The Wnt/β-catenin pathway comprises a family of proteins that play critical roles in embryonic development and adult tissue homeostasis. The deregulation of Wnt/β-catenin signalling often leads to various serious diseases, including cancer and non-cancer diseases. Although many articles have reviewed Wnt/β-catenin from various aspects, a systematic review encompassing the origin, composition, function, and clinical trials of the Wnt/β-catenin signalling pathway in tumour and diseases is lacking. In this article, we comprehensively review the Wnt/β-catenin pathway from the above five aspects in combination with the latest research. Finally, we propose challenges and opportunities for the development of small-molecular compounds targeting the Wnt signalling pathway in disease treatment. Notably, the Wnt/β-catenin signalling pathway is necessary for embryonic development and adult tissue homeostasis regeneration. Abnormal regulation of the pathway is closely associated with different diseases, suggesting that the Wnt/β-catenin signalling pathway is an attractive target for disease treatment. First, this article reviews non-tumour diseases and conditions related to the Wnt/β-catenin signalling pathway, such as hair loss, pigment disorders, wound healing, bone diseases, neurodegenerative diseases, and chronic obstructive pulmonary diseases. Second, we comprehensively summarize tumour diseases based on their biological features, including cancer stem cells, cancer metastasis, cancer metabolism, and cancer immunity. Finally, we review the role of Wnt/β-catenin in the diagnosis and clinical treatment of cancer and non-cancer diseases.Since the first member of the Wnt family was identified in 1982,Drosophila. Because the two genes and functional proteins are similar, researchers combined the terms as the Wnt gene.1 The Wnt signalling pathways include noncanonical and canonical pathways. The noncanonical Wnt pathways are independent of β-catenin-T-cell factor/lymphoid enhancer-binding factor (TCF/LEF), such as the Wnt/Ca2+ pathway and noncanonical Wnt planar cell polarity.2 The canonical Wnt pathway, also known as the Wnt/β-catenin pathway, involves the nuclear translocation of β-catenin and activation of target genes via TCF/LEF transcription factors. The canonical Wnt pathway mainly controls cell proliferation, whereas the noncanonical Wnt pathways regulate cell polarity and migration, and the two main pathways form a network of mutual regulation. Wnt signalling plays an important role in the self-renewal of some tissues in mammals. For example, the Wnt signalling pathway is related to the development and renewal of small-intestinal epithelial tissue and promotes the differentiation of Paneth cells at the base of the crypt. In addition, the Wnt signalling pathway is closely related to liver metabolism and regeneration, lung tissue repair and metabolism, hair follicle renewal, haematopoietic system development, and osteoblast maturation and activity.5The Wnt gene was originally derived from integrase-1 in mouse breast cancer and the wingless gene of 6The Wnt/β-catenin pathway comprises four segments: the extracellular signal, membrane segment, cytoplasmic segment, and nuclear segment. Extracellular signals are mainly mediated by Wnt proteins, including Wnt3a, Wnt1, and Wnt5a. The cell membrane segment mainly contains the Wnt receptors Frizzled (specific sevenfold transmembrane receptor Frizzled protein) and LRP5/6. The cytoplasmic segment mainly includes β-catenin, DVL, glycogen synthase kinase-3β (GSK-3β), AXIN, APC, and casein kinase I (CK1). The nuclear segment mainly includes β-catenin, which translocates to the nucleus, TCF/LEF family members, and β-catenin downstream target genes, such as MMPs and c-Myc.7The canonical Wnt pathway is usually highly conserved and activated via the binding of extracellular Wnt ligands to membrane receptors by autocrine/paracrine methods. Once activated, the typical Wnt pathway induces the stability of β-catenin and transfers it to the nucleus, ultimately facilitating the expression of genes involved in cell proliferation, survival, differentiation, and migration Fig. .7Fig. 1WIn the absence of Wnts, the transmembrane receptors FZD and LRP5/6 are located on the plasma membrane separately. In the cytoplasm, a “destruction complex” comprising adenomatous polyposis coli (APC), AXIN, casein kinase 1 (CK1) and glycogen synthase kinase 3 protein (GSK3 protein) captures β-catenin by phosphorylating CK1 and GSK3, thus activating the process of β-catenin degradation. Therefore, GROUCHOU, which binds to TCF/LEF, inhibits the transcription of target genes.9When Wnts are recognized by FZD and LRP5/6, the “destruction complex” is recruited to the cell membrane by interacting with FZD, which loses the ability to degrade β-catenin. The induced β-catenin translocates to the nucleus and activates the transcription of target genes by interacting with TCF/LEF. The cytoplasmic-nuclear shuttling of β-catenin is considered an important feature of Wnt/β-catenin pathway activation.Here, we review the diseases caused by abnormal Wnt/β-catenin signalling and the underlying mechanism, and we discuss the function of the components and regulators of the Wnt/β-catenin signalling pathway.In this section, we first outline the components Fig.  and func10 Among them, Wnt3a and Wnt1 act as ligands to activate the Wnt/β-catenin signalling pathway after translation and modification. Posttranslational modification of Wnts mainly includes lipid modification and glycosylation, and lipid modification is performed by PORCN. PORCN, located in the endoplasmic reticulum, can add palmitoleate groups to Wnt proteins, and it is a member of the membrane-bound O-acyltransferases (MBOATs). Lipid modification is necessary for Wnt activity, and the opposite is true for glycosylation.11 WLS encodes a multipass transmembrane protein that localizes to the Golgi, is recycled between the Golgi and plasma membrane12 and transports lipid-modified Wnts from the endoplasmic reticulum to the cell surface.Wnts are secreted proteins, and 19 Wnt proteins have been identified in the human body.13 The cysteine-rich domain (CRD) is an extracellular domain at the N-terminal side of FZD proteins and is considered the site of interaction with Wnt ligands. The fully conserved KTxxxW domain is an intracellular domain at the C-terminal side of FZD proteins and interacts with the protein-95/discs-large/zona occludens-1 (PDZ) domains of DvL proteins.14 LRP5/6 are coreceptors of Wnts and are located on the plasma membrane. The intracellular part of LRP5/6 contains multiple phosphorylation sites, the phosphorylation of which is a key step in the initiation of signal transduction via Wnt/β-catenin signalling.15 DVL proteins located in the cytoplasm are pivotal in the transduction of WNT signalling from the receptor level to the intracellular compartment and contain three highly conserved domains: the DIX domain at the N-terminal side (the site interacts with AXIN), the PZD domain in the central part, and the DEP domain at the C-terminal side.16 When the Wnt ligand binds to the receptor, most of the DVL is recruited to the plasma membrane. Activated DvL guides the clustering of FZD and LRP6 and promotes the phosphorylation of LRP6.19 In addition, activated DVL recruits AXIN and GSK-3β to the plasma membrane and inhibits their functions.20 The DEP domain not only binds to FZD but can also change its conformation and thereby dimerize, triggering the WNT/FZD signalosome.14The other main components of the Wnt/β-catenin signalling pathway include FZD, LRP5/6, DvL, AXIN, APC, GSK-3β, CK-1α, β-catenin, and TCF/LEF. FZD proteins are a family of seven transmembrane (TM) receptors and are located on the plasma membrane.14 The APC protein is located in the cytoplasm, and mutations in this protein cause Wnt pathway activation in human cancers. Current models of APC action emphasize its role as a component of DCs to promote β-catenin degradation. Previous studies have shown that APC2 can only compensate for the role of APC as a component of DCs in promoting β-catenin degradation.21 Saito-Diaz et al. found that APCs transduce Wnt signals through clathrin-mediated endocytosis via the clathrin endocytic pathway without the Wnt ligand in APC-depleted cells. Concurrently, they proposed that APC inhibited the formation of constitutive, ligand-independent, clathrin-dependent signalosomes and the unnecessary activation of the Wnt pathway.22 Wnt ligands induce the phosphorylation of LRP6 receptors, mainly mediated by GSK-3β and CK-1α, and dual phosphorylation promotes the binding of LRP6 to AXIN. GSK-3β and CK-1α also induce the phosphorylation of β-catenin in the absence of Wnt ligands. Phosphorylated β-catenin is released from DCs and bound by Trcp-1  for ubiquitination and subsequent degradation.23When the Wnt/β-catenin signalling pathway is in the off-state, a β-catenin destruction complex (DC) is formed in the cytoplasm, comprising AXIN, the APC protein, GSK-3β, CK-1α, and β-catenin. AXIN functions as a scaffold protein because it contains binding domains for other components of the DC, such as the DIX domain at the C-terminal side and RGS domain at the N-terminal side (the site interacts with the APC protein).The canonical Wnt signalling pathway is regulated by many factors. In addition to the canonical molecules in the Wnt/β-catenin signalling pathway mentioned above, many other molecules also affect the signal transduction of the Wnt/β-catenin signalling pathway.25 Glypicans are located on the plasma membrane and bind to Wnt proteins to regulate their extracellular distribution and signal activity. Glypican-3 may have a CRD domain that interacts with the middle region of Wnt3a. Glypican-3 also promotes the coordinated activation of Wnt signalling when FZD is expressed at high levels. This effect is mainly caused by the interaction between GPC3 and FZD through the heparan sulfate (HS) chain.26 Both Tiki and Notum act as enzymes to inactivate Wnt proteins.28sFRPs, WIF, glypicans, Tiki, and Notum are well-known regulators of Wnt/β-catenin signalling that target Wnt proteins to affect signal transduction. sFRPs have a Netrin-related motif in the C-terminal region, WIF has a WIF domain in the C-terminal region, and both bind to Wnt proteins, thereby inhibiting Wnt signalling.30 Norrin interacts with both the CRD domain of FZD4 and ectodomain ECD of LRP5/6 by simulating the finger-like loop of Wnt (the structure of Wnt interacts with the CRD domain of FZD), thereby activating the Wnt/β-catenin signalling pathway.25 DKK-1/3/4 prevent canonical Wnt signalling by binding to LRP5/6 or further forming a tertiary complex with the single transmembrane receptors Kremen 1 and 2, which induce the endocytosis of LRP5/6.31 However, DKKs are downstream targets of Wnt/β-catenin signalling, suggesting a negative feedback mechanism to govern WNT signalling.33In addition to targeting Wnts, some regulators can directly or indirectly activate FZD and LRP5/6 receptors, such as R-spondins, Norrin, and DKKs, and all of them are secreted proteins. The binding of LGR4/5 and R-spondin initiates the formation of a complex with RNF43/ZNRF3 and consequently inhibits the activity of Rnf43/Znrf3. Rnf43/Znrf3 are E3 ubiquitin ligases that are located on the plasma membrane and induce multiubiquitination of lysine. Rnf43/Znrf3 can ubiquitinate FZD, inducing FZD endocytosis and resulting in the downregulation of Wnt signalling.34ROR and RYK are located on the plasma membrane, and both act as coreceptors of Wnt ligands in the Wnt/β-catenin signalling pathway. ROR-1 and ROR-2 have an extracellular CRD domain that closely resembles the CRD domain of the FZD receptor. RYK also has an extracellular domain that is homologous to Wnt inhibitory factor (WIF). Hence, ROR and RYK can compete with the Wnt ligand to inhibit activation of the Wnt/β-catenin signalling pathway.35 In 2004, Kazanskaya et al.36 found that R-spondin2 enhanced Wnt/β-catenin signalling in Xenopus embryos, and mouse Rspodin1-3 also had a similar effect. By inducing the receptor LGR4/5 to bind to Znrf3/Rnf43, R-Spondin, the negative feedback regulator of Wnt, removes the two E3 transmembrane ligases from the membrane, thus weakening the ubiquitination of Frizzled and significantly increasing the transmission of Wnt/β-catenin signals.37 Norrin protein can also bind to Frizzled class receptor 4 (FZD4) and LRP5/6 to form a ternary complex and promote the downstream signal transmission of Wnt/β-catenin.39Spondin is a powerful Wnt agonist. The R-spondin protein family comprises four members with similar domains (Rspodin1-4).40However, the Wnt/β-catenin signalling pathway is not only positively activated by the above factors but also inhibited by many negative feedback regulators, such as Notum, Tiki, DKKs, Wise/SOST, IGFBP-4, sFRPs, WIF1, and ZNRF3/RNF43. Some other molecules also regulate Wnt signalling by processing components in the pathway, such as PP2A and PP1. PP1 and PP2A bind to AXIN and APC, respectively, resulting in the dephosphorylation of AXIN and β-catenin. The dephosphorylation of β-catenin contributes to β-catenin accumulation in the cytoplasm, playing a positive role in Wnt signal transduction.In addition to the above regulatory factors, crosstalk between Wnt/β-catenin and other pathways also regulates canonical Wnt signalling in different ways (Table 4).42MicroRNAs are noncoding endogenous small RNAs. By pairing the 3′-untranslated regions (3′-UTRs) of target mRNAs, microRNAs participate in the regulation of Wnt/β-catenin signalling, and different miRNAs have different effects on the Wnt/β-catenin signalling pathway.43 Similarly, miR-31 directly inhibits the negative regulatory factors of Wnt, namely, DKK1, GSK3β, and AXIN1, to stimulate canonical Wnt signalling.45 In oesophageal squamous cell carcinoma, miR-455-3p participates in the activation of canonical Wnt signalling by simultaneously targeting and inhibiting multiple negative regulatory factors  of the Wnt/β-catenin signalling pathway.46 miR-29 directly inhibits four factors in the Wnt/β-catenin pathway .47 In CD133+ liver cancer stem cells (CSCs), miR-1246 inhibits AXIN2 and GSK3β, two important components of the β-catenin DC.48 MiR-128-3p simultaneously inhibits the expression of AXIN1, SFRP2, and WIF1 to enhance the activity of the Wnt/β-catenin signalling pathway, thus promoting non-small cell lung cancer (NSCLC) metastasis.49 MiR-374a promotes breast cancer metastasis by specifically inhibiting the expression of WIF1, PTEN, and WNT5A, which are negative regulators of Wnt.50 MiR-22-3p and miR-22-5p act together on negative regulatory factors (SFRP2 and PCDH15) of Wnt signalling, resulting in Wnt signalling pathway activation.51 APC is an important component of the degradation complex. However, miR-106a-3p, miR-494, and miR-155 directly target APC, promoting the accumulation of β-catenin in the nucleus and upregulating the active transcription of the target genes c-Myc and cyclin D1.54 Highly expressed miR-92a-3p reduces the ubiquitin-mediated degradation of β-catenin by directly inhibiting FBXW7 and MOAP1 and promoting the progression of colorectal cancer (CRC).55 MiR-182-5p directly inhibits the expression of FOXO3a, preventing its binding to β-catenin, enhancing the interaction between β-catenin and TCF4, and then promoting canonical Wnt signalling.56Some miRNAs enhance the Wnt/β-catenin pathway by inhibiting the negative regulatory factors of the Wnt/β-catenin pathway. miR-100 and miR-125b synergistically inhibit five Wnt antagonists .57 MiR-384-5p suppresses the expression of FZD1, FZD2, Tgrbr1, and LRP6 and decreases the phosphorylation of GSK3βser9 and β-catenin, preventing myocardial fibrosis induced by Wnt activation.58 MiR-4496 directly targets β-catenin to downregulate its expression.59 MiR-181a-5p inhibits the 3′-UTR activity of β-catenin and TCF4, thus reducing the expression of cyclin D1 and AXIN 1, target genes of the Wnt/β-catenin pathway, and plays a tumour suppressor role in CRC.60 In hepatoma cells, miR-125b-5p directly targets the downstream gene STAT3 to enhance β-catenin phosphorylation.61 In infertility and abortion, miR-200c negatively regulates FUT4 to inhibit α1-catenin on CD44, inactivating the downstream Wnt/β-catenin signalling pathway, and reducing the proliferation and adhesion of endometrial cells.62 MiR-708-5p upregulates the expression of the tumour suppressor CDH1 and inhibits the Wnt/β-catenin pathway via the interaction between CDH1 and β-catenin.63Other miRNAs play a negative role in the regulation of the Wnt/β-catenin pathway. MiR-199a-5p acts on Wnt pathway signalling factors , upregulating the expression of AXIN2, MYCA, and CCND1 and inhibiting the Wnt/β-catenin pathway.64 In metastatic tissues of lung cancer, lncRNA-JPX inhibits the expression of GSK-3β and promotes the transfer of β-catenin from the cytoplasm to the nucleus.65 In addition, lncRNA-LALR1 in hepatocytes reduces the expression of AXIN1 by recruiting CTCF from the promoter of AXIN1. The decreased expression of AXIN1 suppresses β-catenin phosphorylation, enhancing the transcription of c-Myc and Cyclin D1.66 The lncRNAs Linc00210 and lncRNA-CCAL inhibit the activity of CTNNBIP1 and activator protein-2α (AP-2α), respectively, and block the binding of CTNNBIP1 and AP-2α to β-catenin, enhancing the interaction between β-catenin and the TCF/LEF complex.68 LncRNA-NEAT1 interacts with DDX5 and promotes the formation of a complex between DDX5 and β-catenin, enhancing gene transcription.69 Lnc-β-Catm is a highly transcribed lncRNA in hepatocellular carcinoma that combines with β-catenin and EZH2 to promote β-catenin methylation and further enhance β-catenin stability.70 In addition, some lncRNAs indirectly regulate the Wnt/β-catenin pathway by targeting microRNAs. The lncRNAs MIR100HG and MIR22HG are the host genes of miR-100/125b and miR-22-3p/5p, respectively, activating the Wnt pathway by upregulating the expression of these miRNAs.51 The lncRNA CRNDE enhances Wnt/β-catenin pathway signalling by inhibiting miR-181a-5p expression.40 In gastric cancer, the novel lncRNA LINC01133 targeting miR-106a-3p plays an indirect role in inhibiting the Wnt signalling pathway.52LncRNAs also have a strong regulatory effect on Wnt/β-catenin, and most lncRNAs, such as FAST, JPX, LALR1, Linc00210, CCAL, NEAT1, and Lnc-β-Catm, directly or indirectly enhance the stability of β-catenin, thus activating the canonical Wnt pathway. In human embryonic stem cells (hESCs), the conserved lncRNA-FAST binds to the WD40 domain of the E3 ubiquitin ligase β-TrCP, inhibiting the ubiquitination of β-catenin by β-TrCP, resulting in the accumulation of β-catenin in the nucleus.Recently, several potential regulators of the Wnt/β-catenin signalling pathway have been gradually identified, such as Twa1, FOXKs, ICAT, and Kdm2a/b.71 found that Twa1 activates the Wnt/β-catenin signalling pathway by facilitating β-catenin nuclear retention in a zebrafish model. Twa1 ) is mainly located in the cytoplasm. Twa1 is targeted by the AXIN complex for ubiquitination and degradation in the absence of Wnt signalling. When Wnt pathway activation occurs, Twa1 translocates to the nucleus, and its conserved CRA (CT11-RanBPM) domain is required for its interaction with β-catenin, which promotes β-catenin nuclear retention and increases Wnt/β-catenin downstream target gene expression.71Lu et al.72 The forkhead-associated (FHA) domain and hydrophobic motif (Leu-137-Phe-145-Phe-154) within its adjacent region on FOXK2 are both required for the interaction of FOXKs and DVL2, and the interaction is regulated by DVL phosphorylation. The association of FOXK with DVL can be enhanced by Wnt signalling and is consistent with the improved DVL nuclear translocation capability, which plays a positive role in regulating the Wnt/β-catenin signalling pathway.72FOXKs promote Wnt/β-catenin signalling by translocating DVL into the nucleus. The forkhead box (FOX) transcription factors FOXK1 and FOXK2 are nuclear proteins that are crucial for the transport of DvL into the nucleus.73Inhibitor of β-catenin and TCF (ICAT) is an 81-amino-acid β-catenin-binding protein initially identified as an inhibitor of β-catenin signalling because its overexpression blocks the formation of the β-catenin-TCF complex. Recently, Ji et al. found that ICAT is a natural inhibitor of APC that plays a positive role in Wnt signal transduction by blocking APC-mediated degradation of β-catenin. On the one hand, when the WNT pathway is activated, ICAT blocks the interaction of β-catenin-APC and inhibits the degradation of β-catenin by competing with APC for the binding site of β-catenin. On the other hand, ICAT also inhibits β-catenin degradation by blocking the β-catenin-mediated APC-AXIN interaction. Considering how Wnt signalling inhibits the function of the β-catenin DC is not entirely clear, Ji et al. conjectured that Wnt might hinder the function of the Wnt-induced DC by inhibiting the direct interaction between APC and AXIN.74Kdm2a/b regulate Wnt/β-catenin signalling by modulating the methylation/demethylation of nuclear β-catenin. Kdm2a/b are demethylation modification enzymes located in the nucleus. When Wnt is activated, nonphosphorylated β-catenin enters the nucleus and is methylated at lysine residues within the fourth and fifth Arm repeats. Subsequently, the modified β-catenin forms a complex with the TCF/LEF transcription factor to activate transcription. To stop signal transduction, Kdm2a/b competes with TCF/LEF for β-catenin binding and removes methyl markers from β-catenin, which is subsequently degraded via ubiquitination. Notably, the destruction of the β-catenin/TCF complex by Kdm2a/b inhibits Wnt signalling even without demethylation.The Wnt/β-catenin pathway plays critical roles in embryonic development and adult tissue homeostasis. The dysregulation of Wnt/β-catenin signalling often leads to many serious diseases, including cancer and non-cancer diseases.The Wnt/β-catenin pathway participates in the physical and pathological processes of the development of different organs and related diseases, including the development of the lung, angiocarcinoma, bone, neurons, and the liver and their corresponding diseases Fig. .Fig. 3WnCommon lung diseases, including chronic obstructive pulmonary disease (COPD), pulmonary inflammation, idiopathic pulmonary fibrosis (IPF), hyperoxia injury, bronchopulmonary dysplasia (BPD) silicosis, and lung cancer, are closely related to the Wnt/β-catenin pathway.75 Two types of epithelial cells are found in alveoli: alveolar type 1 and type 2 cells (AT1 and AT2). AT1 cells mediate gas exchange, and AT2 cells secrete surfactants to maintain alveolar morphology.76 During airway differentiation, inhibition of the Wnt/β-catenin pathway contributes to the rapid directional differentiation of pluripotent stem cells into proximal airway epithelial cells, while activation of the Wnt/β-catenin pathway promotes formation of the distal airway epithelium.80 Alveoli are the main sites for gas exchange in the lungs, and the Wnt/β-catenin pathway plays a positive regulatory role in the development and differentiation of alveoli. LGT5+ mesenchymal cells located in alveoli can secrete Wnt3a to activate canonical Wnt signalling and promote alveolar differentiation of epithelial progenitor cells.81 Notably, Wnt5a, a common nonclassical Wnt pathway activator, after secretion by fibroblasts, activates the classic Wnt signalling pathway in AT2 cells and inhibits the transdifferentiation of AXIN2+ AT2 cells with stem cell activity into AT1 cells, thus maintaining the dryness of AT2 cells.83 When the epithelium is injured, activated Wnt/β-catenin signalling converts AXIN2+ myofibrogenic progenitor cells (AMPs) into mesenchymal alveolar niche cells (MANCs). MANCs and stable β-catenin promote the self-renewal of AT2 cells, facilitating alveolar regeneration.85 In PM2.5-induced lung injury, knockout of RAB6 activates the Wnt/β-catenin signalling pathway by inhibiting the secretion of DKK1, thus promoting the self-renewal of type II alveolar epithelial cells (AEC2) during lung repair.86 Folliculin (FLCN) deletion in pulmonary mesenchymal cells is the leading cause of cystic lesions in Birt-Hogg-Dubé (BhD) syndrome. A significant decrease in the activity of the Wnt/β-catenin signalling pathway was detected in lung tissues with FLCN knockout, indicating that inhibition of the Wnt signalling pathway is related to the abnormal development of alveoli.87Canonical Wnt signalling plays an important role in the development and differentiation of the lung. It promotes the formation of the airway and alveoli.Together, the results show that the Wnt/β-catenin pathway promotes the differentiation of airway epithelial cells and AT2 cells, as well as the regeneration of alveoli.Normal lung development and differentiation are inseparable from the regulation of the Wnt/β-catenin signalling pathway, but the imbalance of this pathway often promotes the occurrence of some lung diseases, including tumour and non-tumour diseases. This section focuses on the role of the Wnt/β-catenin pathway in non-tumour lung diseases.88 Wnt/β-catenin signalling activity is reduced in the lung epithelial cells of COPD patients, and the mechanisms responsible for its downregulation include cigarette smoke and fibroblast-derived Wnt5a. (1) Cigarette smoke is the main risk factor for COPD. Cigarette smoke can reduce the expression of Frizzled receptor 4 (FZD4) in alveolar epithelial cells and enhance the phosphorylation of β-catenin, inhibiting epithelial proliferation and alveolar repair mediated by the classic Wnt signalling pathway.5 (2) The expression of fibroblast-derived Wnt5a is increased in COPD. However, after Wnt5a glycosylation, it negatively regulates Wnt/β-catenin signal transduction by reducing the stability of β-catenin, weakening wound healing and AT2 transdifferentiation.89 Notably, active Wnt/β-catenin signalling by the activator LiCl promotes the proliferation and survival of alveolar epithelial cells and improves alveolar repair, reducing the occurrence of emphysema, further confirming that Wnt/β-catenin reduces the occurrence of COPD.91 Recently, a Wnt-targeted RNA sequencing study of the COPD airway epithelium showed that the canonical Wnt signalling pathway is activated in COPD, which inhibits airway epithelial differentiation and barrier function and induces the upregulation of EMT-related genes.92COPD is a chronic disease characterized by chronic airway inflammation, airway remodelling, airflow obstruction, and loss of the alveolar parenchyma.93 In IPF, abnormally activated Wnt signalling is detected, which manifests as follows: significantly increased expression of Wnt1, Wnt7b, Wnt10b, FZD2, FZD3, β-catenin, and LEF1 in the lung tissue, thereby promoting fibroblast proliferation and epithelial cell-mesenchymal transition, the pathological process of IPF.94 Abnormal activation of the Wnt/β-catenin pathway occurs in IPF fibroblasts, which stimulates the proliferation of mesenchymal cells and promotes the process of fibrosis.95 In addition, serum Kl6/mucin-1 (Muc1) overexpression in IPF mediates the activation of β-catenin and leads to the formation of the Muc1-cytoplasmic tail (cT)/β-catenin nuclear complex, which promotes the formation and proliferation of myofibroblasts.96In contrast to COPD, the Wnt/β-catenin pathway is activated in the lung epithelium of IPF. IPF is a disease characterized by the progressive loss of lung function distinguished by severe pulmonary epithelial injury, fibroblast activation, extracellular matrix deposition, and distorted lung development.+ perivascular macrophages, causing excessive activation of the Wnt/β-catenin pathway in PCECs. Activated Wnt/β-catenin further promotes the expression of Jag1 in PCECs, stimulating Notch signalling in fibroblasts and enhancing fibrosis.97 Hyperoxia injury during lung development upregulates the expression of Wnt2b, Wnt5a, Wnt9a, and Wnt16, leading to excessive activation of Wnt/β-catenin in fibroblasts and AT2 cells, characterized by the accumulation of β-catenin and an increase in AXIN2 expression.98 Although active Wnt signalling is necessary for alveolar regeneration, it is strictly regulated by the body. Saccular stage hyperoxia injury not only decreased the expression of the alveolar formation genes FOXM1, MYB and MCM2 but also overactivated Wnt/β-catenin signalling. In particular, the highly upregulated Wnt5a in mesenchymal cells further mediates the occurrence of bronchopulmonary dysplasia (BPD) with impaired alveolar formation and septal thickening.100 In acute lung inflammation, the canonical Wnt signalling pathway in alveolar epithelial cells is usually inhibited by platelet-derived Dickkopf-1 (DKK1). Activated Wnt/β-catenin signalling inhibits the inflammatory response specifically as follows: ICAM-1/VCAM-1-mediated adhesion of neutrophils and alveolar epithelial cells is reduced, inhibiting the inflammatory response.101In addition, repeated lung injury inhibits the expression of chemokine receptor CXCR7 on lung capillary endothelial cells (PCECs) and recruits VEGFR1102Cardiovascular diseases, with high morbidity and mortality, are common and threaten human health. Wnt/β-catenin signal transduction is very active in the development and pathological remodelling of the cardiovascular system. The occurrence and development of many cardiovascular diseases are related to abnormal regulation of the Wnt/β-catenin signalling pathway.103 However, several studies have shown that activated Wnt/β-catenin signalling also play a vital role in causing the deterioration of atherosclerosis and coronary heart disease by promoting the calcification of vascular SMCs and valve sclerosis.106 Arterial calcification is an important signal for cardiovascular disease, and inhibition of over-activated Wnt/β-catenin signalling may be a feasible strategy to modify vascular calcification and delay disease progression. Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. R-spondins (RSPOs) are a family of four secretory proteins (RSPO1 to RSPO4). In atherosclerotic arteries, RSPO2 is enriched. Mechanistically, RSPO2 inhibits PI3K-AKT-eNOS signalling via LGR4.107Atherosclerosis is characterized by the accumulation of lipid and fibre components on the arterial wall and leads to clogged or narrowed arteries. The CXCL12/CXCR4 axis can prevent atherosclerosis by regulating vascular permeability and the contractile phenotype in smooth muscle cells (SMCs). Activation of Akt/Wnt/β-catenin signal transduction by CXXL12/CXCR4 induces the expression of VE-cadherin and stabilizes its interaction with VE-cadherin-associated phosphatases to maintain the integrity of endothelial cells and favours a contractile phenotype over a synthetic phenotype in arterial SMCs, thus inhibiting atherosclerosis.Myocardial infarction is defined as acute myocardial damage confirmed by abnormal cardiac biomarkers accompanied by acute myocardial ischaemia.108LRP5/6 deletion promotes cardiac ischaemia, while its downstream target, β-catenin, is beneficial in ischaemic injury. DKK-1 induces LRP5/6 endocytosis in cardiomyocytes, causing GPCR signalling disorder and promoting ischaemia-induced DNA damage in cardiomyocytes. IGFBP-4 binds to LRP5/6 in the presence of Wnt ligands to inhibit activation of the Wnt/β-catenin signalling pathway, which has a protective effect on myocardial ischaemic injury.110The pathogenesis of arrhythmic cardiomyopathy mainly includes haemodynamic changes, myocardial mechanical remodelling, and myocardial electrical remodelling.110 The localization of β-catenin in cardiomyocytes is related to the direct interaction with AnkB. AnkB-deficient animals have sinus bradycardia, a prolonged QT interval, and other myocardial electrical phenotype changes, promoting myocardial mechanical remodelling as well as premature death, which is related to the low expression of β-catenin in the intercalated disc and high expression in the cytoplasm caused by the loss of AnkB. GSK-3β inhibitors improve the cardiac function of AnkB-deficient ACM mouse models, and the use of these inhibitors may become a new prevention and treatment strategy for ACM.111Ankyrin-B regulates arrhythmic cardiomyopathy through the Wnt/β-catenin signalling pathway.112 The Wnt/β-catenin signalling pathway is a key downstream molecular pathway of TGF-β-mediated myocardial fibrosis. TGF-β stimulates Wnt secretion and activates the Wnt/β-catenin signalling pathway through the TAK1 pathway to promote myofibroblast differentiation, which leads to myocardial fibrosis.114Cardiac fibrosis, which is common in various heart injuries, can significantly reduce tissue compliance and disrupt cardiac conduction. The absence of β-catenin in cardiac fibroblasts can reduce myocardial hypertrophy and post-TAC fibrosis and improve cardiac function.115LRP5 regulates body fat distribution through the canonical Wnt signalling pathway and affects the occurrence of cardiometabolic disorders, which is consistent with the increased prevalence of cardiovascular disease in subjects with abdominal obesity.116 In AVS patients, DVL2, GSK3β, β-catenin, and SFRP2 are upregulated in the stenotic aorta.118 Further investigations are encouraged to explore changes in downstream Wnt/β-catenin gene expression to eventually develop potentially propitious pharmacological drugs for AVS.Aortic valve stenosis (AVS) is characterized by leaflet calcification and stiffening, eventually inducing left ventricular obstruction and ischaemic injury.122 The canonical Wnt signalling pathway affects the proliferation and differentiation of mesenchymal stem cells (MSCs) and osteoblast progenitor cells, as well as the bone resorption of osteoclasts, contributing to bone formation and playing an indispensable role in the maintenance of bone homeostasis.119 In addition, other signalling pathways, such as the noncanonical Wnt, JAK/STAT, and Hedgehog (Hh) pathways, exhibit signalling crosstalk with the Wnt/β-catenin signalling pathway, jointly achieving the regulation of bone homeostasis. The Wnt signalling pathway has become a promising target for the development of new bone synthesis drugs.124Osteocytes play a central role in regulating bone metabolism and can regulate the balance of bone formation and bone resorption in bone modelling and remodelling through endocrine and autocrine methods.Regulation of bone formation and differentiation by Wnt players: Doxycycline induces Wnt7b expression in an osteoblast cell line, and the high expression of Wnt7b in aged bone promotes the formation of trabecular and endosteal bone and increases the mineral density of the bone callus during fracture healing.125 Wnt1 in late osteoblasts and osteocytes regulates the function of osteoblasts and promotes bone homeostasis.126127 CXXC5 inhibits bone formation and osteoblast differentiation via interactions with Dvl, which blocks the signal transduction of Wnt/β-catenin.128Bioactive oxidized phospholipids (oxPLs) bind to LRP6 in bone marrow mesenchymal stem cells (BMSCs) and induce their endocytosis, contributing to the inhibition of Wnt signalling and diminishing osteoblast differentiation ability.129 In human osteoblasts, miR-483-3p directly binds to DKK2, increases the expression of β-catenin and cyclin D1, and affects the bone formation process by impacting osteoblast proliferation, preosteoblast differentiation into mature osteoblasts, and new bone matrix formation.130 In BMSCs and preosteoblast MC3T3-E1 cells, miR-376b-3p directly targets YAP1 to suppress YAP1 expression, and circ_0024097 serves as a ceRNA to rescue YAP1 by absorbing miR-376b-3p, leading to activation of the Wnt/β-catenin signalling pathway and cell differentiation, which attenuates osteoporosis.131 In osteoblast cell lines, YAP stabilizes β-catenin and promotes nuclear β-catenin-mediated osteogenesis.In mouse femur tissues, the expression of miR-129-5p is negatively correlated with osteoblastic differentiation markers. MiR-129-5p binds to TCF4, inhibiting osteoblast differentiation.133ZBP1 increases osteogenesis but suppresses the adipogenesis of MSCs, participating in a positive feedback process in bone differentiation regulation by the canonical Wnt signalling pathway. The absence of Wnt signalling inhibits the nuclear transport of β-catenin mediated by ZBP1, resulting in a decrease in Runx2 and Sp7 (osteogenic factors) expression in mouse bone marrow-derived MSCs.134 R-spondin2 (RSPO2), an activator of the canonical Wnt signalling pathway, plays a vital role in regulating the production and mineralization of osteoblasts. We showed that RSPO2-deficient mice have reduced active β-catenin signalling and a decreased mineral deposition rate and osteoblast number, similar to the phenotype observed with the knockout of LRP5/6.135 Therefore, the canonical Wnt signalling pathway is considered the main mechanism of RSPO2 regulation, and it has been proposed that it may be a more precise bone mass regulator.136 Gαs signalling simultaneously inhibits Hedgehog (Hh) signalling and enhances Wnt/β-catenin signalling. Bone formation depends on the maintenance of HH and Wnt/β-catenin signalling levels in a certain range.139DHCR7 and INSIG1/2 are related to the effect of cholesterol metabolism on bone formation. Primary osteoblasts deficient in DHCR7 accelerate osteogenic differentiation by upregulating the expression of Col1a1 (osteogenic factor), while INSIG1/2-deficient cells have the opposite effect. β-Catenin binds to the Col1a1 promoter region, and this combination is related to the activity of Wnt/β-catenin signalling, indicating that the canonical Wnt signalling pathway is involved in the bone phenotype of DHCR7 and INSIG1/2 defects.141 Inflammation plays an important role in tissue regeneration and bone loss. The current treatment for osteoporosis is mainly the inhibition of bone resorption.142 The regulation of some molecules in bone diseases can provide new ideas for the treatment of osteoporosis and other bone-related diseases.Osteoporosis is the reduction in bone formation and increase in bone resorption. Osteoporosis has the characteristics of low bone mass, bone structure degradation, and easy fracture. Cortical bone determines bone strength; thus, cortical bone fragility is also a susceptibility factor for fractures.Positive bone mass regulators: Local COX-2 overexpression promotes the differentiation of MSCs into osteoblast progenitor cells and inhibits their differentiation into chondrocyte progenitor cells. Studies have shown that local COX-2 overexpression increases the number of CD90+ mouse skeletal stem cells (mSSCs) at the fracture site and targets CD90 + mSSCs to enhance canonical Wnt signal transduction, which results in the promotion of intramembranous osteogenesis and inhibition of intrachondral osteogenesis.143144 A study by Wu et al. showed that the transcription level of Wnt3a in GMSCs was high, and the CD39-specific inhibitor POM-1 inhibited the expression of Wnt3a and reduced the level of β-catenin, ultimately leading to loss of the osteogenic ability of GMSCs. They also confirmed that transplanted GMSCs could be distributed in the bone marrow and might represent an effective method for the treatment of osteoporosis and other bone-related diseases.145CD39 secreted by gingiva-derived mesenchymal stem cells (GMSCs) promotes bone formation through the Wnt/β-catenin signalling pathway. GMSCs are progenitor cells with immune regulation, repair, and regeneration capabilities. Previous studies have shown that GMSCs play an important role in the treatment of autoimmune diseases or inflammatory diseases, such as inhibiting the production of osteoclasts and bone erosion in autoimmune arthritis.146 Ann-Kristin Picke et al.147 found that sulfated hyaluronic acid (sHA3) also had a strong affinity for SOST and verified that the TriLA scaffold coated with collagen/sHA3 improved the regeneration of bone defects in type 2 diabetic rats.Highly sulfated glycosaminoglycans (sGAGs) regulate bone homeostasis by interfering with the formation of the sclerostin/LRP5/6 complex. Sclerostin (SOST) is a secreted protein produced by osteocytes that binds to LRP5/6 to inhibit Wnt signal transduction, thereby inhibiting bone formation. sGAGs combine with SOST to restore the signal transduction effect of Wnts. GAGs are used to coat biomaterials, making it possible for implants used for fracture treatment to better fuse with bone structures.149NELL-1 activates Wnt/β-catenin signalling in osteoblast and osteoclast precursor cells. In bone marrow stromal cells (BMSCs), NELL-1 induces an increase in β-catenin, thereby promoting osteogenic differentiation and inducing OPG expression. In osteoclast precursor cells, activation of the canonical Wnt pathway also has an anti-osteoclast generation effect, inhibiting bone resorption. Therefore, NELL-1 may become a potential target for bone formation/anti-bone resorption combined treatment for osteoporosis.Negative bone mass regulators: Treatment of osteoblasts using JAK inhibitors downregulates the expression of STAT3 and SOC3 and promotes Wnt/β-catenin signal transduction, improving the functions of osteoblasts and reducing bone loss.150 STAT3 induces the binding of SOC and β-catenin to promote its degradation. Downregulation of STAT3 and SOC3 expression promotes the stability of β-catenin and signal transduction.152154PHD2 deletion in osteocytes in osteoporosis models increases bone formation and decreases bone resorption. In a hypoxic environment, the activity of PHD2 in osteocytes is reduced and HIF signalling is enhanced, improving the activity of the canonical Wnt signalling pathway through the HIF-SIRT1-SOST-β-catenin signalling axis, thereby promoting bone formation mediated by the activated Wnt/β-catenin signalling pathway and inhibiting osteoclast bone resorption mediated by RANKL and OGP.155Cortical bone fragility is a common feature of osteoporosis. Kiper et al. found that the lack of sFRP4 is one of the causes of cortical bone thinning. Deletion of sFRP4 activates both canonical and noncanonical Wnt signalling pathways. Activation of noncanonical Wnt signalling activates BMP signalling pathways, reducing the formation of cortical bone.156Psoriasis is an inflammatory skin disease mediated by elevated IL-17A levels in patient serum. IL-17A effectively inhibits the function of osteoblasts and osteocytes in patients with psoriasis. Studies have shown that IL-17A downregulates the expression of downstream target genes in the Wnt/β-catenin signalling pathway, such as AXIN2 and CCND1, in osteoblasts and osteocytes, leading to bone loss by inhibiting the activity of osteoblasts and osteocytes.157Myelodysplastic syndrome (MDS) includes a series of clonal haematopoietic stem cell (HSC) diseases, which may be caused by changes in the bone marrow microenvironment. Stoddart et al. (2019) found that inhibiting canonical Wnt signal transduction in bone marrow stromal cells prevented the development of MDS and proposed that activation of β-catenin signalling in the bone marrow of MDS patients may be a new treatment strategy that reduces β-catenin levels or inhibits Wnt/β-catenin signalling.158Fluoride induces osteogenesis to cause bone fluorosis. β-Catenin mediates fluoride-induced aberrant osteoblast activity, and osteogenesis and may be a therapeutic target for skeletal fluorosis.Neurodegenerative disease includes a group of diseases characterized by the loss of cells and neurons of the brain and spinal cord. Parkinson’s disease (PD), Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS) are the most common neurodegenerative diseases.Activation of the Wnt/β-catenin pathway has positive significance for the treatment of PD and AD. However, aberrant activation of the Wnt signalling pathway is related to the pathogenesis of ALS.159 Multiple Wnt/β-catenin signalling-related genes are hypermethylated in the brains of PD patients, including its receptor LRP5, the transcription factor TCF7L2, the inhibitors FRZB, SFRP1, and SFRP2, and multiple target genes. The protein expression of 4 Wnt and neurogenesis-related genes  in midbrain dopamine (DA) neurons is significantly reduced in rat models of PD.160Wnt/β-catenin pathway activity is related to abnormal morphology and neuronal mitochondrial dysfunction.161In another study, AXIN-2 shRNA lentivirus particles were stereotaxically injected into the substantia nigra pars compacta (SNpc) of parkinsonian rats. Activated Wnt/β-catenin signalling enhanced net DAergic neurogenesis in parkinsonian rats by regulating pro-neural genes  and mitochondrial biogenesis.162 Ghrelin, an endogenous ligand for growth hormone secretagogue receptor 1a, plays a fundamental role in regulating energy homeostasis in PD. Wnt/β-catenin signalling is relevant to DAergic neuron differentiation induced by ghrelin.163The development of mesodiencephalic dopaminergic (mdDA) neurons and mdDA progenitors, including the nigrostriatal subset that preferentially degenerates in PD, depends on Wnt/β-catenin signalling.164AD is mainly characterized by neuronal loss, the deposition of amyloid-beta plaques, and the formation of hyperphosphorylated tau protein in neurons, particularly the cytotoxic effect of amyloid beta-peptide (Abeta).165 LRP6 is downregulated in the AD brain, and deficiency in LRP6-mediated Wnt/β-catenin signalling contributes to synaptic dysfunction and amyloid pathology in AD.165 Second, DKK1 upregulation suppresses Wnt/β-catenin signalling in the AD brain. DKK1 is induced by β-amyloid (Aβ) and shifts the balance from canonical Wnt signalling to noncanonical Wnt signalling. Activation of noncanonical Wnt signalling enhances Aβ production, while activation of canonical Wnt signalling suppresses Aβ production.166 Third, GSK3β is activated in the AD brain, and the hyperphosphorylation of tau protein in AD patients is mediated by GSK-3β. In addition, the Wnt/β-catenin pathway in the prefrontal cortex of AD patients is impaired.168 Activation of Wnt signalling rescues neurodegeneration and behavioural impairments induced by beta-amyloid fibrils. This phenomenon indicates that compounds that mimic the Wnt/β-catenin signalling cascade may be candidates for therapeutic intervention in patients with AD.170The Wnt/β-catenin pathway intervenes in the pathological process of AD. The Wnt/β-catenin pathway is downregulated in the brains of AD patients. First, LRP6 is dysregulated in the AD brain.171ALS is a neurodegenerative disease characterized by the progressive loss of motor neurons. Wnt/β-catenin signalling is involved in the neurodegenerative process. Three key signalling molecules in the Wnt/β-catenin signalling pathway—Wnt3a, β-catenin, and Cyclin D1—are upregulated at the mRNA and protein levels in the adult spinal cord of SOD1 (G93A) ALS transgenic mice and are related to gliosis of the adult spinal cord in ALS transgenic mice.+ astrocytes is observed in the borderline between the grey and white matter at the ventral horn in ALS samples. The Wnt family of proteins—more specifically, FZD2 and Wnt5a—may be involved in human ALS pathology.172The levels of Wnt3, Wnt4, FZD 2, FZD 8, Wnt2b, Wnt5a, FZD3, LRP5, and sFRP3 are increased in the human spinal cord tissue of ALS patients. An increase in the number of FZD2The liver is a “metabolic factory” in humans. The Wnt/β-catenin pathway is associated with several common liver diseases, including cholestasis, liver fibrosis, fatty liver, and polycystic liver disease.173 The Wnt/β-catenin pathway is an important regulator that controls liver growth, metabolic liver zonation, and liver regeneration in liver injury.175A distinctive physical feature of the liver is that differentiated hepatocytes proliferate to regenerate the liver during acute liver injury.176 This finding demonstrated that the Wnt/β-catenin pathway restores tissue integrity after acute liver injury. In addition, during liver zonation, knocking down LRP5 and LRP6 causes defective liver zonation in mice during liver regeneration. This result indicated that the cell membrane segment of the Wnt/β-catenin pathway regulates liver zonation.177 In a two-thirds partial hepatectomy (PHx) model, a widely studied liver regeneration model, β-catenin is rapidly enriched and is then transported to the nucleus, helping to induce cell proliferation and target gene expression.178 Importantly, stable regulation of Wnt/β-catenin activity is the key to maintaining liver regeneration. When Wnt/β-catenin continues to be abnormally activated, it often leads to liver cancer development.175Acute liver injury induced by carbon tetrachloride leads to activation of the Wnt/β-catenin signalling pathway, which manifests as an increase in the level of the Wnt target gene AXIN2. Lineage tracing of peri-injury AXIN2 hepatocytes has shown that, during recovery, AXIN2+ liver cells regenerate and repair damaged substances.180The cholestasis Wnt/β-catenin pathway decreases bile acid (BA) synthesis, preventing the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL).181Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease that regularly recurs when immunosuppression is reduced. Aberrant activation of hepatic dendritic cells (HDCs) is observed in AIH, and the deficiency of canonical Wnt/β-catenin signalling in HDCs is the main cause of abnormal HDC function. Reactivation of the Wnt/β-catenin signalling pathway by enhancing binding to the Wnt ligand restores the immunomodulatory phenotype of HDC, reducing the severity of AIH. Targeting the Wnt/β-catenin pathway may become a new therapy for AIH.183Under normal conditions, the growth of mammalian hair is cyclical and is divided into the growth, anagen, and resting phases. The maintenance of hair circulation depends on the activity of hair follicle stem cells (HFSCs) in the bulge area of the hair follicle. The canonical Wnt signalling pathway plays an important role in regulating the proliferation and fate determination of HFSCs.184 Hairless (HR) is a transcription inhibitor that is mainly expressed in the skin. It inhibits the expression of specific target genes by interacting with nuclear receptors, thereby regulating the development of hair follicles and the hair cycle. The 5′-UTR mutation of the human HR gene causes a rare autosomal dominant alopecia disease: Marie Unna hereditary hypotrichosis (MUHH), which is characterized by an abnormal hair density on the scalp, eyebrows, eyelashes, or body. In a study of Hairpoor (HRHp) heterozygous mice , HR overexpression in epithelial cells and keratinocytes was found to block the Wnt/β-catenin signalling pathway by upregulating DKK1 expression during the growth phase, promoting the degenerative progression of hair follicles.185An imbalance in Wnt/β-catenin signalling causes HFSCs to lose dryness or not be activated correctly, leading to hair disease. Deletion of the LGR4 gene in skin epithelial cells inhibits the activation of canonical Wnt signalling in hair follicles, manifesting as a significant reduction in the expression of β-catenin, c-Myc, AXIN2, and LEF-1, inhibiting the activation of HFSCs and delaying the growth phase.189 In addition, the transcription factor Twist1 upregulates the expression of downstream target genes of TCF4, such as HGF, VEGF, and IGF-1, by forming complexes with TCF4 and β-catenin, promoting the proliferation of dermal papilla cells and inducing hair follicle regeneration.190Wnt3a and β-catenin in hair stromal cells accelerate the hair follicle cycle and promote hair regeneration by activating the Wnt/β-catenin signalling pathway.191 In addition, O3,4,5-tri-caffeoylquinic acid enhances pigmentation by activating β-catenin in follicular melanocytes and upregulating the expression of MITF, a target gene, in caffeoylquinic acid.192The Wnt/β-catenin signalling pathway plays an important role in regulating pigmentation during hair growth. For example, hepatocyte growth factor (HGF) secreted by dermal white adipose tissue activates the Wnt/β-catenin signalling pathway and enhances the pigmentation of hair follicles by upregulating the expression of AXIN2, LEF-1, Wnt10b, and Wnt6.152 Dysregulation of Wnt/β-catenin signalling is often caused by mutations of various components in the pathway, particularly mutations or silencing of the Wnt tumour suppressor, which occurs frequently in various cancer types, such as adenomatous polyposis coli (APC) mutations,193 Axin1/2 mutations,195 GSK3β deletions,196 inactivation mutations of E3 ubiquitin ligase RNF43,196 and Znrf3.197 β-Catenin mutation is the most common alteration in cancers.198 The APC gene was first identified as a mutated gene in familial adenomatous polyposis coli (FAP). APC mutations are also found in 80% of colorectal adenomas and CRCs and are one of the earliest mutations in the progression of colon cancer.201 APC allele deletion activates Wnt/β-catenin signalling by inhibiting the formation of the degradation complex, which drives CRC development, endows cancer cells with self-renewal growth characteristics and is related to therapeutic drug resistance.204In 1982, the first WNT gene was identified and cloned as an oncogene in mouse breast cancer.208 Patients with hereditary Axin2 mutations develop CRC.209 Generally, APC mutations and classic mutations of β-catenin are more common in CRC,211 gastric cancer (GC), and endometrial carcinoma,212 while AXIN mutations primarily occur in hepatocellular carcinoma (HCC)213 and medulloblastoma with dysfunction of the Wnt pathway.214 AXIN mutations are present in ~10% of hepatocellular carcinomas (HCCs).194 In addition, adenovirus-mediated gene transfer of wild-type AXIN1 induces HCC cell and CRC cell apoptosis, which is blocked by APC, CTNNB1, or AXIN1 mutations.215 The infiltration level of CD4+ and CD8+ T cells decreases in HCC tissues because of AXIN1/CTNNB1 mutations, resulting in immune escape.217 Several new potentially functionally relevant AXIN1 mutations have also been found in advanced prostate cancer.195 GSK-3β inhibits Wnt signalling by promoting β-catenin phosphorylation and then inducing the degradation of β-catenin mediated by E3 ligase. Studies have found that GSK3B deletion leaves HSCs in a precancerous state, and progression to acute myeloid leukaemia (AML) is facilitated after GSK3A deletion.196 Although the mutation frequency of E3 ubiquitin ligase is lower than that of other WNT inhibitors, mutations of RNF43 (which mediates ubiquitin degradation of Frizzled) are still detected in the adenoma tissues of patients with GC,218 early-onset metastatic CRC,219 and Lynch syndrome.220 RNF43 mutations are highly enriched in GC with microsatellite instability (MSI), promoting the development of GC cell resistance to DDR radiation and chemotherapy.221 Accordingly, BRAF, ARID1A, RNF43, and KM2B with higher mutation frequencies can be used to predict the prognosis and distinguish MSI from MSS tumours, and they provide guiding significance for the clinical effect of immunotherapy in MSI CRC.222 Simultaneously, RNF43 and KRAS mutations show a synergistic effect in CRC progression.223 Wnt signalling activated by RNF43 mutation promotes tumour growth and a high recurrence rate in CRC patients.224 Recent studies have further shown that some mutations in RNF43, such as p.G659fs, do not affect β-catenin signal transduction, while the C-terminal truncated mutant has a WNT signal transduction function similar to that of wild-type RNF43.225 Znrf3 inactivation mutations have been found in pancreatic cancer226 and adrenocortical carcinoma,227 but relatively few studies have examined the role of Znrf3 inactivation mutations in tumours.AXIN is a scaffold protein of the degradation complex, and mutation of AXIN1/2 substantially affects Wnt signal transduction activity. In addition to the APC mutation, an AXIN2 mutation is also common in CRC.229 In medulloblastomas, CTNNB1 is a common mutation .230 In patients with childhood medulloblastoma, CTNNB1 gene mutations are only found in patients who are nuclear β-catenin positive, while the nuclear accumulation of β-catenin seems to be a sign of a good prognosis in medulloblastomas.231 Mutation of CTNNB1 exon 3  may be the driver of low-grade and low-stage endometrioid endometrial carcinoma in young women.212 In elderly patients with CRC, the expression of nuclear CTNNB1 may be associated with higher mortality.210 Wnt/β-catenin signals are dysregulated in almost all stages of tumorigenesis, from malignant transformation to metastasis, spread, and drug resistance. This signalling may disrupt cancer immune surveillance, promoting immune evasion and resistance to immunotherapies, including immune checkpoint blockers.233 In addition to the inactivating mutation of Wnt/β-catenin pathway components, abnormal secretion of Wnt ligands in many tumours can also lead to overactivation of the Wnt/β-catenin pathway .6Activation of the Wnt/β-catenin pathway includes not only classic mutations in suppressor molecules but also mutations in CTNNB1. In hepatocellular carcinoma, CTNNB1-activating mutations [28% (34/123) to 40% (18/45)] are the most significant genetic changes in the Wnt/β-catenin pathway.238Wnt/β-catenin signalling is essential to maintain the undifferentiated state of the stem cell population in the intestinal epithelium239 Recently, researchers have reported that the Wnt/β-catenin pathway collaborates with AHCTF1  to act as a superenhancer-mediated gene to drive colon cancer.241 In a study of 955 patients with stage I, II, III, or IV colon and rectal cancer from 1980 through 2004, CTNNB1 was associated with improved CRC-specific survival and overall survival only in patients with a BMI ≥ 30.242Abnormal activation of the Wnt/β-catenin pathway by mutations in adenomatous polyposis coli, AXIN2, or β-catenin (CTNNB1) is responsible for the initiation of almost all colon cancers.A more comprehensive experimental evaluation is needed for the functional assessment of different derived cancer suppressor mutations, improving auxiliary diagnosis and treatments.Constitutive activation and inactivation of the Wnt/β-catenin pathway extensively participate in the physiopathological process of numerous diseases, indicating that this pathway has practical value in the clinic, particularly in the diagnosis, treatment, and prognosis of diseases.Because of the rapid development of molecular biology in recent years, the application of genetic testing for tumour diagnosis has gradually increased. Wnt/β-catenin signal transduction is an important regulator of embryonic development and adult homeostasis and is also closely related to the occurrence and development of many diseases. Related components of the canonical Wnt signalling pathway play potential roles in disease diagnosis.Wnt/β-catenin pathway in the cell-free DNA (cfDNA) test for CRC The traditional diagnosis of CRC has many limitations,245 such as low sensitivity, poor specificity, and painful and traumatic procedures. However, cell-free DNA (cfDNA) analysis, which overcomes these limitations, can provide direct evidence of residual disease and can be used as a minimally invasive detection method for patients with CRC.246 Li et al. found that plasma cfDNA-based testing can capture specific genetic changes in CRC. Studies have shown that, in the cfDNA of patients with stage III or IV CRC, the copy number of key genes related to CRC  is increased, and the copy number of cfDNA changes (CNVs) is positively correlated with tumour progression. The copy number of RSPO2 related to the canonical Wnt signalling pathway is increased. Therefore, the alteration of Wnt and its related gene copy number in cfDNA can be used to diagnose CRC. This diagnostic method is simpler and more readily accepted by patients and can be used for the treatment monitoring and clinical care of CRC patients.Wnt/β-catenin pathway in the DNA methylation of tumour-related diseases Abnormal DNA methylation is an important cause of epigenetic gene silencing. In many human cancers, tumour suppressor gene methylation is widespread.247248Samaei et al. detected the methylation status of the gene promoters of 10 negative regulators of the Wnt/β-catenin signalling pathway in 125 groups of colon cancer and adjacent tissue samples. They found that the promoter methylation level of these genes was higher in cancer tissues than in adjacent tissues, and a correlation was found with different clinicopathological characteristics, such as age, sex, and other factors.250 The four DNA methylation subgroups of medulloblastoma in the study included Wnt, SHH, Group 3, and Group 4. All samples in the Wnt subgroup had mutations in the third exon of the CTNNB1 gene, which is the most reliable single marker to identify Wnt-driven medulloblastoma. Therefore, methylation analysis and CTNNB1 mutation analysis may be helpful for the reliable identification of Wnt-driven medulloblastoma.251 Genome-wide analysis can also be used to detect the methylation status and aberrant activation of Wnt/β-catenin signalling in primary plasma cell leukaemia.252Pietsch et al. proposed a diagnostic method for Wnt-driven medulloblastoma. Studies have shown that activation of the canonical Wnt signalling pathway is mainly evaluated by detecting the β-catenin immunophenotype, and methylation subgroups are consistent with IHC.P < 0.001) and overall survival . The results indicate the role of abnormal Wnt signalling in ALL and establish a group of patients with a significantly worse prognosis (methylated group).253Promoter hypermethylation of Wnt inhibitors is related to activation of the Wnt pathway. In ALL-derived cell lines and bone marrow mononuclear cells from ALL patients, 7 Wnt antagonists  showed abnormal promoter methylation. Clinically, in a group of 261 patients with newly diagnosed ALL, abnormal methylation of Wnt inhibitors was associated with decreased 10-year disease-free survival .257 Fourteen bone-related biomarkers in elderly people with memory problems were not clinically diagnosed with asymptomatic AD, and the expression of TRAIL and DKK1 was elevated in the brain.258 Wnt signalling has a neuroprotective effect, and DKK1, as an antagonist of the canonical Wnt signalling pathway, is negatively correlated with changes in cognitive function.259 Therefore, TRAIL and DKK1 most likely predict cognitive decline and may become potential biomarkers for the diagnosis of AD.260The bone density of patients with AD was lower than that of the control group.261 After using TPD to treat osteoporosis for more than 12 months, the serum DKK1 level increased, and the therapeutic effect of TPD decreased. This finding confirmed that chronic stimulation of PTH in the osteoblast cell line might reduce its anabolic effects through excessive secretion of DKK1. The detection of serum DKK1 levels plays an important role in the formulation of further treatment options for postmenopausal osteoporosis.262Teriparatide (TPD) is a PTH analogue that can be used to treat osteoporosis.263 Nuclear β-catenin accumulation increases in keratinocytes on the epidermal base of patients with psoriasis, upregulating the activity of the transglutaminase 1 (TGase-1) promoter and leading to abnormal proliferation and differentiation of keratinocytes in the skin of psoriasis patients. TGase1 and β-catenin, with abnormally high expression in the skin, serve as potential biomarkers and targets for the diagnosis and treatment of psoriasis.264TGase-1 is a keratinocyte differentiation marker related to the differentiation of keratinocytes, and its expression increases in psoriatic skin.265A meta-analysis showed that the genes associated with childhood pneumonia include the top networks related to Wnt signal transduction. The diagnosis of pneumonia susceptibility factors is meaningful for the prediction, diagnosis, and treatment of lung-related diseases.266The Wnt/β-catenin signalling pathway and renin-angiotensin system may participate in the remodelling of subcutaneous adipose tissue in the initial stage of human weight gain. After overeating, Wnt/β-catenin signal transduction in adipose tissue is inhibited, manifesting as increased expression of inhibitors of the canonical Wnt signalling pathway, such as SFRP2, FRZB/SFRP3, and DKK3, and increased phosphorylation and total GSK-3β levels. These factors lead to a decrease in the level of β-catenin and promote adipogenesis or adipose tissue remodelling. Therefore, the detection of related genes may become a potential target for the pathological diagnosis of adipose tissue in humans, including those with obesity.Abnormal activation of the Wnt/β-catenin signalling pathway promotes the progression of many human diseases, including cancer and non-cancer diseases. Hence, the canonical Wnt pathway has become a very attractive therapeutic target in recent years Table .Table 3AThe canonical Wnt signalling pathway is a therapeutic target for many non-cancer diseases, usually by inhibiting Wnt signal and β-catenin activity.Agents targeting the β-catenin-destruction complex chronic kidney disease (CKD)-mineral and bone disorder (CKD-MBD) begins early in the course of kidney disease and is clinically detectable in stage 2 CKD.267 DKK1 antibody are involved in the pathogenesis of CKD-MBD, neutralization of DKK1 in CKD-2 mice after renal injury stimulated bone formation rates, corrected the osteodystrophy, and prevented CKD-stimulated vascular calcification.268 A multi-centre osteoporosis study conducted in five European countries found that DKK1 positively associated with whole-body bone mineral density (WBMD) in subjects, regardless of age and sex.269 several clinical studies on osteoporosis showed that both zoledronic acid and denosumab reduced bone resorption through upregulation the endogenous Wnt-inhibitors Sclerostin (SOST) and DKK1.271 Meanwhile, Teriparatide (TPD) stimulate bone formation also via increasing SOST and DKK1 in postmenopausal osteoporosis treatment.262 In addition, in solid organ transplant recipients (SOTr), low DKK-1 and increased secreted frizzled related protein-3 levels causing dysregulation of Wnt signalling were closely associated with cytomegalovirus (CMV) infection.272Inhibitors targeting β-catenin/TCF transcription complex PRI-724 is a first-in-class small molecule compound that inhibits the interaction between β-catenin and CBP, and it inactivates resting hepatic stellate cells in vitro and causes macrophages to migrate to the liver during liver fibrosis. A phase I trial showed that patients with Hepatitis C Virus-related Cirrhosis were well tolerated by intravenous infusion of 10 or 40 mg/m2/d of PrI-724, and the liver histology and Child-Pugh (CP) scores were improved in several patients.273 The agent C82 suppresses the activity of Wnt/β-catenin through blocking the interaction of the protein CBP with β-Catenin. Although the clinical trial showed C82 had few clinical effects on systemic sclerosis (SSc), microarray analysis C-82 treatment provide the possibility that C-82 with longer treatment to promote fat regeneration in SSc skin.274 Similarity, in another clinical trials of SSc, the researchers found that rituximab (RTX) relieved symptoms of patients with SSc by upregulation of Dkk-1.275277Further, a novel Wnt inhibitor, SM04690 were proved to effectively relieve the pain of severe symptomatic knee osteoarthritis (OA) and appeared safe and well tolerated through phase I/II clinical trial.281 All above indicate that targeting Wnt signalling is an attractive strategy for the treatment of multiple diseases.In addition, numerous studies have shown that the therapeutic effect of drugs that are not direct activators or inhibitors of Wnt signalling pathway is also depend on the activity of Wnt signalling.The dysregulation of Wnt/β-catenin signalling pathway is closely correlated with the development of cancers. Thus, medication targeting the pathway were developed in cancers. Fig.  Figure 4283 In melanoma mice model, PKF115-584 restored immunocompetence which suppressed by β-catenin activation.284 TNIK is a key regulator of the β-catenin and TCF4 transcription complex, its pharmacological inhibition seems to be a promising therapeutic approach.285 In Apc min/+ mice, the TNIK inhibitor NCB-0846 effectively attenuated the frequency of intestinal tumorigenesis driven by Wnt signal. Also, NCB-0846 significantly reduced tumour formation by the same numbers of HCT116 and DLD-1 cells in an immunodeficient mouse model and PDXs (patient-derived xenografts). Importantly, the body weight of mice (immunodeficient xenograft model) decreased after treatment with NCB-0846, but gradually recovered.286 Besides, β-catenin responsive transcription (CRT) is another crucial therapeutic target in combating various cancers and CRT-inhibitory drugs  are specifically cytotoxic to human colon cancer cells and enhance the infiltration of T and NK cells in syngeneic mouse models of CRC by blocking β-catenin/TCF interaction.288 In the vitro experimental of head and neck cancer289 and triple-negative breast cancer (TNBC)290 also verified the antitumor effects of iCRTs. Moreover, in mantle cell lymphoma (MCL) which is resistant to genotoxic agents vincristine, iCRTs eliminated MCL-initiating cells very well.291 However, there are lacking enough vivo evidences to evaluate the antitumor potential of iCRTs. cAMP-responsive element binding (CREB)-binding protein (CBP) is a coactivator of Wnt/β-catenin-mediated transcription, and is another attractive target for cancer treatment. ICG-001, a preclinical agent, binding CBP and disrupting its interaction with β-catenin, is efficacious killing tumour cells in both vitro experiments and mouse xenograft models of colon cancer and pancreatic ductal adenocarcinoma (PDAC).293 PRI-724, an active enantiomer of ICG-001, has already entered Phase I clinical trials for treating colon cancer and advanced-stage pancreatic adenocarcinoma.294 Moreover, E7386 is the first-in-class orally active β-catenin-CBP antagonist that inhibits Wnt/β-catenin pathway in patient-derived tumour xenograft (PDX) model of HCC and now is in phase I clinical trials..295CPG049090, PKF115-584 and PKF222-815 are natural compounds that have been identified to disrupt the interaction of β-catenin/TCF complex, suppressing the proliferation of colon cancer cells in vitro assays.299 Importantly, in these in vivo studies, niclosamide have shown limited toxicity providing hopeful optimism for Wnt inhibition in human cancer therapy.In all inhibitors of WNT signalling, niclosamide is FDA-approved drug and was used to treat taeniasis, showing inhibiting effect on tumours proliferation, stemness and metastasis by causing FZD1 receptor internalization and decreasing the protein level of DVL2, as well as targeting LRP6.300 Using a mouse melanoma model, the researchers confirmed that the intrinsically active β-catenin signal of the tumour led to T cell rejection and resistance to anti-PD-L1/anti-CTLA-4 monoclonal antibody treatment.301 Simultaneously, the analysis of primary melanoma lesions with BRAF mutations showed that T cell infiltration was negatively correlated with β-catenin in tumour cells.302 Transmission of Wnt3a-β-catenin signal exhausted tumour infiltrating T cells, thus weakening their anti-tumour effects, and inhibiting the production of effector memory T cells.303In addition to directly affecting the function of tumour cells themselves, inhibitors of the Wnt signalling pathway also promote the activation and infiltration of immune cells in the tumour microenvironment and assist in improving the tumour-killing effect. Wnt/β-catenin signalling pathway has shown strong immunosuppressive function in a variety of tumours, and potentially become a novel target of tumour treatment.cluster) improved the tumour response to the PD1/PD-L1 immune checkpoint blockade in melanoma and colon cancer.300 In the parental ID8 murine ovarian cancer model harbouring a knock-out of p53 (ID8p53) and MISIIR-Tag spontaneous ovarian cancer models, CGX-1321(Wnt inhibitor) increased infiltrating CD8+ T cells in the TME and decreased tumour burden.304some Wnt inhibitors strengthen the Immune checkpoint blockade (ICB) therapy by combination therapy. A new specific Wnt inhibitor, a size-tuned nanocluster ,Because Wnt/ β-catenin signalling plays an important role in the aetiology of non-tumour diseases, small-molecule drugs targeting the Wnt/β-catenin signalling pathway have shown unique advantages in the treatment of these non-tumour diseases. DKK1 antibody effectively corrects renal osteodystrophy, while PRI-724, an inhibitor of CBP/β-catenin, inhibits the activation of hepatic astrocytes and delays the progression of liver cirrhosis. Wnt/β-catenin is also a potential therapeutic target for common but refractory diseases such as psoriasis, viral infections, and obesity.315 kidney injury; chronic kidney disease-associated complications316 and toxicities such as bone toxicity317 and intestinal toxicity.318 In addition, the appropriate timing of drug administration is critical, particularly in patient stratification and drug delivery.320A complete understanding of the specific role of Wnt/β-catenin in disease occurrence and progression can help guide the accurate use of these small-molecule drugs to maximize their efficacy and benefit more patients. Although Wnt/β-catenin signalling is an attractive target evaluated by clinical trials, there is a risk of off-target effects, such as the following: side effects such as diarrhoea, fatigue, nausea dysgeusia, and vomitingA more integrated experimental evaluation of mutations from different patient-derived tumours can be used to identify driver mutations from passenger mutations to guide treatment.


Methods for the conversion of human induced pluripotent stem cells (hiPSCs) into motor neurons (MNs) have opened to the generation of patient-derived in vitro systems that can be exploited for MN disease modelling. However, the lack of simplified and consistent protocols and the fact that hiPSC-derived MNs are often functionally immature yet limit the opportunity to fully take advantage of this technology, especially in research aimed at revealing the disease phenotypes that are manifested in functionally mature cells. In this study, we present a robust, optimized monolayer procedure to rapidly convert hiPSCs into enriched populations of motor neuron progenitor cells (MNPCs) that can be further amplified to produce a large number of cells to cover many experimental needs. These MNPCs can be efficiently differentiated towards mature MNs exhibiting functional electrical and pharmacological neuronal properties. Finally, we report that MN cultures can be long-term maintained, thus offering the opportunity to study degenerative phenomena associated with pathologies involving MNs and their functional, networked activity. These results indicate that our optimized procedure enables the efficient and robust generation of large quantities of MNPCs and functional MNs, providing a valid tool for MNs disease modelling and for drug discovery applications. Motor neurons (MNs) are cells located in specific areas of the central nervous system (CNS), including the cerebral cortex (upper MNs), brain stem and spinal cord (lower MNs) . They arSeveral protocols published during the last years reported a high variability in terms of the efficiency and time required for MNs generation, thus proving the importance for the field to develop a rapid, efficient and robust strategy process of differentiation ,13,14.To date, MNs have been successfully differentiated from human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs) by direct differentiation, or from somatic cells  by an induction process based on the overexpression of specific transcription factors for the conversion of somatic cells towards MNs. Most of the xeno-free chemically defined available protocols for the generation of hiPSC-derived neurons are based on floating EB formation, a step that makes the process longer and that, due to the complex EB structure, does not allow proper monitoring of the differentiation ,19,20,212+ concentration ([Ca2+]i) when exposed to drugs acting on the ion channels, thus further proving their mature functional nature. Finally, this protocol was exploited to differentiate hiPSCs from a SMA patient and a healthy individual control.In this study, we report a new optimized monolayer procedure for the rapid and efficient differentiation of hiPSCs into mature MNs. The protocol is based on fully defined culture conditions that allow a nearly pure MNPC population to be obtained in 13 days. Moreover, we tested the molecules that allow stable MNPC expansion to rapidly produce large stocks of cryopreservable MNPCs starting from limited amounts of hiPSCs. By applying this protocol, within 30–40 days in vitro (DIV) the MNPC population can mature into functionally active MNs with ~85% of efficiency. The MNs population showed HB9, ISLET-1 and ChAT immunoreactivity, and the expression of their relative transcripts. These hiPSC-derived MNs also exhibited spontaneous firing as assayed by multielectrode arrays (MEAs) and action potential production in response to a current-clamp setting current injection. Also, these MNs exhibited variation in their intracellular CaOn the whole, our study provides an optimized approach to robustly induce a rapid and highly efficient conversion of hiPSCs into enriched populations of MNPCs and fully mature MNs exploitable for biochemical and functional studies, and for the modelling of human CNS pathologies affecting MN populations.https://www.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=GM24474&Product=CC) and GM24468 (https://www.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=GM24468&Product=CC) fibroblast-derived hiPSC lines were obtained from the Coriell Institute for Medical Research with verified pluripotency and contamination-free. All the hiPSC lines used in this study were cultured in feeder-free conditions in chemically defined Essential 8 (E8) medium on Geltrex-coated tissue culture plastic plates. Medium was changed every 24 h and colonies were passaged by chemical-based procedure with dissociation solution (DPBS without Ca2+/Mg2+ supplemented with 0.5 mM EDTA). Briefly, 70–80% confluent cultures were washed twice with DPBS W/O Ca2+/Mg2+, incubated for 3–5 min at 37 °C with dissociation solution and then detached in E8 medium, collected and spun at 200 g for 5 min. Pellet was resuspended in E8 medium and 1–1.5 × 104 cells/cm2 were plated on Geltrex-coated tissue culture plastic. Cultures were maintained at 37 °C, 5% CO2. All cell culture reagents were purchased from ThermoFisher Scientific .Human hiPSCs were obtained from ThermoFisher Scientific with verified pluripotency and normal karyotype. GM24474  composed of Essential 6 (E6) medium (ThermoFisher Scientific) supplemented with 1% GlutaMAX (ThermoFisher Scientific), 10 μM Y-27632  and maintained at 37 °C for 48 h. After 48 h, the medium was replaced with NIM without Y-27632; medium was entirely renewed every other day. At day 6, cultures were shifted to E6 medium supplemented with 100 nM all-trans retinoic acid  to induce the caudalization of the neuroepithelial cells. Cells were maintained under these conditions for two days and then dissociated and replated on vitronectin-coated plastic in MNP patterning medium (MNP-PM) composed of E6 medium supplemented with 2% B27 supplement (ThermoFisher Scientific), 100 nM RA, 2 μM purmorphamine , and 10 μM Y-27632 for the selection of MNPCs. Cells were maintained in these conditions for two days and then medium was replaced with MNP-PM without Y-27632; medium was then renewed every other day.Once confluent, MNP cultures can be passaged for further expansion by dissociation with StemPro Accutase (ThermoFisher Scientific) and replated on vitronectin-coated plastic in MNPCs expansion medium (MNP-EM) consisting of MNP-PM supplemented with 0.5 mM valproic acid , 3 μM CHIR99021  and 10 μM Y-27632. Medium was changed two days after plating with MNP-EM without Y-27632 and renewed every other day. Cells can be expanded to confluency for at least 3 passages; cultures can be cryopreserved at this stage.4 cells/cm2 seeded on vitronectin-coated plastic in MN maturation medium (MN-MM) composed of neurobasal medium (ThermoFisher Scientific) supplemented with 1% GlutaMAX, 2% B27, 100 nM RA, 1.5 μM PMN, 1% N2 supplement (ThermoFisher Scientific), 0.2 μM cAMP (Santa Cruz Biotechnology), 10 ng/mL BDNF (Peprotech), 10 ng/mL GDNF (Peprotech), 10 ng/mL IGF  and 10 μM Y-27632. After 2 days, the medium was completely renewed with MN-MM without Y-27632 and then partially (50%) renewed twice a week. MNs in these conditions can be long-term cultured to induce full functional maturation.To induce MNs maturation, MNPCs were dissociated with StemPro Accutase and 2–4 × 102+/Mg2+, fixed with 4% PFA for 15 min at room temperature (RT), permeabilized with permeabilizing solution (0.5% Triton X-100 in PBS) for 15 min at RT and blocked 1 h at RT with blocking solution . Cells were incubated at 4 °C overnight with antibody solution  containing the specific primary antibodies, rinsed twice with PBS and incubated with relative secondary antibodies for 2 h at RT. Primary antibodies used in this study included polyclonal anti-aPKC , polyclonal anti-Zo-1 , monoclonal anti-ISLET1 , polyclonal anti-ISLET1 , polyclonal anti-OLIG2 , monoclonal anti-HB9 , monoclonal anti-TuJ1 , polyclonal anti-MAP2 , monoclonal anti-NKX2.2 , monoclonal anti-ChAT , monoclonal anti-NESTIN , and monoclonal anti-phospho-histone H3 . Secondary antibodies used in this study were Alexa Fluor 488 and 568 . Nuclei were counterstained with Hoechst 33342 (ThermoFisher Scientific). Negative controls were obtained by omitting the primary antibody. Images were visualized on Leica DM IL LED fluo microscope equipped with 5×/10×/20×/40× objectives and images acquired with Leica DFC450 C camera. For the evaluation of the number of immunofluorescence-labelled cells, 5–8 photographic fields for each sample  were analyzed and quantified by using Image J software (NIH) or by manual counting.Cells were washed once with cold DPBS with Ca2O. Amplification reaction was performed on RealTime System CFX96TM (Bio-Rad) using the following program: 95 °C for 15 min and then 40 cycles  followed by 60 cycles of 5 s from 65 °C to 95 °C increasing the temperature by 0.5 °C per cycle. Quantitative levels for all genes were normalized to housekeeping genes (GAPDH or beta-actin). For MNPC and MN genes, levels were expressed as relative to hiPSCs. Primer sequences used are as follow: Beta-Actin_Fw: 5′GACAGGATGCAGAAGGAGATTACTG, Beta-Actin_Rev: CTCAGGAGGAGCAATGATCTTGAT; hGapdh_Fw: CCACTCCTCCACCTTTGAC, hGapdh_Rev: ACCCTGTTGCTGTAGCCA; hChAT_Fw: CCCTGATGCCTTCATCCA, hChAT_Rev: GTAGGTGGGCACCAGTCTTC; hOlig2_Fw: AGCTCCTCAAATCGCATCC, hOlig2_Rev: ATAGTCGTCGCAGCTTTCG; hHb9_Fw: TGCCTAAGATGCCCGACTT, hHb9_Rev: AGCTGCTGGCTGGTGAAG; hIslet1_Fw: CGTGCCCGCTCCAAGGTGTATCA, hIslet1_Rev: CATTGGGCTGCTGCTGCTGGAGTT; hNestin_Fw: GGAGAAGGACCAAGAACTG, hNestin_Rev: ACCTCCTCTGTGGCATTC-3′; hβ3-tubulin_Fw: TCAGCGTCTACTACAACGAGGC, hβ3-tubulin_Rev: GCCTGAAGAGATGTCCAAAGGC.Total RNA was isolated using TriFast (Euroclone) and subsequently used to synthesize cDNA by iScript kit (Bio-Rad). qPCR was then performed using the reaction mix containing SsoAdvanced SYBR green (Bio-Rad), primers (200 nM), cDNA (2.5 ng/μL) and nuclease-free H5 cells were replated in maturation medium on vitronectin-coated 12-mm plastic coverslips (SPL) and allowed to mature for 30 or 60 days. Patch-clamp recordings were obtained from a total number of 40 hiPSC-derived motor neurons in the whole-cell configuration i variation was evaluated by means of optical fluorimetric recordings with Fura-2AM fluorescent probe i, following bath application of glutamate receptor agonists on the 4-weeks and 8-weeks mature cultures. The NMDA treatment triggered a fast increase in [Ca2+]i at both maturation time points. Notably, the higher ratio (1.48 ± 0.04) of the peak rapidly achieved following stimulation and the biphasic response (ratio at 30 min after treatment: 1.80 ± 0.06) in the 8-weeks mature MNs was suggestive of increased receptor functional activity, and more neuronal mature identity, compared to the 4-weeks mature MNs. Indeed, the latter exhibited only a minor peak with a ratio that progressively increased up to 1.60 ± 0.02 by the end of the 30-min observation . However, the higher cell density was detrimental for the long-term cell cultures (data not shown). Another characteristic reported to alter the neuronal differentiation process is the presence of astrocytes in culture, which is known to increase the MNs maturation and spontaneous spike frequency . We therPatient-specific MNs represent valuable in vitro model systems to decipher the pathogenesis of a variety of movement diseases. We thus tested our differentiation procedure to generate MNPCs and mature MNs, starting from an SMA patient-derived and a related  control hiPSC line. Both the populations rapidly generated neuroepithelial cells organized in neural rosettes and were efficiently patterned into MNPCs. The immunofluorescence analysis on the DIV13 cultures indicated that the cells in culture homogenously co-expressed NESTIN and OLIG2 A.+ve neurons co-expressing HB9, ISLET-1 and ChAT MN markers . Following 4 weeks of exposure to the neuronal maturation conditions, the cultures were predominantly populated by MAP2 markers B.These results confirmed that the differentiation procedure is applicable to different hiPSC lines to generate nearly pure populations of expandable MNPCs to be further robustly differentiated towards large numbers of MNs to be exploited for in vitro disease modelling approaches.We have reported an optimized procedure for generating a highly enriched population of MNPCs in 11–13 days in fully defined monolayer conditions and with a reduced use of small molecules. We showed that these MNPCs can be expanded for at least three passages in culture, allowing the production of large lots of cryopreservable cells retaining their original competence to efficiently generate fully mature functional MNs in culture. Besides the purity and yield of hiPSC-derived MNs, an important challenge in modeling neurological diseases is how to culture these cells physiologically, allowing for long-term cultures. We showed that in our conditions, the MNs cultures can be long-term maintained , thus providing the means to study the molecular processes guiding progressive functional maturation, and to analyze the degenerative phenomena associated with the pathologies involving MNs and their functional, networked activity. To this end, we showed for the first time the increasing glutamate receptors functional activity during human MNs maturation, opening to new opportunities for studying the pharmacological excitability over time on these cell populations.Our results further confirm the important role of the time and range of exposure to key signals during progenitor cells specification to achieve the best efficiency for cell identity maintenance and subsequent neuronal differentiation. The use of fully defined media and the reduced complexity of the differentiation strategy that reduces the number of factors needed for neuralization and patterning, exhibiting a completely xeno-free environment during the whole process, provides a fast and accessible method to be used in human motor neuron disease modelling studies. Given these features, our protocol offers a valid alternative that may be adapted in GMP conditions for translational and clinical purposes and in applications requiring large amounts of nearly homogeneous well-defined target cells, such as for high-throughput screening.Future efforts will be devoted to generating spinal cord astrocytes to better reproduce a completely controlled environment that leads to MNs maturation and to study glial cells implication and contribution in healthy and pathological conditions, providing additional means to unravel the pathophysiological aspects not yet fully understood.


Background: Androgen alopecia (AGA), the most common type of alopecia worldwide, has become an important medical and social issue. Accumulating evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in the progression of various human diseases, including AGA. However, the potential roles of lncRNAs in hair follicle stem cells (HFSCs) and their subsequent relevance for AGA have not been fully elucidated. The current study aimed to explore the function and molecular mechanism of the lncRNA AC010789.1 in AGA progression.Methods: We investigated the expression levels of AC010789.1 in AGA scalp tissues compared with that in normal tissues and explored the underlying mechanisms using bioinformatics. HFSCs were then isolated from hair follicles of patients with AGA, and an AC010789.1-overexpressing HFSC line was produced and verified. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to verify the molecular mechanisms involved.Results: AC010789.1 overexpression promoted the proliferation and differentiation of HFSCs. Mechanistically, we demonstrated that AC010789.1 overexpression promotes the biological function of HFSCs by downregulating miR-21-5p and TGF-β1 expression but upregulating the Wnt/β-catenin signaling pathway.Conclusion: These results reveal that overexpression of AC010789.1 suppresses AGA progression via downregulation of hsa-miR-21-5p and TGF-β1 and promotion of the Wnt/β-catenin signaling pathway, highlighting a potentially promising strategy for AGA treatment. Functional data suggest that they play essential regulatory roles in multiple biological processes, such as cell development, differentiation, disease, subcellular localization, and cell structure maintenance . Intrigugression . Moreovegression  demonstrβ-catenin signaling. Therefore, this study provides insights into the mechanisms underlying AC010789.1 regulation of AGA progression to provide a new theoretical and experimental basis for the prevention and treatment of AGA.The current study aimed to explore the potential function of the lncRNA AC010789.1 in AGA progression. We demonstrate that AC010789.1 expression was downregulated in AGA scalp tissues, and overexpression of AC010789.1 promoted the proliferation and differentiation of HFSCs. Mechanistically, overexpression of AC010789.1 was shown to delay AGA progression by interacting with miR-21 and activating Wnt/Hair follicle tissues extracted from patients with AGA were obtained from the Shanghai East Hospital affiliated with the Tongji University School of Medicine, and informed consent was obtained from all patients. Ethical approval was obtained from the Shanghai East Hospital Ethics Committee. Tissue specimens were snap-frozen and stored in liquid nitrogen until further use.https://www.ncbi.nlm.nih.gov/geo). The GSE84839 data set was based on the GPL21827 platform (Agilent-079487 Arraystar Human LncRNA microarray V4) and included three pairs of male AGA scalp tissues and adjacent normal tissues. The GSE36169 data set was based on the GPL96 platform (Affymetrix Human Genome U133A Array) and contained AGA and adjacent normal scalp tissues from five individuals. The lncRNA and mRNA expression data in the AGA and adjacent normal tissues were downloaded and used for this study.Gene expression microarray data sets (GSE84839 and GSE36169) were downloaded from the Gene Expression Omnibus (GEO) database (http://www.bioconductor.org/). A p-value < .05, and a base-2 logarithm of fold change (log FC) < −1 or >1 were used as selection criteria to screen differentially expressed lncRNAs and mRNAs  provides a comprehensive annotation tool to help investigators better clarify the biological function of the submitted genes , a database that documents scientific literature supporting crosstalk between pairs of signaling pathways, was used to explore the relationship between pathways.Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8 (ed genes . In thishttp://genemania.org/) is a data set that provides a series of functional association information to identify the relation between genes of the submitted set in terms of their genetic interactions, pathways, expression patterns, localization, and protein domain similarity. In this study, protein-protein interaction (PPI) networks were analyzed using the GeneMANIA data set, and Cytoscape (version 3.7.2) was used to visualize the PPI networks.GeneMANIA  of HFSCs was characterized by immunofluorescence with an anti-K15 antibody  and was used for follow-up studies.Isolation of HFSCs was performed as previously described . Follicu3 cells/well in 96-well plates, and cell attachment was allowed for 12 h. Then, a 10 μl CCK8 test solution was added to each well at 24, 48, 72, and 96 h, and the cells were incubated in a humidified incubator with 5% CO2 at 37°C for 2 h at each time point to evaluate the cell growth viability. The optical density (OD) was measured at 450 nm with a microplate reader . For EdU assays, the experiments were performed according to the manufacturer’s instructions (Cell Counting Kit 8 (CCK8) assays  and 5-ethynyl-2 deoxyuridine (EdU) assays  were performed to assess the cell proliferation ability. For CCK8 assays, cells were seeded at approximately 2×10ructions . Then, t® Reagent  and reverse transcribed into cDNA using a cDNA synthesis kit (Thermo Scientific) according to the manufacturer’s instructions. qRT-PCR was performed as previously reported , and β-catenin , and the secondary antibody was horseradish peroxidase-conjugated goat antimouse . Protein expression was observed and visualized by chemiluminescence using an Alpha Imager scanner .The cells from each sample were collected and lysed in radioimmunoprecipitation (RIPA) buffer (Thermo Scientific), followed by centrifugation at 1,200×escribed . The prit-test. All statistical analyses were performed using SPSS (version 17.0). Statistical significance was set at p < 0.05.All data are expressed as the mean ± standard deviation, and statistical significance was determined using Student’s p < 0.05 and |log FC| > 1 revealed a total of 4,939 differentially expressed lncRNAs . A volcano map was used to show the distribution of all differentially expressed lncRNAs , which were represented on a volcano map . Subsequepidermis development), GO:0001942 (hair follicle development), and GO:0007010 (cytoskeleton organization). In terms of CCs (Staphylococcus aureus infection), and the downregulated DEGs were mainly enriched in hsa05144 , hsa04151 , and hsa04350 .To further explore the pathogenesis of AGA, we performed GO and KEGG enrichment analyses to further reveal the enrichment status of the DEGs in terms of their MFs, BPs, CCs, and pathways. With regard to BPs , the uprs of CCs , the majs of CCs , the uprs of CCs . The mosNext, the PPI networks of the common DEGs were analyzed using GeneMANIA, and the 20 most relevant genes were identified. GPM6B, SOX10, MBP, PIM2, CDH19, PMP2, GPM6A, PTPRN, MAG, TNFRSF17, ENPP6, ENPP2, SP2, ITGAV, SPI1, PIGO, CD79A, ENPP7, PIGG, and ENPP5 were primarily associated with the PPI network of the upregulated common genes , and COLβ1, Wnt10b, and β-catenin in AC010789.1-OE. The results showed that the expression of DKK-1 and TGF-β1 was significantly downregulated, whereas the expression of Wnt10b and β-catenin was significantly upregulated in AC010789.1-OE compared with control cells (β-catenin pathway.According to the results of KEGG pathway enrichment analysis, a majority of the enriched pathways were highly associated with Wnt/β-catenin signaling . Thus, wol cells . These dAGA is an androgen-dependent genetic hair loss disorder characterized by progressive microencapsulation of hair follicles and continuous shortening of the hair follicle growth period . It is cSeveral studies indicate that lncRNAs play essential roles in the occurrence and progression of various diseases and that they could be used as new diagnostic and treatment markers. For example, α-dihydrotestosterone-induced HFSC apoptosis by inhibiting the PI3K-Akt pathway, thereby delaying the progression of AGA. HFSC aging, characterized by a reduction in stemness signatures and a concomitant increase in epidermal commitment, leads to a progressive miniaturization of hair follicles and ultimately, to the hair loss characteristic of AGA . RecentlEmerging evidence indicates that a large number of lncRNAs participate in a variety of biological functions by interacting with miRNAs and regulating their target genes . In thisβ1 and Wnt signaling pathways are the most crucial pathways for maintaining a quiescent niche and regulating the proliferation and differentiation of HFSCs (β1 promotes telogen-to-anagen transition in hair follicles, whereas the transition from anagen to telogen is significantly delayed in the hair follicles of TGF-β1 knockout mice (β-catenin signaling pathway. Previous studies reveal that inhibiting the expression of miR-21 can lead to upregulation of the Wnt/β-catenin pathway, thereby promoting cell activity (β-catenin signaling (β signaling pathway, and a majority of the pathways were highly correlated with the Wnt/β-catenin signaling pathway. Further analysis showed that AC010789.1 overexpression induced the upregulation of Wnt10b and β-catenin, and downregulation of DKK-1 and TGF-β1. In summary, our results suggest that AC010789.1 regulates Wnt/β-catenin pathway activation, thereby enhancing the proliferation and differentiation of HFSCs and participating in AGA progression.The TGF-of HFSCs . Previouout mice . The Wntout mice . In the out mice . Moreoveout mice  found thactivity . A recenignaling . In thisβ-catenin signaling pathway (In summary, our data shows that AC010789.1 overexpression delayed AGA progression through the downregulation of hsa-miR-21-5p and promotion of the Wnt/ pathway . Our fin