Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic profiling of LAMA5-deficient human stem cell–derived chondrogenesis reveals a human-specific ECM–WNT signaling axis

ABSTRACT: This study investigates the transcriptional consequences of Laminin-α5 (LAMA5) loss in human urine-derived stem cells (USCs) to understand the molecular etiology of idiopathic short stature. The experiment consists of two components: 1. Differentiation Profiling: CRISPR/Cas9-mediated LAMA5 knockout (KO) USCs and wild-type (WT) controls were profiled in three conditions: undifferentiated state, osteogenic differentiation, and chondrogenic differentiation. This analysis aims to interpret how cell-matrix interactions regulate lineage-specific transcriptional programs and spheroid architecture. 2. Pharmacological Rescue: To assess the functional role of canonical WNT signaling in LAMA5-mediated regulation, LAMA5 knockout USCs were undergoing chondrogenic differentiation were treated with the WNT agonist Lithium Chloride (LiCl) or left untreated. Data analysis identifies a LAMA5-dependent gene regulatory network enriched for limb morphogenesis factors (including WNT7A, PITX1, and FLI1) and demonstrates partial restoration of this network upon β-catenin stabilization via LiCl. Study Design / Samples Total Samples: 24 Organism: Homo sapiens Cell Type: Urine-derived stem cells (USCs) Detailed Sample List: Condition 1 (Genotype x Lineage): Comparison of WT vs. LAMA5 KO across three differentiation stages (n=18). 3x Wild-type (WT) - Undifferentiated 3x Wild-type (WT) - Osteogenic 3x Wild-type (WT) - Chondrogenic 3x LAMA5 KO - Undifferentiated 3x LAMA5 KO - Osteogenic 3x LAMA5 KO - Chondrogenic Condition 2 (Rescue Treatment): Effect of LiCl treatment on LAMA5 KO cells (n=6). 3x LAMA5 KO (Clone 2) - Untreated (Control) 3x LAMA5 KO (Clone 2) - Treated with LiCl

INSTRUMENT(S): Illumina NovaSeq X, Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Alexander Schulz

PROVIDER: E-MTAB-16566 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mutations in the RMRP gene are the origin of cartilage-hair hypoplasia. Cartilage-hair hypoplasia is associated with severe dwarfism caused by impaired skeletal development. However, it is not clear why mutations in the RMRP gene lead to skeletal dysplasia. Viperin is a known substrate of RMRP. Since chondrogenic differentiation of the growth plate is required for development of the long bones, we hypothesized that viperin functions as a chondrogenic regulator downstream of RMRP. Viperin protein is expressed throughout the stages of chondrogenic differentiation in vivo. Viperin gene expression is increased during knockdown of Rmrp RNA in the ATDC5 model for chondrogenic differentiation. Viperin is expressed during ATDC5 chondrogenic differentiation. Viperin knockdown reduces, while viperin overexpression increases overall protein secretion, with CXCL10 identified as a potential target via mass spectrometry-proteomics. CXCL10 protein expression is reduced during knockdown and increased during overexpression of viperin and CXCL10 protein expression coincides with viperin expression in ATDC5 chondrogenic differentiation. Viperin knockdown induces, while viperin overexpression reduces TGFβ activity. Furthermore, viperin knockdown conditioned media increases, while viperin overexpression conditioned media reduces chondrogenic differentiation of ATDC5 cells. TGFβ target genes Pai1 and Smad7 are increased during knockdown and reduced during overexpression of viperin. Moreover, TGFβ activity is reduced when differentiating ATDC5 cells are exposed to CXCL10 and, acting as a viperin overexpression mimic, CXCL10 similarly reduces chondrogenic differentiation of ATDC5. Lastly, we show that in CHH patient cells, RMRP expression is reduced and viperin expression is increased, coinciding with reduced chondrogenic differentiation and increased CXCL10 expression, possibly explaining the CHH phenotype. Together our data show that viperin may play a pivotal role in chondrogenic differentiation, with potential consequences for cartilage-hair hypoplasia pathobiology.

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Transcriptomic profiling of LAMA5-deficient human stem cell–derived chondrogenesis reveals a human-specific ECM–WNT signaling axis

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