Brockmann, E. M. | bioRxiv Science

bioRxiv2025

LAMA5 deficiency disrupts ECM-WNT crosstalk in chondrogenesis and contributes to idiopathic short stature

Idiopathic short stature (ISS) affects 2%-3% of the population and is genetically heterogeneous, with emerging evidence implicating the extracellular matrix (ECM) of the growth plate. We identify LAMA5, encoding laminin-5, as a candidate ISS gene, with rare heterozygous variants present in 1.2% of affected individuals. To define its functional role, we generated CRISPR/Cas9-mediated LAMA5-knockout (KO) urine-derived stem cells (USCs) and induced chondrogenic differentiation in two- and three-dimensional culture systems. Loss of LAMA5 impaired chondrogenesis, with disruption of cell-cell junction programs and abnormal architecture of chondrogenic spheroids. Bulk RNA sequencing combined with weighted gene co-expression network analysis revealed WNT7A and FLI1 as key dysregulated genes within the module most strongly associated with the KO phenotype. Gene Ontology enrichment of this module highlighted embryonic limb morphogenesis as the top biological process, and WNT7A was assigned to canonical WNT signaling. Pharmacologic activation of WNT signaling using lithium chloride (LiCl) partially restored expression of WNT7A, FLI1, TFAP2A, GRHL2, and PITX1 toward wild-type levels, indicating that attenuated WNT activity is a principal downstream consequence of LAMA5 deficiency. Consistent with this, we identified an individual with ISS carrying a heterozygous PITX1 missense variant, supporting convergence of ECM (LAMA5) and transcriptional (PITX1) perturbations on a shared WNT-centered limb-morphogenesis network. Together, these findings demonstrate that laminin-5 is required for proper ECM-WNT signaling integration during human chondrogenesis and suggest that dysregulated WNT activity represents a mechanistic link between LAMA5 dysfunction and impaired endochondral growth. Partial rescue by WNT pathway re-activation highlights a potentially targetable downstream mechanism in ISS pathogenesis.

genetics↗

bioRxiv2024

High-throughput differentiation of human blood vessel organoids reveals overlapping and distinct functions of the cerebral cavernous malformation proteins

Cerebral cavernous malformations (CCMs) are clusters of thin-walled enlarged blood vessels in the central nervous system that are prone to recurrent hemorrhage and can occur in both sporadic and familial forms. The familial form results from loss-of-function variants in the CCM1, CCM2, or CCM3 gene. Despite a better understanding of CCM pathogenesis in recent years, it is still unclear why CCM3 mutations often lead to a more aggressive phenotype than CCM1 or CCM2 variants. By combining high-throughput differentiation of blood vessel organoids from human induced pluripotent stem cells (hiPSCs) with a CCM1, CCM2, or CCM3 knockout, single-cell RNA sequencing, and high-content imaging, we uncovered both shared and distinct functions of the CCM proteins. While there was a significant overlap of differentially expressed genes in fibroblasts across all three knockout conditions, inactivation of CCM1, CCM2, or CCM3 also led to specific gene expression patterns in neuronal, mesenchymal, and endothelial cell populations, respectively. Taking advantage of the different fluorescent labels of the hiPSCs, we could also visualize the abnormal expansion of CCM1 and CCM3 knockout cells when differentiated together with wild-type cells into mosaic blood vessel organoids. In contrast, CCM2 knockout cells showed even reduced proliferation. These observations may help to explain the less severe clinical course in individuals with a pathogenic variant in CCM2 and to decode the molecular and cellular heterogeneity in CCM disease. Finally, the ability to differentiate blood vessel organoids in a 96-well format will further facilitate their use in drug discovery and other biomedical research studies. STATEMENTS AND DECLARATIONSO_ST_ABSConflicts of interest statementC_ST_ABSThe authors declare no competing interests. The here described protocol for high-throughput organoid synthesis has been filed as a patent application at the European Patent Office (Process number: EP24213596.0) Author contribution statementMR, DSk, and UF designed the study. DSk, VS, LM, and RAP performed most of the functional experiments. SH and TA performed the CAM assays. SR performed the immunohistochemical stainings. SB, DSi, DSk, and VS performed the confocal microscopy and high-content imaging analyses. AE, CB, and EMB performed the scRNA sequencing analysis. AW and CAH performed and analyzed the karyotyping of the hiPSC clones. DSk, RAP, VS, KC, MR, and SB analyzed the data. DSk, VS, LM, and MR prepared figures. All authors contributed to the interpretation of the results. DSk, RAP, VS, and MR drafted the manuscript, and all authors contributed to writing. Ethics statementThis study does not involve human participants or animal subjects. Availability of data and materialsAll relevant data are published within the paper and the supplementary files. ScRNA sequencing data can be accessed through the Gene Expression Omnibus (GEO) database (record number: GSE276497).

molecular biology↗