Ander Izeta

Background: Secondary lymphedema is a chronic complication of oncologic surgery and radiotherapy for which effective disease-modifying therapies remain lacking. While mesenchymal stem cells (MSCs) have shown partial benefit in experimental models, direct functional comparison with lineage-committed lymphatic endothelial progenitor cells (LEPCs) remains limited. Methods: Secondary lymphedema was induced in C57BL/6J mice by circumferential tail skin excision with disruption of superficial lymphatics. Animals received intradermal phosphate-buffered saline (PBS), MSCs, or adipose-derived LEPCs on postoperative days 1 and 7. Lymphatic function was longitudinally quantified using IVIS-based near-infrared indocyanine green (ICG) imaging with standardized region-of-interest analysis. Tail diameter was measured serially throughout follow-up as a complementary morphometric parameter. Tissue remodeling was assessed by Picrosirius Red staining and qualitative LYVE-1 immunofluorescence. Statistical analysis incorporated mixed-effects modeling to evaluate treatment group, sex, time, and their interaction terms, with estimation of effect sizes and 95% confidence intervals. Results: LEPC-treated mice demonstrated higher IVIS signal intensity than MSC- and PBS-treated animals, consistent with improved lymphatic transport at the injury site. Longitudinal tail diameter analysis showed a more favorable temporal profile in the LEPC group relative to controls, with exploratory analysis suggesting potential variations in temporal profiles between sexes that warrant further investigation in larger cohorts. Histologic analysis showed reduced non-tissue/void area and a more favorable qualitative pattern of LYVE-1 staining in LEPC-treated tissue. Conclusions: LEPC administration enhanced functional and structural recovery in a murine model of secondary lymphedema. These findings support further translational evaluation of LEPC-based approaches as a regenerative strategy for secondary lymphedema.

Developing effective treatments for malignant melanoma remains a complex challenge, requiring models that accurately replicate the tumor microenvironment to study metastasis and drug response. Whereas in vitro models have become essential tools due to their tunability and controllability, systems incorporating both biomimetic extracellular matrices (ECM) and vascular components are more complex and therefore remain challenging. In this study, we present an in vitro melanoma model comprising a dermal decellularized ECM (dECM) together with both blood and lymphatic vessels, cultured within a commercial microfluidic platform that enables high-throughput testing. Our system features a triculture melanoma spheroid embedded within a dermis dECM hydrogel and flanked by vascular structures, effectively recreating key aspects of the melanoma microenvironment, as confirmed by immunostaining for canonical markers and morphological resemblance. Proof-of-concept drug testing demonstrates the model's suitability for high-throughput drug screening, as validated by cytotoxicity assays. The complexity and biomimetic nature of this model provides a robust platform for studying melanoma progression, tumor-vascular interactions, and therapeutic responses, ultimately offering a promising alternative for in vitro melanoma research and drug development.

The lymphatic system plays critical roles in fluid homeostasis, immune regulation, and lipid transport, making its dysfunction a contributor to numerous pathological conditions. Lymphatic tissue engineering aims to develop therapeutic strategies for lymphatic regeneration and repair, depending on the availability of suitable cell sources for lymphatic endothelial cell (LEC) generation. Key cell sources explored for lymphatic tissue engineering include the dermis, bone marrow and stromal vascular fraction. To our knowledge, no existing differentiation protocols can accurately generate lymphatic endothelial progenitor cells (LEPCs). This study presents a novel in vitro differentiation protocol for generating LEPCs from stromal vascular fraction, dermis and bone marrow-derived mesenchymal stem cells (MSCs). The feasibility of using them as tissue sources was first evaluated leading to dermal cells exclusion due to low yield post-isolation, and highlighting the limited differentiation efficiency of bone marrow MSCs. However, the stromal vascular fraction emerged as the optimal source, exhibiting robust LEPCs differentiation and superior scalability. In vitro characterization confirmed that LEPCs maintain a lymphatic endothelial phenotype, exhibiting high migratory ability, robust angiogenic structure formation, and consistent expression of key lymphatic markers in both 2D and 3D cultures, as validated by RNA-Seq analysis. The protocol provides a consistent platform for studying lymphatic endothelial biology and potential applications in regenerative medicine and therapeutic angiogenesis.

Neuromuscular junctions (NMJs) are complex multicellular structures that convey motor neuron-induced responses in the skeletal muscle. Their cellular composition is well characterized, but interactions between the different cell types and with the surrounding microenvironment remain underexplored. Here, by using a panel of newly discovered mouse cell lineage markers, single-cell RNA sequencing analyses and iDisco tissue clarification, we demonstrate the existence of contacts between adjacent NMJs through their kranocytes, assembling kranocyte network-like structures within the muscle interstitium, which is essential for regulating blood flow and supporting muscle regeneration. Indeed, kranocytes do contact with the surrounding microvasculature as well, both of them uncovering a previously unexplored, expansive interactive system. Moreover, unlike the robustness shown by their counterparts, the terminal Schwann cells, we strikingly observed that kranocytes rapidly detected stressful conditions, leading to structural changes and even the loss of their connections in response to acute damage, such as muscle injury or neuromuscular disease-induced denervation. Therefore, we propose kranocytes as the interactive sensory platforms of the NMJs, working in a perpendicular axis of nerve-transmission, as key NMJ-connectors by, interconnecting adjacent NMJs and interacting with the microvasculature and the microenvironment, and also as NMJ-sensors by detecting microenvironmental inputs. Together, our data postulate kranocytes as triple connectors of the nervous, musculoskeletal and vascular systems.

Regenerative medicine and tissue engineering approaches based on the use of 3D-bioprinted decellularized extracellular matrix (dECM) present the advantage of a relatively biomolecule-rich matrix, which directs cell growth and differentiation in a tissue-specific manner. However, little is known about the composition changes that occur with standard processing of dECM-based inks. To characterize this process, six porcine tissues/tissue layers (artery, breast, dermis, epidermis, muscle and nerve) were independently decellularized via chemical, mechanical and enzymatic processes and the resulting dECMs formulated into biocompatible inks, to serve as source biomaterials for 3D printing. A comparative liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based proteomic analysis was carried out for native tissue, decellularized and formulated ECMs, and the resulting complexity of the matrisome analyzed. En este protocolo se encuentra descrito la información relativa a las muestras (information and classification), al análisis mediante LC-MS/MS y posterior identificación de las muestras, el procesamiento y curado de los resultados así como la generación de las figuras publicadas.

Regenerative medicine and tissue engineering approaches based on the use of 3D-bioprinted decellularized extracellular matrix (dECM) present the advantage of a relatively biomolecule-rich matrix, which directs cell growth and differentiation in a tissue-specific manner. However, little is known about the composition changes that occur with standard processing of dECM-based inks. To characterize this process, six porcine tissues/tissue layers (artery, breast, dermis, epidermis, muscle and nerve) were independently decellularized via chemical, mechanical and enzymatic processes and the resulting dECMs formulated into biocompatible inks, to serve as source biomaterials for 3D printing. A comparative liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis was carried out for native tissue, decellularized and formulated ECMs, and the resulting complexity of the matrisome analyzed. A core matrisome was found to overlap in all decellularized tissues, as well as tissue-specific components that correlated with predicted functional (gene ontology-based) definitions. The proportion of collagens (mostly the α1 chains of collagen type I and III) increased in the final processing step (inks) as compared to the native ECM and dECM stages. Overall, a median of 55 matrisomal proteins (range 45-126) was detected in the dECM-derived inks. This complexity is far superior in terms of mimicking the composition of native tissue to non-dECMbased inks. Our results support the use of dECM-based inks and biomaterials in mimicking native tissue ECM complexity, demonstrating tissue specific composition, which can improve future therapeutic approximations.

Single-cell RNA sequencing (scRNAseq) studies have unveiled large transcriptomic heterogeneity within both human and mouse dermal fibroblasts, but a consensus atlas that spans both species is lacking. Here, by studying 25 human and 9 mouse datasets through a semi-supervised procedure, we categorize 15 distinct human fibroblast populations across 5 main axes. Analysis of human fibroblast markers characteristic of each population suggested diverse functions, such as position-dependent ECM synthesis, association with immune responses or structural roles in skin appendages. Similarly, mouse fibroblasts were categorized into 17 populations across 5 axes. Comparison of mouse and human fibroblast populations highlighted similarities suggesting a degree of functional overlap, though nuanced differences were also noted: transcriptomically, human axes seem to segregate by function, while mouse axes seem to prioritize positional information over function. Importantly, addition of newer datasets did not significantly change the defined population structure. This study enhances our understanding of dermal fibroblast diversity, shedding light on species-specific distinctions as well as shared functionalities.