Maintaining the native morphology and characteristic staining patterns of dissected tissues is critical for histological analysis. Reliable preservation enables accurate assessment of structural integrity and cellular components, which is fundamental to identifying viable avenues for tissue regeneration and improving clinical outcomes. Acknowledging the constraints of conventional paraffin embedding, we propose an alternative method employing gelatin as an embedding medium. Gelatin is a natural, water-soluble protein derived from collagen, known for its biocompatibility. As a hydrogel, it provides a supportive matrix that closely mimics the natural extracellular matrix of soft tissues facilitating excellent preservation of structural and molecular features during the histological process. Here we demonstrate that this strategy offers superior support for fragile tissues, including fibrocartilage and hyaline cartilage across various anatomical sites, such as the temporomandibular joint, the knee joint, the long bone growth plate, and the intervertebral disc. To assess the maintenance of tissue morphology and matrix composition, histological sections were subjected to various staining techniques, including hematoxylin and eosin, Masson's trichome, safranin O, immunofluorescence, and von Kossa staining. Gelatin embedding resulted in superior maintenance of fibrocartilage architecture, as evidenced by histological evaluation. In addition, quantification of safranin O staining showed significantly greater glycosaminoglycan content in gelatin-embedded samples.

Gastric cancer primarily originates from gastric stem/progenitor cells and is driven by somatic mutations. Although Helicobacter pylori is the main risk factor, how it drives malignancy is still not well understood. Analysis of single-cell RNA sequencing data reveals that human gastric cancer correlates with suppressed BMP signalling, a crucial niche signal for gastric stem cells, in stromal cells rather than epithelial cells. Genetic disruption of BMP signalling in Col1a2+ stromal cells or Acta2+ myocytes/pericytes alone, but not in gastric stem cells themselves, triggers mutations in gastric stem cells and initiates cancer development. Mechanistically, loss of BMP signalling increases stromal production of Wnt ligands, which dose-dependently drive transcription-replication collisions, R-loops, and DNA damage in gastric stem cells. The resulting DNA damage and carcinogenesis can be prevented by small-molecule inhibitors targeting Wnt pathway. Importantly, we identify inflammation as a key disruptor of stromal BMP signalling, as seen in patient samples with chronic atrophic gastritis and H. pylori-infected mouse gastric samples, which is associated with DNA damage. Together, these findings show how chronic inflammation derails niche signalling to drive stem cell mutations and gastric cancer and highlight promising avenues for early prevention.

KSQ-4279 CryoEM workflow and resolution distribution

USP1/UAF1 CRISPR dependency profiles

CRISPR dropout scores for USP1 and UAF1

The temporomandibular joint (TMJ), essential for jaw movements, is susceptible to osteoarthritis, impacting a significant portion of the population. This study introduces an innovative genetic mouse model to explore TMJ development, maintenance, and interactions with the mechanical environment. We exploited Evc2/Limbin conditional KO (Evc2 cKO) mice, specifically targeting neural crest cell-derived tissues (Wnt1Cre), to observe TMJ development. Disruption of Evc2 in neural crest cells contributed to morphological changes in the TMJ growth plate cartilage layers, predisposing the joint components to defects. Condyle defective regions presented a unique environment composed of cartilage, bone, stem cells, and an augmented polymorphic layer. Our findings further revealed that the Evc2 cKO mice presented TMJ components degeneration clinically like those observed in human TMJ-osteoarthritis (OA). Mandible condyle gene expression analysis showed augmented expression of general inflammatory and OA markers. Supplying the mice with regular chow worsens the phenotype, but soft chow fed partially rescued both condyle morphology and intra-articular space. The data suggest that changes in the loading environment critically affect the integrity and functionality of the TMJ, with direct implications for its preservation and disease management.

IHC staining of KSQ-4279 treated tumors

Western blot analysis of sensitive and insensitive cell lines to USP1 inhibition

List of antibodies used for immunoblotting

Craniosynostosis is a debilitating congenital anomaly characterized by the premature fusion of cranial sutures in the skull, resulting from the abnormal fate specification of critical skeletal stem cells. This leads to disrupted craniofacial development and dysmorphology. As an alternative to invasive cranial vault remodeling surgery, we propose a tissue engineering approach to effectively restore the damaged or absent suture stem cell niche using a biomaterial capable of regioselective stem cell maintenance, marking a significant paradigm shift. By harnessing the role of biomaterial scaffold pore design to direct cell fate, we developed a “bone-suture-bone” design within a triphasic scaffold. We demonstrate that this unique scaffold design maintains stemness in a central region while promoting osteogenic differentiation in the surrounding areas, replicating the interfaces and function of the native suture. The scaffold’s ability to reconstitute an engineered skeletal stem cell niche and facilitate the functional recovery of the craniosynostosis phenotype is validated in a caBmpr1a; Wnt1-Cre murine model of midline craniosynostosis, the most common nonsyndromic clinical presentation in humans. This work shows significant promise for improving patient outcomes through a rational tissue engineering strategy for reconstituting a native stem cell niche.

The immune system is not only essential for host defense, but it is also involved in tissue maintenance and disease pathogenesis. Macrophages play a key role in tissue repair, fibrosis, and tumorigenesis, but the mechanisms underlying their multifunctionality have not been fully explored. Here, we identified Mrep (Ly6ChiCX3CR1loPDPN+CD9+) as a crucial subset of macrophages for muscle regeneration after muscle injury. Muscle regeneration required Mrep-derived activin A, which was produced via the TLR4/TIR domain-containing adapter-inducing interferon-β/TANK-binding kinase 1/interferon regulatory factor 3/7 signaling pathway in response to muscle injury. Mrep exerted pathological effects by secreting activin A in a model of genetically induced heterotopic ossification (HO), which was suppressed by TLR4 inhibition. Thus, this study elucidates the context-dependent functions of macrophages and the link between injury and HO, suggesting that Mrep is a potential therapeutic target for regenerating muscles and suppressing HO.

The pathologic, osteogenic differentiation of fibroadipogenic progenitor cells (FAPs) is the primary recognized contributor to ectopic bone formation in fibrodysplasia ossificans progressiva (FOP) and trauma-induced heterotopic ossification (HO). Both conditions are characterized by up-regulated BMP signaling - the former by a gene mutation rendering the BMP receptor ACVR1 susceptible to activation by inflammatory ligands (Activin A), and the latter by up-regulated presence of BMP2 ligand in the setting of unmutated BMP receptor. We performed an unbiased assessment of FDA-approved therapies which would optimally target the transcriptional aberrations observed in developing FOP and HO lesions based on publicly-available datasets. This analysis uncovered rosiglitazone, a peroxisome proliferator-activated receptor gamma (PPARγ) agonist as the highest scoring therapeutic option across three data sets for both conditions. Rosiglitazone treatment eliminated ectopic bone lesions in a mouse model of FOP, and replaced these lesions with ectopic adipose tissue; similarly, systemic and local rosiglitazone treatment eliminated ectopic bone lesions in a mouse model of trauma-induced HO and replaced these lesions with ectopic adipose tissue. Our findings were corroborated by a single case report from 2010 showing positive results with rosiglitazone in a non-diabetic patient with FOP, with no subsequent studies. Overall, our findings suggest that a previously FDA-approved therapeutic is likely to be a successful therapeutic agent for both FOP and trauma-induced HO, both conditions for which current therapeutic options remain inadequate.

Combination studies using KSQ-4279 and PARP inhibitors

Tuberous sclerosis complex 1 (TSC1) is a negative regulator of mTORC1 signaling, a key pathway in skeletal homeostasis, but its region- and compartment-specific effects on bone remain unclear. Here, we examined the skeletal impact of Tsc1 deletion using the 8-kb Dmp1-Cre line, which targets mature osteoblasts and osteocytes. Cre activity in Ai14 reporter mice showed comparable recombination in craniofacial, appendicular, and axial bones. Conditional Tsc1 deletion resulted in greater bone mass at multiple sites, but with marked regional and compartmental variations. In the craniofacial skeleton, the frontal bone showed the most robust elevation in thickness and volume, the parietal bone displayed intermediate elevation, and the mandible exhibited the smallest elevation but reduced bone volume fraction and tissue mineral density, consistent with greater porosity. In femurs, both trabecular and cortical bone mass was augmented. Histomorphometric analysis of femoral trabecular bone revealed increased osteoblast number and surface together with reduced osteoclast parameters. In vertebrae, Tsc1 deletion produced modest trabecular bone accrual but > 100

The temporomandibular joint (TMJ) relies on specialized progenitor cells for tissue maintenance and repair. We characterized TMJ-derived progenitor cells in mice and investigated the role of Evc2-mediated Hedgehog signaling. Progenitor cells from the anterior TMJ exhibited greater colony-forming capacity and an elongated morphology, while posterior cells were cuboidal, highlighting regional heterogeneity. TMJ-derived progenitors demonstrated multipotency, differentiating into osteogenic and chondrogenic lineages. Gli1-expressing, slow-cycling cells localized to the ligament attachment regions, initially accumulating there and not overlapping with specialized cells (Col1+ cells). Conditional Evc2 disruption in Gli1-expressing cells paradoxically augmented expression of Gli1 and mechanosensors (Yap, Wwtr1, Piezo1), and produced more confluent, rapidly expanding colonies. We hypothesize that these colonies are primarily composed of transit amplifying cells (TACs), which may proliferate robustly but face challenges in terminal differentiation. These results reveal critical roles for EVC2 and regional progenitor cell diversity in TMJ regenerative biology and suggest that targeting cell signaling and mechanical factors may inform novel strategies for TMJ disorder therapies.

Clonogenic assays in the presence of KSQ-4279 and olaparib in combination

Mouse body weight measurements following treatment with KSQ-4279 and olaparib

Abstract X chromosome inactivation (X-inactivation) is generally regarded as a dosage-compensation mechanism restricted to female mammals. Here we show that BMP signaling induces X-inactivation through upregulation of Xist and promotes chondrogenesis in both sexes. Remarkably, augmented BMP signaling induced ectopic X-inactivation: transiently inactivating both X chromosomes in females and one in males in a tissue-specific manner. In cranial neural crest cells, ectopic X-inactivation suppressed X-linked gene Tmsb4x , leading to ectopic cartilage formation. Genetic reduction of Xist or pharmacological restoration of the Tmsb4x product suppressed this phenotype. Moreover, we identified ectopic X-inactivation in SOX9-positive chondroprogenitors during wild-type forelimb development in both sexes. Inhibition of X-inactivation disrupts proximodistal limb patterning ex vivo . These findings establish X-inactivation as a signaling-dependent developmental mechanism linking chromosome-scale gene alterations to skeletal fate specification.

CRISPR dropout scores for USP1 and UAF1

Engineered cell therapies present an opportunity for endogenous, site-specific production of therapeutic agents. Fibrodysplasia ossificans progressiva (FOP) is a morbid condition in which patients develop extensive heterotopic bony lesions in response to aberrant sensitivity to Activin A, through a mutation in the type I bone morphogenetic protein (BMP) receptor ACVR1 (ACVR1 R206H). We designed a transposon plasmid containing the transgene encoding ActR2A-Fc, with expression controlled by the BMP-responsive element (BRE), which is pathologically activated upon exposure to Activin A in FOP cells. FOP-derived marrow cells modified with this plasmid exhibited closed-loop functionality, with increased ActR2A-Fc expression upon exposure to Activin A and reduced expression upon its withdrawal. Bone marrow transplantation of labeled engineered FOP marrow cells into FOP mice resulted in the trafficking of engineered cells to sites at risk of FOP and a reduction of heterotopic bony lesions. These data provide proof-of-concept and a blueprint for marrow-derived cell therapies across the disease spectrum.