Biology subjects

van Hoolwerff, M.

Publications and source records attributed to van Hoolwerff, M..

4 recordsLinked to original sources

bioRxiv2026

Nuclear Factor I genes drive chondrogenic cell-fate commitment

Human induced pluripotent stem cells (hiPSCs) offer a powerful platform to model chondrogenesis and enable regenerative strategies, yet regulation of cell-fate commitment remains elusive. Here, we systematically mapped cell-fate trajectories from 7 time points during a 49-day chondrogenic hiPSC differentiation protocol using single-nucleus multimodal transcriptomic and chromatin accessibility profiling (scRNA-seq and scATAC-seq). Integrative analysis of dynamics revealed branching differentiation trajectories with clear bifurcation points and distinct cell-fates. Notably, the chondrogenic trajectory originated at day 6 as a neurogenic development and branched off at day 21 to a chondrogenic cell-fate. Through transcription factor activity analysis (TFAA) and cis-co-accessibility networks, we found that NFIA and NFIB drove chondrogenic distinction, exhibited in both modalities as directly targeting chondrogenic genes such as COMP, FIBIN, VIM. This was then confirmed by experimental validation as modulation of NFIA expression at this point further enhanced chondrocyte formation. Together, our study provides a high-resolution multimodal atlas of chondrogenic differentiation and identified critical transcriptional regulators that can now be leveraged to implement regenerative cartilage therapies from hiPSCs.

cell biology↗

bioRxiv2022

WWP2 osteoarthritis risk allele rs1052429-A confers risk by affecting cartilage matrix deposition via hypoxia associated genes

ObjectiveTo explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage. MethodsCo-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N=35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N=8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways. ResultsUpon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (|{rho}|>0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1, and WNK4. Subsequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2, and GJA1. Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregulation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1. ConclusionsMimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detrimental processes in the cartilage shown by a response in hypoxia associated genes EPAS1,GDF10, and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN.

cell biology↗

bioRxiv2020

Elucidating another level of epigenetic regulation in osteoarthritis by identifying functional cis-acting long non-coding RNAs and their targets in articular cartilage

ObjectiveTo identify robustly differentially expressed long non-coding RNAs (lncRNAs) with osteoarthritis (OA) pathophysiology in cartilage. Moreover to explore potential target mRNAs by establishing co-expression networks, followed by functional validation. MethodsRNA sequencing was performed on macroscopically lesioned and preserved OA cartilage of patients who underwent a joint replacement surgery due to OA (N=98). Differential expression (DE) analysis was performed on lncRNAs that were annotated in GENCODE and Ensembl. To identify potential interactions, correlations were calculated between the identified DE lncRNAs and previously reported DE protein-coding genes in the same samples. Modulation of chondrocyte lncRNA expression was achieved using LNA GapmeRs. ResultsBy applying our in-house pipeline we identified 5,053 lncRNAs to be robustly expressed, of which 191 were FDR significant differentially expressed between lesioned and preserved OA cartilage. Upon integrating mRNA sequencing data, we showed that intergenic and antisense DE lncRNAs show high, positive correlations with their flanking, respectively, sense genes. To functionally validate this observation we selected P3H2-AS1, which was downregulated in primary chondrocytes, resulting in downregulation of P3H2 gene expression levels. As such, we can confirm that P3H2-AS1 regulates its sense gene P3H2. ConclusionBy applying an improved detection strategy, robustly differentially expressed lncRNAs in OA cartilage were detected. Integration of these lncRNAs with differential mRNA expression levels in the same samples showed insight into their regulatory networks. Our data signifies that intergenic, as well as antisense lncRNAs play an important role in regulating the pathophysiology of OA.

genomics↗

bioRxiv2020

RNA sequencing reveals interacting key determinants of osteoarthritis acting in subchondral bone and articular cartilage.

ObjectiveThe aim of this study was to identify key determinants of the interactive osteoarthritis (OA) pathophysiological processes of subchondral bone and cartilage. MethodsWe performed RNA sequencing on macroscopically preserved and lesioned OA subchondral bone of patients that underwent joint replacement surgery due to OA (N=24 pairs; 6 hips, 18 knees, RAAK-study). Unsupervised hierarchical clustering and differential expression analyses were performed. Results were combined with previously identified, differentially expressed genes in cartilage (partly overlapping samples) as well as with recently identified OA risk genes. ResultsWe identified 1569 genes significantly differentially expressed between lesioned and preserved subchondral bone, including CNTNAP2 (FC=2.4, FDR=3.36x10-5) and STMN2 (FC=9.6, FDR=3.36x10-3). Among the identified genes, 305 were also differentially expressed and with same direction of effects in cartilage, including IL11 and CHADL, recently acknowledged OA susceptibility genes. Upon differential expression analysis stratifying for joint site, we identified 509 genes exclusively differentially expressed in subchondral bone of the knee, such as KLF11 and WNT4. These exclusive knee genes were enriched for involvement in epigenetic processes, characterized by for instance HIST1H3J and HIST1H3H. ConclusionTo our knowledge, we are the first to report on differential gene expression patterns of paired OA subchondral bone tissue using RNA sequencing. Among the most consistently differentially expressed genes with OA pathophysiology in both bone and cartilage were IL11 and CHADL. As these genes were recently also identified as robust OA risk genes they classify as attractive druggable targets acting on two OA disease relevant tissues.

genomics↗