Biology subjects

Sheyn, D.

Publications and source records attributed to Sheyn, D..

9 recordsLinked to original sources

bioRxiv2026

Tendon-derived injectable bio-instructive gel augments tenogenic differentiation of iMSC-SCX cells and extracellular matrix remodeling

In the US, 33 million musculoskeletal injuries have been reported per year, with 50% involving tendons and ligaments in both athletic and aging populations. Tendon repair often results in the formation of biomechanically inferior scar tissue rather than functional regeneration. Local cell therapy is garnering significant interest in tendon repair because it provides a targeted means to repopulate defects with potent therapeutic cells. Here, we developed a porcine tendon-derived thermoresponsive extracellular matrix hydrogel (TG) as an injectable, bio-instructive carrier for scleraxis-overexpressing induced mesenchymal stem cell derived tenocytes (iTenocytes), with the goal of improving cell retention and overall transplantation success. TG was compositionally distinct from purified collagen (PC, from rat tail type 1 collagen) and exhibited favorable material properties for a minimally invasive percutaneous strategy, including thermoresponsive gelation, shear-thinning injectability, retention at the injection site, and controlled biodegradation. In vitro, TG supported three-dimensional cell residence, promoted cell interconnectivity and redistribution at the matrix interface, and increased collagen type I release from iTenocytes compared to those embedded in PC. Transcriptomic and proteomic analyses further showed that TG enhanced programs associated with tenogenic maturation, extracellular matrix assembly, focal adhesion, mechano-transduction, and remodeling. In a rat Achilles tendon partial defect model, both optical imaging and histological analyses demonstrated retention of iTenocytes at the injection site. Additionally, confocal imaging demonstrated retention of iTenocytes within the defect site for up to 10 days. Together, these findings identify TG as a biofunctional injectable carrier that supports the tenogenic characteristics of iTenocytes and enhances their local persistence after transplantation.

cell biology↗

bioRxiv2026

Large oncosomes reprogram bone marrow mesenchymal stem cells to establish a pre-metastatic niche

Metastatic progression depends on the systemic remodeling of distant tissues before tumor cell arrival, yet the cancer-derived signals that orchestrate this process remain poorly understood. Large oncosomes (LOs) are atypically large (>1 um), tumor-derived extracellular vesicles shed by invasive cancer cells. Here, we demonstrate that LOs function as systemic mediators of pre-metastatic niche formation by activating innate immune sensing in bone marrow mesenchymal stem cells (BM-MSCs). Systemic administration of prostate- and breast cancer-derived LOs to immunocompetent tumor-bearing mice did not affect primary tumor growth but increased metastatic burden. LOs induced a robust, dose-dependent interferon-driven inflammatory program, characterized by interferon-stimulated genes and neutrophil chemokines. Mechanistically, this response was driven by LO-associated nucleic acids activating convergent cytosolic DNA- and RNA-sensing pathways in recipient stromal cells. LO-conditioned BM-MSCs promoted the accumulation and polarization of neutrophils toward an immunosuppressive, polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) phenotype. In vivo, genetic suppression of LO shedding in tumor cells reduced PMN-MDSC accumulation in the bone marrow, which was reverted by systemic LO administration. Together, these findings establish LOs as specialized carriers of immunomodulatory signals that reprogram the bone marrow microenvironment to support metastatic colonization, identifying a previously unrecognized mechanism linking tumor vesiculation to immune remodeling at distant sites.

cancer biology↗

bioRxiv2026

Age-Dependent Fibroblast Programs Govern Regenerative and Fibrotic Tendon Repair

Tendon injuries often result in fibrosis, compromising function and predisposing to re-injury. Here, we used a full-width, non-repair Achilles tendon transection model in young (3 weeks old) and adult (18-20 weeks) rats to elucidate the cellular mechanisms governing regenerative versus fibrotic healing. Functional and biomechanical analyses revealed that young tendons recovered motion and load-bearing capacity more rapidly but exhibited more fibrotic early healing. Single-nuclei RNA sequencing identified seven major cell populations within the connective tissue compartment. Adult tendons maintained a "synthetic fibroblast" population marked by upregulated ECM synthesis, reduced stress-related gene expression, whereas young tendons favored expansion of Cxcl12/Lrp6/Gas6 fibrotic fibroblasts linked to oxidative and pro-angiogenic signaling. The young group showed sustained activation of Nox4-Gas6 pathways driving a self-reinforcing fibrotic circuit. These findings define a fibroblast lineage bifurcation that dictates oxidative stress signaling as a key regulator of fibrotic remodeling, highlighting potential therapeutic targets to promote regenerative tendon repair.

developmental biology↗

bioRxiv2026

Modulating Inflammation in Post-Traumatic Osteoarthritis using iPSC-derived Anti-inflammatory Macrophages

Post-traumatic osteoarthritis (PTOA) is a common long-term consequence of joint injury and a major cause of chronic pain and disability, yet no disease-modifying therapies are currently available. A central barrier to effective intervention is the persistence of maladaptive synovial inflammation, driven in part by macrophage-mediated signaling that sustains tissue degeneration and pain. Here, we developed a scalable, chemically defined platform to generate human induced pluripotent stem cell (iPSC)-derived anti-inflammatory macrophages (iMac-M2) as an off-the-shelf cell therapy designed to restore joint immune homeostasis after injury. These cells maintained a stable anti-inflammatory phenotype and function under osteoarthritis-relevant inflammatory conditions and suppressed inflammatory and catabolic responses in human joint cell co-culture systems. In a preclinical model of PTOA, intra-articular delivery of iMac-M2 after injury improved functional and structural outcomes while modulating synovial inflammatory and pain-associated transcriptional programs. Treatment was well tolerated, with no evidence of systemic immune activation or ectopic tissue formation. Together, these findings support iPSC-derived macrophage therapy as a clinically translatable immunomodulatory strategy to interrupt early inflammatory drivers of PTOA and preserve joint health following injury.

cell biology↗

bioRxiv2025

A Porcine Model of Intervertebral Disc Injury Recapitulates Human Discogenic Pain via Notochordal Cell Loss and Pain-inducing Nucleus Pulposus Cell Emergence

Lower back pain (LBP) is one of the most common causes of disability, with up to 40% of LBP cases attributed to intervertebral disc (IVD) degeneration. While small animal models are widely used to study IVD and LBP, the small size of their IVDs limits translational and biological relevance. Large animal models more accurately emulate human disease; however, methods of measuring LBP are not well established. Pigs were also considered unfit for LBP research, due to notochordal cells (NCs) persistence through life, unlike humans. We developed a comprehensive porcine model with quantitative measure of discogenic pain via biobehavioral testing (BBT), MRI, and multi-omics tissue analysis of IVD and DRGs. MRI demonstrated the progression of IVD degeneration beginning at 4 weeks post-injury. BBTs showed the development of significant pain responses by week 4 post-injury, supported by transcriptomics of injury matched DRGs. Single cell transcriptomics, trajectory and cell-cell communication analyses suggest that, with injury, NCs are differentiating to nucleus pulposus cells (NPCs). Furthermore, NPCs showed upregulation of cellular stress, neural outgrowth, and inflammation pathway, consistent with pain-inducing distress signals found in human samples. This study establishes novel MRI and BBT-based methods for quantifying LBP in pigs and supports its translational relevance to human discogenic LBP. The identification of LBP-associated clusters mirrors our previous finding in humans. Moreover, the shift of NC to NPC phenotype further supports that the porcine model is relevant to human pathology, as the injury induced accelerated aging and loss of NCs with IVD degeneration and discogenic pain.

developmental biology↗

bioRxiv2023

Single Cell Transcriptomics-informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineage

During vertebrate embryogenesis, axial tendons develop from the paraxial mesoderm and differentiate through specific developmental stages to reach the syndetome stage. While the main roles of signaling pathways in the earlier stages of the differentiation have been well established, pathway nuances in syndetome specification from the sclerotome stage have yet to be explored. Here, we show stepwise differentiation of human iPSCs to the syndetome stage using chemically defined media and small molecules that were modified based on single cell RNA-sequencing and pathway analysis. We identified a significant population of branching off-target cells differentiating towards a neural phenotype overexpressing Wnt. Further transcriptomics post-addition of a WNT inhibitor at the somite stage and onwards revealed not only total removal of the neural off-target cells, but also increased syndetome induction efficiency. Fine-tuning tendon differentiation in vitro is essential to address the current challenges in developing a successful cell-based tendon therapy.

developmental biology↗

bioRxiv2023

Environmentally stressed human nucleus pulposus cells trigger the onset of discogenic low back pain

Low back pain (LBP) is often associated with the degeneration of human intervertebral discs (IVDs). However, the pain-inducing mechanism in degenerating discs remains to be elucidated. Here, we identified a subtype of locally residing nucleus pulposus cells (NPCs), generated by the environmental stress in degenerating discs, that triggered the onset of discogenic LBP. Single-cell transcriptomic analysis of human tissues showed a strong correlation between this specific pain-triggering subtype and the pain conditions in human degenerated discs. Next, we recreated this pain-triggering subtype by applying known exogenous stressors to healthy NPCs in vitro. The recreated pain phenotype activated functional sensory neurons response in vitro and induced local inflammatory responses, hyperalgesia, and mechanical sensitivity in a healthy rat IVD in vivo. Our findings provide strong evidence of a previously unknown pain-inducing mechanism mediated by NPCs in degenerating IVDs. This newly defined pathway will aid in the development of NPC-targeted therapeutic strategies for clinically unmet need to attenuate discogenic LBP. One Sentence SummaryDiscogenic low back pain can be initiated by a stress-induced subtype of nucleus pulposus cells present in human degenerating intervertebral discs

molecular biology↗

bioRxiv2021

Directing iPSC Differentiation into iTenocytes using Combined Scleraxis Overexpression and Cyclic Loading

Regenerative therapies for tendon are falling behind other tissues due to the lack of an appropriate and potent cell therapeutic candidate. This study aimed to induce cell tenogenesis using stable Scleraxis (Scx) overexpression in combination with uniaxial mechanical stretch of mesenchymal stromal cells (MSCs) of different origins. Scleraxis (Scx) is the single direct molecular regulator of tendon differentiation known to date. Mechanoregulation is known to be a central element guiding tendon development and healing. Cells explored were bone marrow-derived (BM-)MSCs as well as MSCs differentiated from induced pluripotent stem cells (iMSCs). Mechanical stimulation combined with Scx overexpression resulted in morphometric and cytoskeleton-related changes, upregulation of early and late tendon markers, increased ECM deposition and alignment, and tenomodulin perinuclear localization in iMSCs, which was greater compared to BM-MSCs and controls. Our findings suggest that these cells can be differentiated into tenocytes and may be a better candidate for tendon cell therapy applications than BM-MSCs.

cell biology↗

bioRxiv2021

Single-cell Atlas Unveils Cellular Heterogeneity and Novel Markers in Human Neonatal and Adult Intervertebral Discs

The origin, composition, distribution, and function of cells in the human intervertebral disc (IVD) has not been fully understood. Here, cell atlases of both human neonatal and adult IVDs have been generated and further assessed by gene ontology pathway enrichment, pseudo-time trajectory, histology, and immunofluorescence. Comparison of cell atlases revealed the presence of two sub-populations of notochordal cells (NCs) and their associated markers in both the neonatal and adult IVDs. Developmental trajectories predicted 7 different cell states that describe the developmental process from neonatal to adult cells in IVD and analyzed the NCs role in the IVD development. A high heterogeneity and gradual transition of annulus fibrosus cells (AFCs) in the neonatal IVD was detected and their potential relevance in IVD development assessed. Collectively, comparing single-cell atlases between neonatal and adult IVDs delineates the landscape of IVD cell biology and may help discovering novel therapeutic targets for IVD degeneration.

developmental biology↗