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Trancsriptomic analyses reveal aberrant differentiation trajectory with WNT1 Loss-of-Function in type XV osteogenesis imperfecta

ABSTRACT: Osteogenesis imperfecta (OI) is a serious genetic bone disorder characterized by congenital low bone mass, deformity and frequent fractures. Type XV OI is a moderate to severe form of skeletal dysplasia caused by WNT1 mutations. In this cohort study from southern China, we summarized the clinical phenotypes of patients with WNT1 mutations and found the proportion of type XV patients was around 10.3% (25 out of 243) with diverse phenotypic spectrums. Functional assays indicated that mutations of WNT1 significantly impaired its secretion and effective activity, leading to moderate to severe clinical manifestations, porous bone structure and enhanced osteoclastic activities. Analysis of proteomic data from human skeleton indicated that the expression of SOST was dramatically reduced in type XV patients. Single-cell transcriptome data generated from human tibia samples revealed aberrant differentiation trajectory of skeletal progenitors and impaired maturation of osteocytes, resulting in excessive CXCL12+ progenitors and abnormal cell populations with adipogenic characteristics. The integration of multi-omics data from human skeleton delineates how WNT1 regulates the differentiation and maturation of skeletal progenitors, which will provide a new direction for the treatment strategy of type XV osteogenesis imperfecta and relative low bone mass diseases such as early onset osteoporosis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE262092 | GEO | 2024/08/13

REPOSITORIES: GEO

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Project description:Osteogenesis Imperfecta (OI) is a genetic, rare disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of the cases are due to mutations in COL1A1 or COL1A2 genes which code for the type I collagen molecule. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cell type in the bone, have been proposed and tested as an innovative therapy for OI with promising but transient outcomes. In this study, we performed a phase I clinical trial based on reiterative infusions of histocompatible MSCs in two pediatric patients affected by severe and moderate OI. The aim of this study was to assess the safety and effectiveness of this cell therapy in non-immunosuppressed OI patients. The host response to MSCs was also evaluated by collecting the sera from OI patients before, along and after the cell therapy. The results of this clinical trial are twofold: the sequential administration of MSCs is safe and improve the bone parameters and quality of life of OI patients. Moreover, MSCs therapy elicits a pro-osteogenic paracrine response in patients, especially noticeable in that affected by severe OI. These results indicate the feasibility and potential of reiterative MSCs infusion for OI and highlight the paracrine response shown by OI patients as a consequence of MSCs treatment. Study design and patients: The Mesenchymal Stem Cell Therapy for the Treatment of Osteogenesis Imperfecta (TERCELOI) study (www.clinical trials.gov: # NCT02172885, EudraCT number: 2012-002553-38) is an independent multi-center Phase I clinical trial to evaluate the feasibility, safety and potential efficacy of infused sibling HLA-matched MSCs in non-immunosuppressed children with OI. PBMCs isolation and serum collection: Venous blood samples were collected before the cell therapy (basal serum), along the cell therapy (1 week, 1 month and 4 months after each MSCs infusion) and during the follow-up period (1 and 2 years after the fifth and last MSCs infusion). MLR assay: PBMCs isolated from patients were resuspended in PBS+FBS (5%) and stained with carboxyfluoroscein succinate-ester (CFSE) (Molecular Probes, USA). Bone mineral density: Bone mineral density (BMD) at the lumbar spine (LS) from L1 to L4, was measured by dual energy x-ray absorptiometry (DXA) on a whole-body scanner within a pediatric platform (Hologic QDR densitometer). Raw measurements were converted to Z-scores for analysis using reference standards for age and pubertal status. OI-MSCs isolation and characterization and culture: OI-MSCs were derived from discarded and donated bone fragments of OI pediatric patients undergoing corrective surgery. All patients suffered Type III-IV OI, with mutations in either COL1A1 or COL1A2 genes. The donation was approved by the Basque Clinical Research Ethics Committee. Briefly, bone chips were mechanically flushed with PBS and then cut into small pieces to extrude cells without the use of enzymes. The cut bone pieces were let undisturbed during 14 days until OI-MSCs migrated to cell culture plate. OI-MSCs were assessed for the expression of CD105, CD90 and CD73 and the absence of CD14, CD34, CD45, CD19 and HLA-DR. MSCs were also tested for their potential to differentiate to osteoblasts and adipocytes using specific cell culture media. OI-MSCs were cultured under standard growth medium (DMEM low glucose with glutamax (Gibco), penicillin/streptomycin (Gibco) and fetal bovine serum at 10% (Sigma-Aldrich, USA). Osteogenesis induction medium (OIM) was composed of standard medium plus ascorbic acid 0.2 mM, beta-glycerophosphate 10 mM, and dexamethasone 10 nM (all from Sigma-Aldrich). When specified, FBS was replaced by P01 and P02 serum samples at 2.5%. RNAseq and Q-PCR validation: OI-MSCs from 10 OI pediatric patients were seeded in 96-well plates (1 000 cells/well) and the following day cultured in the presence of OIM. Next, total RNA was isolated with the AllPrep kit (QIAGEN, USA). cDNA library (TrueSeq stranded Total cDNA library, Illumina, USA) and sequencing at HiSeq 4000 (PE100nt, 50 million reads/sample).

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Trancsriptomic analyses reveal aberrant differentiation trajectory with WNT1 Loss-of-Function in type XV osteogenesis imperfecta

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