Stem cell vesicles reverse blood vessel ageing to speed diabetic wound healing

A study in human cells, mice and miniature pigs has found that extracellular vesicles from a novel three-dimensional stem cell culture system can restore ageing blood vessel linings and accelerate the healing of diabetic wounds

Prolonged inflammation, poor circulation and defective angiogenesis – the growth of new blood vessels – can leave wounds such as diabetic foot ulcers remaining open for many months increasing the risk of both infection and amputation. Extracellular vesicles, tiny particles that cells release to carry proteins and other regenerative cargoes, have shown promise as a treatment when derived from adipose-derived mesenchymal stem cells. 

However, the conventional two-dimensional methods used to grow these stem cells in the laboratory gradually weaken their activity and their capacity to secrete useful cargo. Many laboratory studies have previously relied on generic endothelial cells that do not fully reproduce the processes seen in the behaviour of the skin’s microvasculature when placed under diabetic stress. These limitations meant that further work was needed to develop more potent vesicles and to establish how they rescue the affected human dermal microvascular endothelial cells found in diabetic wounds.

The research team was drawn from scientists at Lanzhou University Second Hospital, Xijing Hospital at the Fourth Military Medical University, the Affiliated Hospital of Jiangnan University and the Key Laboratory of Stem Cells and Gene Drugs, in Gansu Province China. The researchers set out to determine whether extracellular vesicles released by adipose stem cells – grown using a self-feeder layer three-dimensional culture system – could reverse microvascular endothelial ageing and improve the repair of diabetic wounds.

This culturing method allows the cells to generate their own supportive matrix rather than relying on a separate synthetic scaffold, and the team found that it preserved the stem cells’ regenerative character and enriched the beneficial cargo carried in the vesicles they released. To test this, the researchers combined an analysis of human tissue, cultured endothelial cells, diabetic mouse wounds, fat graft experiments and a diabetic Bama miniature pig wound model. (Bama pigs are a breed whose skin closely resembles that of humans, making them a useful large-animal model for wound research.)

The team first used single-cell RNA sequencing to map the endothelial populations present in diabetic foot ulcer tissue, and this revealed pronounced signs of ageing within the microvascular compartment. They then produced three-dimensional adipose stem cells and isolated three-dimensional adipose stem cell-derived extracellular vesicles (tdASC-EVs) from these.

When human dermal microvascular endothelial cells were exposed to high glucose levels, tdASC-EVs reduced senescence-associated beta-galactosidase activity – a standard marker of cell ageing – lowered oxidative damage, restored mitochondrial membrane potential and improved the cells’ ability to form tubes, an assay that indicates capacity for blood vessel formation.

Mechanistic experiments showed that the vesicles reactivated the phosphatidylinositol 3-kinase, protein kinase B, mechanistic target of rapamycin and 4E-binding protein 1 (4EBP1) pathway. Increased phosphorylation of 4EBP1 released the eukaryotic translation initiation factor 4E (eIF4E), which was then able to bind the eukaryotic translation initiation factor 4G (eIF4G) and restart cap-dependent protein translation, the process by which cells build proteins from their genetic instructions.

Inhibitors of this pathway weakened the vesicles’ benefits, while a phosphorylation-mimicking form of 4EBP1 restored them. Proteomic profiling identified 2,097 differentially expressed proteins, including cargo linked to regenerative signalling and to protein translation.

In diabetic mice, treatment with tdASC-EVs accelerated wound closure, improved blood perfusion, increased the number of microvessels positive for cluster of differentiation 31, reduced expression of the senescence marker p16, narrowed the resulting scars and produced better-organised collagen.

In diabetic miniature pigs, tdASC-EVs outperformed extracellular vesicles from conventionally cultured adipose-derived mesenchymal stem cells and produced faster wound closure, denser neovascularisation, less scarring and more complete tissue regeneration.

“Diabetic wounds are not only short of new blood vessels; the endothelial cells that should build those vessels become prematurely aged and lose the ability to translate proteins needed for repair,” the study’s authors said.

“By improving the way adipose stem cells are cultured, we generated extracellular vesicles with stronger regenerative activity. These vesicles reopened a key translation pathway, restored endothelial function, and improved both the speed and quality of healing across several preclinical models.

“The large-animal results are especially encouraging, although clinical safety and manufacturing consistency must still be established,” they added.

The findings support further development of tdASC-EVs as an acellular treatment for diabetic foot ulcers, and possibly for other diabetes-related tissue injuries marked by microvascular ageing. Because extracellular vesicles can deliver regenerative signals without the need to transplant living cells, they may offer practical advantages for storage, dosing and standardised production. However, the therapy remains at a preclinical stage.

Future studies need to identify which vesicle proteins are essential, to verify the pathway using models with precise genetic controls, such as gene knockouts, and to test long-term biodistribution, clearance, immune responses and any potential to promote tumour growth.

Larger human tissue datasets and well-designed clinical trials will also be needed to determine effective doses, treatment schedules and manufacturing standards, and to establish whether the approach can reduce recurrence, amputation and reconstructive surgery among patients with more than a passing risk of diabetic foot ulcers.

For further reading please visit: 10.1093/burnst/tkag042