthase kinase-3β; TCF: T-cell factor; LEF: lymphoid enhancer factor. FGF, fibroblast growth factor; HGF: Hepatocyte growth factor; IGF: Insulin-like growth factor; TGF, transforming growth factor.

TABLE 1. Summary of the role of Wnt signaling in pluripotent stem cell fate decisions.

Applications	The role of Wnt signaling	Characterization	Ref.
Pluripotency and reprogramming	Activation of Wnt signaling with Wnt3a or GSK inhibitor BIO maintains self-renewal of hESCs	Expression of pluripotent markers in a short-term culture.	Sato et al, 2004 [49].
	Wnt activation (Wnt3a) promotes the reprograming of the fibroblasts into iPSCs	Addition of Wnt3a can reprogram fibroblasts in the absence of c-Myc. About 20-fold more drug resistant colonies were formed in the presence of Wnt3a.	Marson et al, 2008 [50]
	Wnt/β-catenin promotes mouse Episc (epiblast stem cells) and hESC self-renewal when stabilized β-catenin is retained in the cytoplasm.	Using Wnt activator CHIR or Wnt inhibitor IWR-1 to determine how β-catenin localization affects the pluripotency of hESCs. β-catenin translocation into the nucleus induces differentiation.	Kim et al, 2013[51]
Mesoderm and cardiovascular differentiation	Temporal modulation of Wnt signaling promotes mesoderm and cardiac differentiation of PSCs	CHIR activation of Wnt signaling followed by the Wnt inhibition with IWP4 results in the efficient generation of high purity of cardiomyocytes (>90%).	Lian et al, 2012 [17]
	Efficient differentiation of hPSCs to endothelial progenitors by Wnt activation.	CHIR activation of Wnt signaling without the following Wnt inhibition results in the efficient generation of endothelial progenitors. Additional magnetic purification step is required.	Lian et al, 2014 [27]
Ectoderm and neural differentiation (early induction)	Inhibition of Wnt signals promotes anterior character and enhances neurectodermal differentiation in embryoid bodies (EB).	Using Wnt3a for activation and DKK1 for inhibition. Characterized by EB structure (polarity) and the expression of Pax6, Otx2, Sox1 ectoderm genes.	ten Berge et al, 2008 [16]
	Wnt^low hPSCs are prone to neuroectodermal induction.	Wnt^high cells differentiated into endodermal and cardiac cells (FOXA2^+), while Wnt^low cells differentiated into neuroectodermal cells (PAX6^+ and Otx2^+)	Blauwkamp et al, 2012 [9]
Neural tissue patterning (from rostral to caudal identity)	Wnt inhibition (using DKK1 or XAV-939) in combination with SHH signaling promotes ventral patterning of telencephalic neural precursors	In combination with the dual SMAD inhibitors, XAV-939 treatment increases FOXG1 and Nkx2.1 expression (with forebrain/midbrain identity).	Nicoleau et al, 2013 [18]
	Regulate the heterogeneity of hPSC-derived NPCs and the subsequent neural differentiation along rostral and caudal identity.	With high endogenous Wnt signaling, NPCs adopt a posterior neural fate by expressing PITX2, FGF8, IRX3, HOXB4, the markers for hindbrain/spinal cord, low Wnt signaling enriches forebrain neurons.	Moya et al, 2014 [19]
	Activation of Wnt signaling promotes motor neuron differentiation from hPSCs	In the presence of Wnt activator CHIR, motor neuron progenitors (>95% Oligo2^+) were generated in 12 days. Spinal motor neurons (74% ISL1^+HB9^+, use CHIR 3 μM and RA 1 μM) were generated in 14 days.	Du et al, 2015 [20] Maury et al, 2015 [22]

TABLE 2. Wnt antagonists and agonists.

Category	Molecules	Action mechanism	References
Wnt antagonists	Dickkopf (DKK) family, e.g., DKK1	Inducing internalization and degradation of low-density lipoprotein receptor-related protein 6 (LRP6) through transmembrane Kremen proteins	MacDonald et al, 2009 [23]
	WISE/SOST family	The LRP5/6 ligands/antagonists, disrupt Wnt-induced Frizzled-LRP6 complex	MacDonald et al, 2009 [23]
	Inhibitors of Wnt-production, e.g., IWP2, IWP4	Prevents palmitoylation of Wnt proteins by Porcupine	Chen et al, 2009 [24]
	XAV939	A Tankyrase inhibitor that inhibits Wnt/β-catenin signaling.	Davidson et al, 2012 [14]; Nicoleau et al, 2013 [18]
	Inhibitors of Wnt-response, e.g., IWR-1	An antagonist which inhibits Wnt/β-catenin through the stabilization of Axin	Chen et al, 2009 [24]
Wnt agonists	Wnt ligands (canonical), e.g., Wnt3a	Bind to Frizzled receptor or its coreceptor LRP6 to activate Wnt pathway	Azzolin et al, 2014 [12].
	Wnt ligands (non-canonical), e.g., Wnt5a	Wnt activation independent of β-catenin	Park et al, 2015 [13]
	GSK inhibitors, such as CHIR99021	Activate Wnt through GSK-3β inhibition	Lian et al, 2012 [27]; Maury et al, 2015 [22]
	Norrin	A specific ligand for Frizzled 4	MacDonald et al, 2009 [23]
	R-spondin	Exhibit synergy with Wnt, Frizzled receptor, and LRP6; a ligand for Kremen and antagonizes DKK/Kremen interactions	Astudillo et al, 2014 [29]

Figure 2. Illustration of the interactions of extracellular microenvironment (matrix rigidity, geometry etc.) with Wnt/β-catenin signaling through biophysical regulation. Rigid matrix increases the RhoA and Rac which activate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and phosphatidylinositol 4-kinase (PI4K). These kinases synthesize the phosphoinositide lipid (PIP2) which enhances Wnt signaling. Rigidity also results in elevated membrane type 1 matrix metalloproteinase (MT1-MMP) expression, a transcriptional target of β-catenin/T-cell factor (TCF), and thereby promotes Wnt signaling. Dvl: Dishevelled.

TABLE 3. Summary of the role of YAP in stem cell fate decisions.

Function	Culture systems	Characterization	Ref.
Cell density and YAP (organ size control)	Heart General organs	High cell density induces YAP phosphorylation and cytoplasmic translocation, reduce proliferation and restrain organ size.	Zhao et al, 2011 [11] Heallen et al, 2011 [45]
Stress fibers and YAP	NIH 3T3 cells, MTD-1A cells. YAP regulates cell density-dependent control of proliferation.	Disruption of stress fibers using cytochalasin D reduces nuclear YAP. Inhibiting myosin II contractility using Blebbistatin or the inhibition of ROCK signaling using Y27632 to decrease the actomyosin contractility also reduce nuclear YAP	Wada et al, 2011 [41]
YAP/TAZ in hPSC self-renewal and reprogramming	hESC self-renewal YAP mediated by Rho signaling	Stiff surface based on glycosaminoglycan-binding hydrogels promoted the nuclear localization of co-activator YAP, which supports the self-renewal of hPSCs	Musah et al, 2012; [35]. Ohgushi et al, 2015 [40]
	Reprogramming	Activation of YAP can increase reprogramming efficiency and prevents differentiation of PSCs	Ramos et al, 2012 [36]
YAP/TAZ can control the switch between cardiac and endothelial lineages	The proliferation and differentiation of Sca-1^+ adult cardiac progenitor cells on substrates with different stiffness	YAP silencing (or soft substrates) correlated to the down-regulation of cardiomyocyte genes and the up-regulation of endothelial-specific genes as well as matrix metalloproteinase genes.	Mosqueira et al, 2014 [39]
YAP cytoplasmic retention to promote neural differentiation	Polydimethylsiloxane micropost arrays to control the stiffness of substrates. hPSC motor neural differentiation.	Inhibition of Hippo/YAP signaling on soft surface accelerates motor neuron differentiation (PAX6^+ cells at day 6: 60–70% vs. 20–30% for 5 kPa vs. 1200 kPa; 33% Tuj^+ and 18% HB9^+ at day 23 vs. 10% Tuj^+ and 3% HB9^+ cells).	Sun et al, 2014 [37]
	hPSC neural differentiation on the soft substrate.	Neuronal specification of hPSCs was enhanced in the absence of neurogenic factors on soft substrate (0.7 kPa vs. 3–10 kPa) that inhibits nuclear YAP localization.	Musah et al, 2014 [38]
YAP for mechanical memory behavior	Poly(ethylene glycol) hydrogels with different elastic modulus (stiffness) (2–10 kPa), MSC osteogenic differentiation and adipocyte differentiation.	Mechanical dosing of MSC on stiff surface bias the differentiation into osteogenic lineage. YAP/TAZ acts as intracellular rheostat, storing the information from past physical environments.	Yang et al, 2015 [42]
Bio-directional regulation of YAP and Wnt	Wnt activation (Wnt3a, GSK3 inhibitor) leads to the nuclear YAP localization. β-catenin dependent. Tested HEK293, MSCs, and ESCs.	In the absence of Wnt signaling, YAP/TAZ is sequestered to the destruction complex in the cytoplasm, which consists of a central protein named Axin, GSK3, β-catenin, as well as YAP/TAZ. This destruction complex is used to degrade β-catenin.	Azzolin et al, 2014 [12]
	Wnt activation (by Wnt5a) leads to nuclear YAP localization. β-catenin independent. Tested osteogenic and adipocyte differentiation. HEK293A cells, 3T3-L1 cells.	Activation of YAP/TAZ inhibits Wnt/β-catenin signaling. YAP/TAZ depletion abolishes Wnt4-induced enhancement of osteogenic differentiation. YAP/TAZ are mediators of alternative Wnt signaling.	Park et al, 2015 [13]

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