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2.3. Signaling pathway activation crucial role in the differentiation of urothelial epithelium.
In fallopian tube organoids, inhibiting Notch signaling,
2.3.1. Wnt
through the γ-secretase inhibitor dibenzazepine (DBZ),
The Wnt/β-catenin pathway regulates stem cell pluripotency affects organoid growth and cell fate decisions. After DBZ
and plays a crucial role in cell proliferation, differentiation, treatment, 78 genes were significantly downregulated,
and embryonic development. Wnt, a secreted glycoprotein, including Eph4A, RNF43, SMO, and FZD receptors, FZD2
is synthesized intracellularly and transported to the and FZD7. The downregulated genes were enriched in
membrane, forming a protein complex with the membrane- “stem cell characteristic” genes essential for maintaining
bound Frizzled (FZD) receptors and LRP5/6 co-receptors. stem cell states, resulting in significant fold changes and
The dimerization of this receptor activates kinases that alterations in global gene expression patterns within the
phosphorylate the LRP tail and recruit the scaffold protein organoids. These findings suggest that Notch-dependent
Axin. β-catenin is a protein that mediates cell-cell adhesion regulation in fallopian tube organoids overlaps with the
43
through E-cadherin and participates in signal transduction. ISCs network, indicating shared regulatory mechanisms
In its inactive state, it is sequentially phosphorylated by CK1 between the two systems. 51
and GSK3, followed by ubiquitination and degradation by
β-TrCP/Skp. Typically, free β-catenin levels are maintained 2.3.3. BMP
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at low intracellular levels to prevent excessive malignant BMP4 are signaling proteins critical for cell growth,
proliferation. However, in the membrane-bound complex, differentiation, and development. Initially recognized
phosphorylated LRP inhibits GSK3 activation, stabilizing for their key functions in bone and cartilage formation,
intracellular β-catenin levels. This stabilization promotes its BMPs are now known to be essential in organ development
nuclear translocation, a critical step in initiating the Wnt/β- and stem cell regulation. BMP signaling is primarily
catenin signaling pathway. During the determination of categorized into two; the classical BMP/Smad pathway and
45
cortical neuron subtype proportions, downregulation of the non-classical pathways involving MAPK and PI3K.
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β-catenin increases SATB2 expression in the subcortical Bustamante-Madrid et al. analyzed the effects of BMP4
53
+
region of the brain, aligning its proportion with TBR1 and Notch signaling blockade on organoids generated
neurons and affecting the layered expression patterns from adjacent normal tissue of patients with colorectal
between the cortical and subcortical regions of the brain. cancer. They verified BMP4 responsiveness by detecting
46
In liver organoid culture, single Lgr5 stem cells can clonally phosphorylated SMAD1/5/8 proteins and the expression
+
expand into organoids that retain many characteristics of of BMP4 target genes, ID2 and DKK1. The activity of DBZ
the original epithelial tissue. This expansion requires the was validated by detecting the decreased expression of the
Wnt agonist Rspo1 in the culture medium, which serves as Notch target gene HES1. The results showed that BMP4
a ligand for Lgr5. 47 plays a primary role in promoting the differentiation of
intestinal cells into intestinal epithelial cells, while Notch
2.3.2. Notch signaling strongly inhibits the mucus-secreting cell
The Notch signaling pathway plays a crucial role in maintaining pathway. In inner ear organoids, BMPs exhibit a dual
53
stem cell gene expression and driving the differentiation role in stem cell differentiation. Under specific conditions
+
of intestinal Lgr5 stem cells. These intestinal stem cells (e.g., SB-431542 [SBB] treatment), BMPs promote the
(ISCs) can be cultured homogeneously under various formation of non-neural epithelial cells, while under other
conditions that provide essential microenvironmental conditions (e.g., SB-431542 [LSB] treatment), they inhibit
signals, such as Notch and Wnt signaling, supporting neural epithelial cell formation. Specifically, treatment with
rapid proliferation and long-term self-renewal. In the LSB (a TGF-β inhibitor) alone upregulates non-neural
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cultivation and formation of multilineage liver organoids, markers, such as TFAP2A and DLX3, without inducing
Kim et al. utilized hepatic stellate cell-like cells to promote CDX2 expression. However, co-treatment with BMP4 and
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the formation of functional microvasculature within the SBB promotes the expression of TFAP2A, DLX3, and the
organoids relying on Notch pathway-mediated endothelial extraembryonic marker CDX2. Conversely, co-treatment
interactions. This microvasculature facilitates the delivery with the BMP inhibitors, LDN-193189 and LSB, leads to
of oxygen and nutrients to the inner layers of the organoids, the upregulation of neuroectodermal markers, such as
thereby preventing necrotic core formation. Similarly, PAX6 and N-cadherin, and the suppression of TFAP2A and
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pharmacological studies analyzed the role of the Notch ECAD expression, indicating that BMP activity plays a role
signaling pathway in organoid differentiation. Notch in inhibiting non-neural differentiation. 54
inhibitors increased the expression of basal markers
mRNA, upregulated TP63, and significantly reduced 2.3.4. TGF-β
luminal cell markers (such as Upk1b, Upk2, Upk3a, and TGF-β is a homodimeric protein primarily composed of
Krt20 mRNA), indicating that Notch signaling plays a two identical subunits, each with a molecular weight of

Volume 1 Issue 2 (2025) 5 doi: 10.36922/or.8262