Speakers

Abstract Title: Medium throughput sCNV-seq and msRRBS towards clinic application
Abstract: Single-cell CNV sequencing (scCNV-seq) is essential for genomic analysis, yet existing methods are limited by high cost, biased calling, and reliance on whole-genome preamplification or specialized equipment - major barriers to clinical application where cell numbers are scarce. To address these challenges, we developed msCNVS, a traceable, medium-throughput method that directly labels cells in a microplate using barcoded Tn5 transposomes. This design enables early pooling, eliminates preamplification, and requires no specialized instruments. msCNVS is adaptable and currently processes up to 48 cells (scalable to 96–384), supported by a two-dimensional fitting algorithm for accurate CNV calling. Overall efficiency is improved by more than tenfold. msCNVS reliably distinguished CNV patterns across five cell lines (292 cells), showing high concordance with bulk sequencing (K562: R = 0.90–0.98; HeLa triplicates: R = 0.99) and no detectable cross-contamination. It achieved superior coverage uniformity over MDA and MALBAC, approached the levels of eMDA and DOP-PCR, and exhibited less fluctuation than PTA. Validation was confirmed using karyotyping and microarray in primary amniotic fluid cells. The method successfully detected CNV deletions in abnormal blastocysts (including one case of mosaicism), as well as CNVs in circulating tumor cells, cancerous pleural effusion cells, and patient-derived xenograft (PDX) nuclei. Together, these results establish msCNVS as a robust and efficient CNV-seq solution for precious and rare cells encountered in reproductive and cancer clinics. Similarly we have also developed a highly efficient approach for single cell DNA methylome sequencing: medium-throughput single-cell Reduced Representation Bisulfite Sequencing: msRRBS.

Abstract: Recapitulating the Niche: Optimized Ovarian Cancer Organoids Preserve CD44⁺/LGR5⁺ CSC Populations

Ovarian cancer remains the most lethal gynecologic malignancy, with recurrence driven by therapy-resistant cancer stem cells (CSCs) that survive initial treatment. Conventional two-dimensional (2D) culture systems fail to preserve CSC niches, limiting preclinical drug development. This study establishes a physiologically relevant three-dimensional (3D) organoid platform to investigate CSC biology and evaluate targeted therapeutic strategies. Organoid culture conditions were optimized using OVCAR-3 cells by testing growth factor cocktails containing Noggin, R-spondin-1, Wnt3a, EGF, FGF2, KGF2, and neuregulin-1. Optimized conditions were validated across three ovarian cancer cell lines (OVCAR-3, SKOV3, A2780) and six primary patient tumors (clear cell carcinoma and high-grade serous carcinoma). CSC marker expression (CD44, LGR5, EpCAM) was characterized by immunofluorescence. Drug sensitivity to carboplatin, paclitaxel, dasatinib (SRC/FAK inhibitor), and panobinostat (HDAC inhibitor) was compared between 2D monolayers and 3D organoids. CD44⁺ and LGR5⁺ subpopulations were isolated by magnetic-activated cell sorting for differential sensitivity profiling. Optimal organoid formation required combined supplementation with Noggin, R-spondin-1, Wnt3a, EGF, and FGF2 (92% establishment efficacy vs. 0% in basal medium, p < 0.001). Organoids were successfully established from all three cell lines (88-95% success) and 5/6 patient tumors (83% success), with morphology reflecting histological subtype. Organoids preserved architecturally conserved CSC niches, with 23.4 ± 4.2% CD44⁺, 15.7 ± 3.8% LGR5⁺, and 8.3 ± 2.1% dual-positive cells spatially localized to organoid peripheries and luminal interfaces. Three-dimensional architecture mediated distinct therapeutic responses: dasatinib and panobinostat induced complete organoid dissolution at clinically relevant concentrations (>95% structural loss), whereas carboplatin and paclitaxel achieved only partial disruption even at supratherapeutic concentrations. CD44⁺ cells exhibited universal multidrug resistance across all models (carboplatin: 1.96-5.77×, dasatinib: 2.75-8.30×, panobinostat: 2.13-4.22× IC50 increase). LGR5⁺ cells showed context-dependent responses, including enhanced panobinostat sensitivity in OVCAR-3 (IC50 reduced to 45% of parental, p = 0.014) but universal dasatinib resistance (1.9-3.2×). Patient-derived organoids revealed both conserved vulnerabilities (dasatinib/panobinostat sensitivity) and interpatient heterogeneity (4-fold difference in carboplatin response). ovarian cancer organoid platform recapitulates CSC niche architecture and reveals microenvironment-dependent therapeutic vulnerabilities invisible to 2D screening. CD44 mediates universal multidrug resistance through physical niche sequestration and pro-survival signaling, while LGR5⁺ CSCs exhibit lineage-specific epigenetic dependencies. Dual targeting of physical niche integrity (dasatinib) and intrinsic epigenetic programming (panobinostat) represents a promising strategy to eradicate therapy-resistant CSCs. Patient-derived organoids capture interpatient heterogeneity, supporting their utility in precision medicine approaches for ovarian cancer.

Abstract: Macrophage–β-Cell Crosstalk in Type 2 Diabetes Mellitus

Type 2 Diabetes Mellitus (T2DM) involves progressive loss of functional β‑cell mass, a critical event worsened by chronic lipid overload. Lipid excess promotes a pro‑inflammatory phenotype in islet macrophages (IMs) through a fatty acid oxidation (FAO)‑dependent pathway, with Carnitine Palmitoyltransferase 1A (Cpt1a) as the rate‑limiting enzyme. However, the direct role of macrophage Cpt1a in β‑cell health remains unclear. To address this, we generated myeloid‑specific Cpt1a knockout (Cpt1a MKO) mice and fed them a high‑fat diet (HFD) to induce obesity and glucose intolerance. Metabolic phenotyping, islet morphology, and β‑cell proliferation were assessed. Bone marrow‑derived macrophages (BMDMs) from WT and Cpt1a MKO mice were treated with palmitic acid (PA), and their conditioned media were tested on β‑cells. Proteomic profiling, Seahorse metabolic analysis, and mitochondrial function assays were performed. Cpt1a MKO mice were protected from HFD‑induced glucose intolerance and showed significantly enhanced β‑cell proliferation. Conditioned media from PA‑treated Cpt1a‑deficient BMDMs promoted β‑cell proliferation, while media from WT BMDMs inhibited it. Proteomics identified a key secreted factor—strongly suppressed in PA‑treated WT macrophages but restored in Cpt1a‑deficient cells. Neutralizing this factor abolished the pro‑proliferative effect, confirming its essential role. Mechanistically, Cpt1a deletion preserved mitochondrial mass and ATP production under lipotoxic stress, preventing AMPK hyperactivation and enabling a protective secretory profile. Collectively, our findings show that macrophage‑specific Cpt1a deletion alleviates metabolic dysfunction in T2DM by promoting β‑cell proliferation through mitochondrial preservation and the subsequent upregulation of a protective secreted factor. Targeting macrophage Cpt1a may offer a novel strategy to preserve β‑cell mass in diabetes.

Abstract: Biomaterial based strategies to promote vascularization in bone tissue engineering

Bone tissue regeneration remains a significant clinical challenge, particularly in large-scale segmental defects where nutrient supply and metabolic exchange are critically restricted. A key bottleneck in successful bone reconstruction is rapid, adequate vascularization; without a functional vascular network, cell survival within thick 3D scaffolds is severely compromised, leading to core necrosis and graft failure. This presentation explores multi-faceted bioengineering strategies designed to overcome vascularization limitations by integrating material chemistry, structural design, and biofabrication technologies to synergistically induce angiogenesis and osteogenesis. A central focus is the therapeutic orchestration of bioactive inorganic ions—such as cobalt, copper, and silicate ions—as stable, cost-effective alternatives or complements to recombinant growth factors like VEGF. By controlling the spatiotemporal release of these therapeutic ions from porous matrices, microparticles, and bioactive glass nanostructures, it is possible to trigger hypoxia-inducible mechanisms (e.g., HIF-1α stabilization) and direct pro-angiogenic cell responses without the high costs, short half-lives, or off-target safety risks associated with biological proteins. In parallel, advanced biofabrication techniques—including coaxial 3D extrusion, core-shell hydrogel encapsulation, and microfluidic-assisted printing—are presented as powerful tools to construct highly organized pre-vascularized tissue constructs. Coaxial extrusion allows the direct patterning of endothelial and mural/smooth muscle cell compartments, mimicking native vessel architecture and promoting rapid lumen formation upon implantation. Furthermore, combining self-setting silica-based inks, biopolymer matrices, and tailored supramolecular hydrogels enables the creation of mechanically robust, macroporous scaffolds that provide structural guidance while delivering dual pro-angiogenic and osteoinductive signals. Beyond direct signaling, the interplay between immunomodulation and vascularization is emphasized. The early host immune response, driven largely by macrophage polarization (M1 to M2 transitions), heavily dictates downstream angiogenic sprout formation and subsequent matrix mineralization. By tuning surface micro/nanotopography and ion-doping profiles, biomaterials can be engineered to attenuate chronic inflammatory pathways and foster a pro-healing microenvironment conducive to capillary sprouting. In conclusion, achieving predictable and rapid vascularization in bone tissue engineering requires a holistic approach combining bioactive ion delivery, immunomodulatory scaffold design, and precise 3D bioprinting. Translating these combined pro-angiogenic strategies into scalable, clinical-grade platforms represents a pivotal step toward functional, long-lasting hard tissue repair

Abstract: Synergistic Effects of Substrate Curvature and Surface Micro-Topography on Human Corneal Endothelial Cell Regeneration

Corneal blindness represents a major global health challenge, affecting approximately 10 million individuals worldwide. Currently, corneal transplantation remains the primary clinical intervention; however, its implementation is severely restricted by a worldwide donor tissue shortage. Transplant failure is frequently driven by dysfunction of the corneal endothelium—a non-regenerative monolayer of corneal endothelial cells (CECs) responsible for maintaining corneal hydration, transparency, and visual acuity through barrier and ionic pump functions. While expansion of primary human CECs in vitro offers a promising alternative to tissue transplantation, standard two-dimensional culture models often result in rapid phenotypic degradation, loss of characteristic cell-cell junctions, and abnormal cell morphology. To overcome these limitations, this study presents a biomimetic tissue engineering strategy designed to recapitulate both the anatomical macro-curvature and micro-topographical features of the native human cornea. Custom 3D-printed molds were fabricated with specific concave and convex curvature profiles and surface topographies, including smooth surfaces and patterned feature sizes (50 µm, 200 µm, and 300 µm). Type I collagen hydrogels were crosslinked with Genipin and cast into these custom molds to yield unpatterned and patterned biomimetic substrates. Crosslinking efficiency and cytocompatibility were evaluated across multiple Genipin dilutions (control, 1:200, 1:100, 1:50) using CCK-8 cell viability assays. Primary human CECs were cultured on flat, concave, patterned, and unpatterned substrates. Cellular responses were evaluated via immunofluorescence (F-actin/Phalloidin and ATP1A1 expression), quantitative gene expression (qPCR) for tight and pump junction markers, and quantitative morphometric analysis (cell area and circularity index) at short (3 days) and extended (6 days) culture periods. Optical and cross-sectional characterization demonstrated that 3D-printed molds successfully transferred uniform micro-patterns and macro-curvature to collagen hydrogels with high structural fidelity. A 1:200 Genipin crosslinker dilution provided optimal hydrogel stability while maintaining ~100% cell viability relative to controls. Culturing primary CECs on patterned curved hydrogels restored native-like cell architecture, with cells rapidly forming tightly packed monolayers featuring hexagonal/polygonal morphology. At Day 3, cells cultured on patterned curved substrates achieved a circularity index of 0.870 ± 0.071 and a mean cell area of 315.9 ± 98.48 µm², closely matching native endothelial parameters. Immunofluorescence confirmed strong cell-surface localization of the functional pump marker ATP1A1. Furthermore, gene expression analysis revealed that macro-scale substrate curvature had a dominant effect on overall CEC behavior, whereas micro-topographical patterning specifically enhanced tight junction expression over pump junctions. Notably, shorter culture durations (3 days) on curved patterned hydrogels preserved significantly higher marker expression and physiological morphology than extended culture periods or traditional flat substrates. This work demonstrates that combining macro-curvature and micro-topography provides critical biophysical cues that promote CEC functional phenotype preservation and structural organization. This biomimetic hydrogel platform offers a robust approach for expanding functional corneal endothelial grafts, addressing key bottlenecks in ocular tissue engineering.