Fat-Derived Stem Cells Transformed Into Lymphatic Vessels in Lab Breakthrough

The lymphatic system has long lived in the shadow of its better-known twin, the blood vasculature. Yet this network of thin vessels draining fluid from every tissue in the body is central to immune surveillance, fat absorption and fluid balance, and its failure underlies conditions ranging from lymphedema to cancer metastasis. Now, a team of researchers in Spain has reported a new laboratory protocol that turns ordinary mesenchymal stem cells into lymphatic endothelial precursor cells, or LEPCs, offering what they describe as a reliable and scalable source of lymphatic-like cells for regenerative medicine. The study, published in the journal Angiogenesis, was led by Inazio Arriola-Alvarez of the Biogipuzkoa Health Research Institute in Donostia/San Sebastián, together with colleagues including senior authors Sonia Alonso-Martín and Ander Izeta.

The central challenge the team set out to solve is deceptively simple to state and notoriously hard to achieve. Lymphatic tissue engineering, whether aimed at repairing damaged vessels, modelling disease or testing drugs, depends on having enough authentic lymphatic endothelial cells to work with. Commercial cell lines are reproducible but physiologically limited, primary cells are authentic but scarce and age quickly in culture, and induced pluripotent stem cells, while powerful, remain expensive and technically demanding. Mesenchymal stem cells from bone marrow, dermis and adipose tissue have all shown lymphangiogenic potential, but until now no differentiation protocol, according to the authors, could accurately generate lymphatic endothelial precursor cells from them.

To find the best starting material, the researchers isolated cells from three mouse tissues: the stromal vascular fraction of adipose tissue, bone marrow and dermis. The dermis was quickly ruled out, yielding only around 500,000 cells per mouse with poor viability of roughly 50 percent. Bone marrow produced abundant cells, approximately 250 million per animal with more than 90 percent viability, while the stromal vascular fraction yielded about 2 million cells with similarly high viability. Crucially, however, the adipose-derived cells proliferated roughly twice as fast as their bone marrow counterparts, reaching useful numbers in nearly half the culture time and displaying less morphological heterogeneity. Both populations passed standard mesenchymal identity tests, expressing characteristic surface markers and successfully differentiating into fat, bone and cartilage lineages under appropriate conditions.

With the cell sources validated, the team applied a differentiation protocol deliberately modelled on embryonic lymphatic development. During embryogenesis, a subset of endothelial cells in the cardinal veins is committed to the lymphatic lineage by the transcription factor PROX1, guided largely by vascular endothelial growth factor C signalling through its receptor VEGFR3. The new protocol recapitulates this molecular choreography in three stages. First, an induction phase of five days exposes the cells to VEGF-C alone. A seven-day differentiation phase then adds CCBE1, a collagen and calcium-binding protein known to potentiate VEGF-C signalling, together with angiopoietin-2. Finally, a seven-day maturation phase introduces insulin-like growth factor 2, fibroblast growth factor 2 and angiopoietin-1, factors required for proper lymphatic vessel development and maintenance.

The results were strikingly source-dependent. Nearly all adipose-derived cells, 97.4 percent on average, turned on podoplanin, a canonical lymphatic endothelial marker, with a fivefold increase in fluorescence intensity compared with their undifferentiated progenitors. Bone marrow-derived cells also increased podoplanin expression, but far more variably, reaching only 76.2 percent positivity with weaker signal intensity. Gene expression analysis reinforced the picture: adipose-derived precursor cells significantly upregulated Pdpn, Lyve1, Vegfr3, Igf2 and Vegf-c, while bone marrow-derived cells upregulated the first three but showed no changes in Igf2 or Vegf-c. Notably, neither population induced Prox1, the master transcription factor of lymphatic identity, a finding the authors discuss at length as an intriguing dissociation between marker acquisition and canonical specification.

Functionally, the adipose-derived LEPCs behaved like genuine lymphatic endothelial cells in a battery of assays. They closed scratch wounds within 48 hours, matching the migratory performance of commercial murine lymphatic endothelial controls, and their migration was significantly stimulated by VEGF-C, rivaling the response to the established chemoattractant FGF2. Their proliferation proved dependent on VEGF-C signalling, since blocking the VEGFR3 kinase with the inhibitor MAZ51 significantly suppressed growth. Transendothelial electrical resistance measurements revealed robust junctional tightness and monolayer integrity, exceeding both controls through seven days of culture. Gene expression of key lymphatic markers remained stable across three further passages after differentiation, an essential property for any cell source intended for scalable production.

Perhaps the most visually compelling evidence came from three-dimensional culture. When embedded in Matrigel hydrogels, the LEPCs rapidly assembled into web-like networks that matured into thick, tube-like structures over ten days, while the commercial lymphatic endothelial cells used as controls remained static. In planar Matrigel assays, the LEPCs likewise formed interconnected elongated structures within hours, outperforming the controls. Immunofluorescence staining confirmed that even after ten days in three-dimensional culture, the cells continued to express the canonical lymphatic markers PDPN, LYVE1 and VEGFR3 alongside F-actin, indicating that the engineered environment did not erode their acquired identity.

Transcriptomic analysis by RNA sequencing provided genome-wide confirmation of the transformation. Principal component analysis cleanly separated differentiated cells from their mesenchymal progenitors, with the first principal component alone explaining 69.2 percent of the variance. Differential expression identified 2,037 upregulated and 1,936 downregulated genes, and among the top 50 lymphatic endothelial marker genes from a published single-cell atlas, 47 were expressed in the differentiated cells, with seven, including Pdpn, Vegfr3, Lyve1, Notch1 and Cd34, significantly upregulated. Gene set enrichment analysis revealed coordinated activation of lymphangiogenesis, lymphatic vessel morphogenesis, VEGFR signalling and sprouting angiogenesis pathways, alongside a marked loss of stemness programs and a metabolic shift toward fatty acid transport and oxidation. Intriguingly, comparison against tissue-specific lymphatic endothelial subpopulations showed the closest alignment with testicular and pulmonary lymphatic endothelial cells.

The final and arguably most important test took place in living animals. The researchers labelled the LEPCs with the red fluorescent protein tdTomato and injected two hundred thousand cells beneath full-thickness skin wounds created on the backs of nude mice. Seven days after transplantation, the labelled cells were detected within and closely associated with lymphatic vessels in the surrounding tissue, their lymphatic identity confirmed by co-localization with PDPN and LYVE1. The commercial lymphatic endothelial cells used as a biological reference localized similarly, suggesting the lab-generated cells can find and integrate into the host lymphatic microenvironment after transplantation into injured tissue.

The authors are careful to frame the achievement honestly. The differentiated cells retain features of an intermediate state, most notably low Prox1 expression, and the team deliberately uses the term precursor rather than progenitor, since classical progenitor properties such as long-term self-renewal were not formally demonstrated. The protocol was also optimized in mouse cells, and its fidelity in human cells remains to be established. Nevertheless, the combination of rapid expansion, reproducible differentiation, coordinated transcriptional change and demonstrated functional competence, together with successful incorporation into a vascularized melanoma model, positions this platform as a practical new tool for lymphatic disease modelling, tissue engineering and, ultimately, therapies aimed at regenerating the body’s most overlooked vascular network.

Subject of Research: In vitro differentiation of mesenchymal stem cells into lymphatic endothelial precursor cells for lymphatic tissue engineering

Article Title: A novel approach for reliable differentiation of lymphatic endothelial precursor cells in vitro

Article References: A novel approach for reliable differentiation of lymphatic endothelial precursor cells in vitro. (n.d.). https://doi.org/10.1007/s10456-026-10097-x

Image Credits: AI Generated

DOI: 10.1007/s10456-026-10097-x

Keywords: lymphatic endothelial cells, mesenchymal stem cells, lymphangiogenesis, stromal vascular fraction, VEGF-C, tissue engineering, regenerative medicine, differentiation protocol, RNA sequencing, lymphedema, PROX1, wound healing