From mesenchymal niches to engineered in vitro model systems: Exploring and exploiting biomechanical regulation of vertebrate hedgehog signalling

Ghuloum, Fatmah I.; Johnson, Colin A.; Galdo, Natalia A. Riobo-Del; Amer, Mahetab H.

doi:10.1016/j.mtbio.2022.100502

Cited by 5 publications

(5 citation statements)

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“…Proliferation rates in 3D-cultured systems better represent in vivo models than 2D cultures [52]. The differences in C3H10T1/2 cell morphologies when cultured on smooth and dimpled microparticles agree with those we previously reported using human MSCs [2], which are hypothesised to be due to preferential use of different mechano-transducers to adapt to the different physical microenvironments [53]. Human MSCs attachment on dimpled microparticles was reported to be mediated primarily via integrins α 5 and α v β 3, unlike those cultured on smooth microparticles [2].…”

Section: Discussionsupporting

confidence: 86%

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Towards Modular Engineering of Cell Signalling: Topographically-Textured Microparticles Induce Osteogenesis via Activation of Canonical Hedgehog Signalling

Ghuloum

Stevens

Johnson

et al. 2023

Preprint

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Polymer microparticles possess great potential as functional building blocks for advanced bottom-up engineering of complex tissues. Tailoring the three-dimensional architectural features of culture substrates has been shown to induce osteogenesis in mesenchymal stem cells in vitro, but the molecular mechanisms underpinning this remain unclear. This study proposes a mechanism linking the activation of Hedgehog signalling to the osteoinductive effect of surface-engineered, topographically-textured polymeric microparticles. In this study, mesenchymal progenitor C3H10T1/2 cells were cultured on smooth and dimpled poly(D,L-lactide) microparticles to assess differences in viability, cellular morphology, proliferation, and expression of a range of Hedgehog signalling components and osteogenesis-related genes. Dimpled microparticles induced osteogenesis and activated the Hedgehog signalling pathway relative to smooth microparticles and 2D-cultured controls without the addition of exogenous biochemical factors. We observed upregulation of the osteogenesis markers Runt-related transcription factor2 (Runx2) and bone gamma-carboxyglutamate protein 2 (Bglap2), as well as the Hedgehog signalling components, glioma associated oncogene homolog 1 (Gli1), Patched1 (Ptch1), and Smoothened (Smo). Treatment with the Smo antagonist KAAD-cyclopamine confirmed the involvement of Smo in Gli1 target gene activation, with a significant reduction in the expression of Gli1, Runx2 and Bglap2 (p less than or equal to 0.001) following KAAD-cyclopamine treatment. Overall, our study demonstrates the role of the topographical microenvironment in the modulation of Hedgehog signalling, highlighting the potential for tailoring substrate topographical design to offer cell-instructive 3D microenvironments. Topographically-textured microparticles allow the modulation of Hedgehog signalling in vitro without adding exogenous biochemical agonists, thereby eliminating potential confounding artefacts in high-throughput drug screening applications.

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Section: Discussionsupporting

confidence: 86%

“…Smo translocates to the primary cilia in response to Hh ligands, with its activation promoting the release of Gli proteins from their carrier protein, Suppressor of Fused (Sufu). Direct transcriptional targets of Hh signalling include Gli1 and Ptch1 [53,92] (Fig. 8).…”

Section: Discussionmentioning

confidence: 99%

Towards Modular Engineering of Cell Signalling: Topographically-Textured Microparticles Induce Osteogenesis via Activation of Canonical Hedgehog Signalling

Ghuloum

Stevens

Johnson

et al. 2023

Preprint

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“…Dicam, as a regulator of primary cilia-mediated Indian hedgehog (Ihh) signaling in chondrocytes, can promote proliferation and maturation of growth plate chondrocytes both in vivo and in vitro (Han et al, 2018). The regulation of Hedgehog signaling in mechanically sensitive bone marrow mesenchymal stromal cells offers a new approach to modeling skeletal diseases and provides new opportunities for targeted therapies against the extracellular matrix (Ghuloum et al, 2022). Given the close relationship between Frontiers in Genetics frontiersin.org the ECM and OA, it is possible that the hedgehog signaling pathway affects OA development by influencing ECM involvement.…”

Section: Discussionmentioning

confidence: 99%

Characterizing hedgehog pathway features in senescence associated osteoarthritis through Integrative multi-omics and machine learning analysis

Wang,

Li,

Zhao

et al. 2024

Front. Genet.

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Purpose: Osteoarthritis (OA) is a disease of senescence and inflammation. Hedgehog’s role in OA mechanisms is unclear. This study combines Bulk RNA-seq and scRNA-seq to identify Hedgehog-associated genes in OA, investigating their impact on the pathogenesis of OA.Materials and methods: Download and merge eight bulk-RNA seq datasets from GEO, also obtain a scRNA-seq dataset for validation and analysis. Analyze Hedgehog pathway activity in OA using bulk-RNA seq datasets. Use ten machine learning algorithms to identify important Hedgehog-associated genes, validate predictive models. Perform GSEA to investigate functional implications of identified Hedgehog-associated genes. Assess immune infiltration in OA using Cibersort and MCP-counter algorithms. Utilize ConsensusClusterPlus package to identify Hedgehog-related subgroups. Conduct WGCNA to identify key modules enriched based on Hedgehog-related subgroups. Characterization of genes by methylation and GWAS analysis. Evaluate Hedgehog pathway activity, expression of hub genes, pseudotime, and cell communication, in OA chondrocytes using scRNA-seq dataset. Validate Hedgehog-associated gene expression levels through Real-time PCR analysis.Results: The activity of the Hedgehog pathway is significantly enhanced in OA. Additionally, nine important Hedgehog-associated genes have been identified, and the predictive models built using these genes demonstrate strong predictive capabilities. GSEA analysis indicates a significant positive correlation between all seven important Hedgehog-associated genes and lysosomes. Consensus clustering reveals the presence of two hedgehog-related subgroups. In Cluster 1, Hedgehog pathway activity is significantly upregulated and associated with inflammatory pathways. WGCNA identifies that genes in the blue module are most significantly correlated with Cluster 1 and Cluster 2, as well as being involved in extracellular matrix and collagen-related pathways. Single-cell analysis confirms the significant upregulation of the Hedgehog pathway in OA, along with expression changes observed in 5 genes during putative temporal progression. Cell communication analysis suggests an association between low-scoring chondrocytes and macrophages.Conclusion: The Hedgehog pathway is significantly activated in OA and is associated with the extracellular matrix and collagen proteins. It plays a role in regulating immune cells and immune responses.

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“…There is also controversy regarding the Hh ligands regulate osteogenesis. The role of Ihh in endochondral ossification is well established [ 50 ]. However, there is also evidence suggesting the crucial role for Ihh in the intramembranous ossification of craniofacial bones [ 51 , 52 ].…”

Section: Discussionmentioning

confidence: 99%

Distraction force promotes the osteogenic differentiation of Gli1+ cells in facial sutures via primary cilia-mediated Hedgehog signaling pathway

Jin,

An,

Wang

et al. 2024

Stem Cell Res Ther

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Background Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown. Methods 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling. Results Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force. Conclusions Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.

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From mesenchymal niches to engineered in vitro model systems: Exploring and exploiting biomechanical regulation of vertebrate hedgehog signalling

Cited by 5 publications

References 321 publications

Towards Modular Engineering of Cell Signalling: Topographically-Textured Microparticles Induce Osteogenesis via Activation of Canonical Hedgehog Signalling

Towards Modular Engineering of Cell Signalling: Topographically-Textured Microparticles Induce Osteogenesis via Activation of Canonical Hedgehog Signalling

Characterizing hedgehog pathway features in senescence associated osteoarthritis through Integrative multi-omics and machine learning analysis

Distraction force promotes the osteogenic differentiation of Gli1+ cells in facial sutures via primary cilia-mediated Hedgehog signaling pathway

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