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.

Abstract: The Essentialome of Human Development and Disease

Human pluripotent stem cells (hPSCs) have a major role in investigating human development, and in modeling genetic disorders. We have previously generated haploid hPSCs carrying only one set of chromosomes. Interestingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers Furthermore, we demonstrated the superior utility of haploid hPSCs for loss-of-function genetic screening. To define the essentialome of hPSCs, we generated a genome-wide loss-of-function library in the haploid cells utilizing CRISPR/Cas9 technology using about 180,000 guide RNAs, targeting virtually all coding genes. This library enabled us to define the genes essential for the normal growth and survival of undifferentiated hPSCs, and discover the clusters of genes regulating human puripotency. We have also mapped the essential genes for the differentiation of hPSCs into ectoderm, mesoderm and endoderm, defining the essentialome of each germ layer separately and identifying the signal transduction pathways regulating the differentiation into these lineages. More recently, we have defined the essentialome of neurogenesis and parental imprinting. Our data enabled generation of an atlas of all neurological disorders, uncovering the stage of their manifestation during human development. In addition to defining the essentialome of early human development, we utilized the genome-wide screening to identify genes that can reverse the phenotype of neurological and imprinting disorders. We focused on chromatin-related genes that their mutation can induce a normal molecular phenotype, and validated their effect in diseased cells, as a way to suggest novel therapies. Overall, our work sheds light on the gene networks regulating pluripotency, early gastrulation, neurulation, and human genetic disorders by utilizing genome-wide screening in hPSCs.

Abstract: Stem Cell-Laden Polycaprolactone–Gelatin Nanoscaffolds for Repair of Critical-Size Radial Bone Defects

Fracture healing complications carry a significant healthcare and economic burden. We evaluated the therapeutic potential of a canine bone marrow mesenchymal stem cell (cBM-MSC)-laden composite nanofibre scaffold to enhance bone repair in a rabbit model. A novel bone-mimicking scaffold was fabricated by electrospinning the synthetic polymer polycaprolactone (PCL) with the natural polymer gelatin. Its morphology was characterised by scanning electron microscopy (SEM) and its chemical properties by FT-IR. The nanoscaffold showed a fibre diameter of 228–341 nm and a pore size of 2 µm, dimensions that promoted cell elongation and proliferation. cBM-MSCs were isolated, cultured and characterised by surface antigen expression and by differentiation into osteogenic, chondrogenic and adipogenic lineages. The cells were then seeded onto the nanoscaffold. Critical-size bone defects in rabbits were treated with the MSC-laden scaffold, the scaffold alone, or left untreated as controls. Healing was monitored by radiography, histopathology and SEM analysis of the healed bone. At day 60, radiological and histological analyses revealed complete bone bridging in animals treated with the MSC-laden composite nanoscaffold, significantly outperforming both the scaffold-only and control groups (p < 0.05). A biodegradable, osteomimetic nanofibrous scaffold laden with cBM-MSCs promotes osteogenesis and bone regeneration in a critical-size defect, and emerges as a promising candidate for the treatment of bone defects.

Abstract: Optimizing Amniotic Membrane-Derived Bioscaffolds for Bone Regeneration: A Comparative Study in a Rabbit Radius Critical-Sized Defect Model

Healing critical-sized bone defects remains a major hurdle in regenerative medicine, highlighting the need for advanced bioscaffolds and cell therapies that boost osteogenesis. Here, we assessed the bone-healing capacity of a rabbit amniotic membrane (rAM) bioscaffold. We then benchmarked its performance against a decellularized buffalo rib periosteum scaffold recellularized with rAM-derived mesenchymal stem cells (MSCs). Following characterization of passage 3 (P3) rAM-MSCs via PCR, immunolocalization, and trilineage differentiation, their osteogenic regenerative capacity was evaluated alongside fresh and three-month cryopreserved rAM. Using an autologous rabbit model with a 10 mm critical-size radial defect, five treatment arms were tested: control (Group I), fresh rAM (Group II), frozen-thawed rAM (Group III), DP alone (Group IV), and DP enriched with rAM-MSCs (Group V). Healing outcomes were tracked by radiography at days 1, 60, and 90, with terminal assessments performed via histomorphology and electron microscopy. Groups II and V demonstrated superior bone healing across radiological and histological parameters. They exhibited the greatest defect size reduction, osteogenesis, and remodeling, as reflected by the highest total radiological scores. Histologically, these groups also achieved the highest total scores, marked by peak levels of bone marrow development along with cortical and medullary bone formation. Overall, the findings highlight both fresh rAM as an abundant source of MSCs and rAM-MSC-seeded DP as optimal bioscaffolds for critical bone defect regeneration.

Abstract: Interplay Between TAZ–SMAD4–ERK Signaling Orchestrates Osteogenic Commitment in Canine Bone Marrow Derived Mesenchymal Stem Cells

Mesenchymal stem/stromal cells (MSCs) are widely investigated for their potential in bone regeneration and regenerative medicine. Osteogenic lineage commitment is governed by a complex network of signaling pathways, among which canonical bone morphogenetic protein (BMP)–SMAD signaling and non-canonical mitogen-activated protein kinase (MAPK) signaling play important regulatory roles. SMAD4 is a central mediator of canonical TGF-β/BMP signaling and has been implicated in osteogenic differentiation through regulation of the osteogenic transcription factor RUNX2. In addition, the transcriptional coactivator TAZ has emerged as an important regulator of MSC fate, with increasing evidence supporting functional crosstalk between TAZ and SMAD4 during osteogenic differentiation. However, the regulatory relationship between these signaling components in canine bone marrow-derived MSCs (cBMSCs) remains insufficiently characterized, despite the translational value of canine models in musculoskeletal research. In the present study, we investigated the contribution of TAZ, SMAD4, and ERK/MAPK signaling to osteogenic differentiation of primary cBMSCs. Canine bone marrow-derived cells were characterized for MSC-associated phenotypic and multilineage differentiation properties prior to mechanistic investigation. The expression and intracellular distribution of TAZ and SMAD4 were evaluated during osteogenic induction using quantitative PCR, immunocytochemistry, and western blotting. The functional roles of TAZ and SMAD4 were examined using targeted shRNA-mediated gene silencing, followed by analysis of osteogenic markers including RUNX2, osteocalcin (OCN), and osteopontin (OPN). Osteogenic differentiation was further evaluated using alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining as functional indicators of osteogenic maturation and mineral deposition. The contribution of ERK/MAPK signaling was investigated through ERK inhibition and subsequent assessment of osteogenic differentiation and marker expression. Osteogenic induction was associated with increased TAZ and SMAD4 expression and a predominantly nuclear distribution of TAZ, consistent with their involvement in osteogenic commitment. Targeted depletion of either TAZ or SMAD4 markedly reduced the expression of osteogenic markers and diminished mineral deposition, demonstrating that both factors are required for efficient osteogenic differentiation of cBMSCs. In contrast, suppression of ERK signaling resulted in enhanced osteogenic differentiation, accompanied by increased osteogenic marker expression and mineralization. These findings indicate that TAZ and SMAD4 positively contribute to osteogenic commitment, whereas ERK/MAPK signaling exerts an opposing influence under the conditions examined. Collectively, this study identifies distinct regulatory contributions of TAZ–SMAD4 and ERK/MAPK signaling during osteogenic differentiation of canine bone marrow-derived MSCs. The findings provide mechanistic insight into signaling networks governing osteogenic fate in cBMSCs and further support the use of canine MSCs as a translational model for investigating strategies aimed at improving bone regeneration and regenerative medicine.

Abstract: Cellular and Differentiation Dynamics of Bovine Adipose-Derived Stromal Cells Under Acute and Chronic Hyperthermia

Rising global temperatures and extreme heat events pose severe threats to mammalian physiology, contributing to hyperthermia, tissue dysfunction, and impaired systemic recovery. At the cellular level, heat stress triggers proteotoxicity and oxidative damage. Mesenchymal stromal cells (MSCs), such as adipose-derived stromal cells (ADSCs), are pivotal for tissue repair and regeneration, yet their functional limits under thermal stress remain insufficiently understood. Using bovine ADSCs (bADSCs) as a translational large-animal model for mammalian stem cell biology, this study systematically evaluated the impact of acute and chronic heat stress on cell viability, antioxidant capacity, and multilineage differentiation potential. Bovine ADSCs (passages 3–4) isolated from subcutaneous adipose tissue were subjected to acute (2 h) or chronic (24 h) thermal stress representing normothermia (37°C), mild physiological fever (39°C), and severe hyperthermia/heatstroke conditions (41°C). Proliferation and metabolic viability were quantified via trypan blue dye exclusion and MTT assays. The cytoprotective heat shock response was evaluated through HSP70 expression, while oxidative stress dynamics were tracked by measuring superoxide dismutase (SOD) activity. Osteogenic and adipogenic differentiation capacities were analyzed morphologically and molecularly via key lineage markers (Osteopontin/OPN and PPAR-gamma). Exposure to mild hyperthermia (39°C) induced a transient metabolic boost at 2 h that normalized by 24 h, reflecting an adaptive physiological response. In contrast, severe hyperthermia (41°C) acutely stimulated metabolic and SOD activity at 2 h, followed by SOD depletion at 24 h, demonstrating eventual antioxidant system exhaustion. Significant time- and temperature-dependent upregulation of HSP70 confirmed robust activation of cellular defense pathways. Crucially, severe thermal stress altered stem cell fate commitment: OPN expression was significantly down-regulated under acute (41°C) and chronic (39°C and 41°C) stress, leading to severely impaired osteogenic matrix mineralization, whereas adipogenic signaling (PPAR-gamma) trended upward at 41°C. Using a bovine stem cell model, this study demonstrates that mammalian ADSC resilience is strictly threshold-dependent. While mild fever (39°C) triggers protective adaptation, severe hyperthermia (41°C) exhausts antioxidant defenses and selectively suppresses osteogenic lineage commitment. These findings elucidate fundamental cellular mechanisms of heat-induced pathology, suggesting that compromised adult stem cell function may be a major driver of impaired tissue repair and regenerative failure during severe thermal stress in both veterinary and translational human medicine.

Abstract: Deciphering the Immunoregulatory and Regenerative Potential of Caprine Amniotic Membrane-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) represent a promising cell-based therapeutic platform owing to their multilineage differentiation capacity, regenerative properties, and ability to modulate immune responses. The present study investigated the immunoregulatory and regenerative potential of caprine amniotic membrane-derived mesenchymal stem cells (cAM-MSCs). MSCs isolated from caprine amniotic membrane were characterized according to the International Society for Cellular Therapy (ISCT) criteria based on plastic adherence, characteristic surface-marker expression, and trilineage differentiation potential. Their immunomodulatory activity was evaluated through co-culture with immune cells, followed by assessment of lymphocyte proliferation and cytokine-associated gene expression. The expression of key immunoregulatory and inflammatory mediators, including indoleamine 2,3-dioxygenase (IDO), interleukin-10 (IL-10), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6), was analyzed. MSC co-culture resulted in marked suppression of lymphocyte proliferation, accompanied by modulation of inflammatory and anti-inflammatory cytokine profiles, supporting the immunoregulatory capacity of cAM-MSCs. The regenerative potential of cAM-MSCs was further investigated using a cutaneous wound-healing model. Full-thickness dorsal wounds (2 × 2 cm²) were established, and wound progression was monitored through serial wound-area assessment. Histological evaluation of regenerated tissue was performed on days 14 and 28 to assess tissue repair and healing. Treatment with cAM-MSCs promoted accelerated wound closure and enhanced tissue regeneration compared with the control group. Collectively, these findings demonstrate the dual immunoregulatory and regenerative potential of cAM-MSCs, highlighting their promise as a cell-based therapeutic strategy for tissue repair and modulation of dysregulated inflammatory responses, with potential applications across veterinary and translational regenerative medicine.

Abstract: RABBIT AMNIOTIC MEMBRANE AS MSC BASED BIOSCAFFOLD: A STREAMLINED REGENERATIVE MEDICINE STRATEGY TO ACCELERATE SUBCUTNEOUS WOUND REPAIR

The current study was conducted to utilize rabbit amniotic membrane as bio-scaffold before and after cryopreservation for subcutaneous wound healing. Simultaneously to understand isolation, in vitro expansion, phenotypic characterization of r-AM MSCs and collection of its conditioned media to use them as a mode of treatment for early and better subcutaneous wound healing. The r-AM MSCs of P3 stage were used in all the experiments for their characterization as per ICST guidelines. The r-AM MSCs adhered to plastic, positively expressed stemness marker genes CD73, CD90, CD105 and lacked expression of CD34 & CD45 genes and also differentiated into mesodermal trilineage (osteogenic, chondrogenic and adipogenic) cells on complementation of respective supplements. The conditioned media was collected from P3 culture of r-AM MSCs which was later lyophilised and stored at -20^oC for further use in topical application. The rabbit amniotic membranes after collection were subjected to cryopreservation where 10% DMSO was used as cryoprotectant along with low glucose DMEM and 40%FBS. Slow freezing method was utilized for cryopreservation where amniotic membranes in freezing media were exposed to 4⁰C for 2hrs, -20⁰C for 2hrs, -80⁰C for 12 hrs and finally stored at - 196⁰C until date of application on wound. On the day of wound creation, the amniotic membranes were thawed in water bath maintained at 37 °C, rinsed thoroughly in PBS and suspended in serum free media before use. Therapeutic property of fresh and frozen thawed amniotic membrane was tested by applying on to full thickness skin wound. Similarly, r-AM MSCs (1x10⁶ rAM MSCs in 100μl PBS) & their conditioned media (15mg LCM in 200μl DMEM) were applied on to the wound to compare their therapeutic potential. These wounds were observed for 28 days with measuring their areas on every 7 days. The results revealed significantly higher rate (p<0.05) of wound contraction in all four treatment groups amongst which r-AM MSCsand conditioned media group had highest wound contraction by day 14 (88.26% and 87.97% respectively) followed by cryopreserved AM and fresh AM (80.42% & 78.7% respectively). Histologically, healing was better in group receiving fresh amniotic membrane followed by groups receiving r-AM MSCs and their conditioned media. Histological evaluation revealed r-AM MSCs and conditioned media had better epithelialization scores compared to amniotic membrane groups, cryopreserved AM group had least score for epithelialization. The group which received fresh rabbit amniotic membrane had significantly maximum neovascularization scores followed by cryopreserved AM group and conditioned media group. Collagen density scores didn’t differ significantly among groups. This study concluded that r-AM MSCs and conditioned media are better mode of treatment for subcutaneous wound healing. The fresh AM is better bio-scaffold compared to cryopreserved AM as cryopreservation can alter viability of cells in amniotic membrane compromising quality of wound healing. Further, refining the cryopreservation protocols of amniotic membrane, to make it a ready to use bio-scaffold is required.