With the power of self-renewal and multipotency, mesenchymal stem cells (MSCs) offer the possibility of regenerative treatment for a variety of medical conditions, including spinal cord injury and damage following a heart attack.1
A leading hypothesis is that the therapeutic benefits of MSCs are conferred by their secretion of extracellular vesicles (EVs): membrane-derived structures that contain a variety of important bioactive molecules.2 Therapeutic cells like MSCs have variable EV production, contributing to inconsistent outcomes that hinder their clinical translation. Yet, scientists are unable to select cells based on their EV secretion levels.
“How do you know how many extracellular vesicles the [MSCs are] secreting? How do you know this batch of cells is better than this batch?” said Dino Di Carlo, a bioengineer at the University of California, Los Angeles.
In a new study published in Nature Communications, Di Carlo and his colleagues applied their expertise in microfluidics and nanotechnology to answer these questions.3 They developed a method to identify subpopulations of MSCs that secrete high levels of EVs, an approach that could allow for the selection of more therapeutically active cells for clinical applications.
Previously, Di Carlo and his team developed hydrogel-based microcontainers, which they called nanovials, that act as test tubes for single cells.4 By coating the inside of the bowl-shaped nanovials with antibodies that are specific to proteins released by a cell of interest, the researchers could capture individual cells and quantify their secretions. In an earlier study, the team used this technology to measure protein secretion from individual B cells, which they then linked to cell surface markers and gene expression data from the same cell.
When Di Carlo set out to measure EV secretion from individual MSCs he found that it wasn’t as straightforward.
“The first challenge was that measuring secreted extracellular vesicles is a little different than measuring secreted proteins,” remarked Di Carlo. “Extracellular vesicles are a lot larger [and] they also [express] some common proteins and cell surface markers.”
Di Carlo’s team needed to identify markers that they could use to stain secreted EVs, but not the cells that released them. They knew that membrane-bound scaffold proteins called tetraspanins were good markers of EVs, but some of these could also be expressed by MSCs. After assessing several combinations, they settled on dual targets: CD63 and CD9. These tetraspanins are expressed in combination on the surface of EVs, but the cells that secrete the EVs only express one or the other.
By lining their microscopic test tubes with anti-CD63 antibodies, they laid the bait for any cell expressing the marker. However, the addition of anti-CD9 antibodies ensured that they only captured EVs. Using this approach on single immortalized MSCs (iMSCs), they found that some cells released significantly more EVs than others.
“It's very exciting,” said Joy Wolfram, a nanoscientist at the University of Queensland who specializes in the therapeutic application of extracellular vesicles and was not involved in this work. “This study shows that even if it's the same cell line, there's still this heterogeneity.”
Wolfram explained that primary human MSCs would exhibit even greater disparity in the secretion of EVs owing to donor differences in genetics and lifestyle. “Being able to perform a high throughput method to capture the ideal donor cells, which could vary depending on the application, that’s something we are very interested in—how can we capitalize on that and select the optimal clones, or even human donors, for this type of therapy?” Wolfram commented.
The team also identified several key features of the captured high-secreting cells: they displayed increased proliferation, maintained the high-secretion phenotype over multiple generations, and retained their multipotency. This allowed the researchers to identify therapeutically potent candidates and then enrich for these cells.
Because their goal is to improve clinical outcomes for regenerative MSC therapies, Di Carlo and his team wanted to put their method to the test in an in vivo model. To do so, they selected high- and low-secreting primary mouse MSC populations and compared their therapeutic efficacies in a mouse model of myocardial infarction (heart attack). Twenty-eight days after treatment, mice injected with high-secreting MSCs displayed higher rates of vascular regeneration and improved heart function compared to those injected with low-secreting cells.
Di Carlo and his team are pursuing preclinical studies of myocardial injury in pigs, the preferred large animal model for cardiovascular research. They are also exploring other potential therapeutic applications for high secretors—an enthusiasm shared by Wolfram.
“This is just the beginning in terms of what type of future applications can come from this technology,” said Wolfram. “If we find that a certain type of EV is more therapeutic, you could use the same technology to select for cells that release that type.”
References
1. Kou M, et al. Mesenchymal stem cell-derived extracellular vesicles for immunomodulation and regeneration: A next generation therapeutic tool? Cell Death Dis. 2022;13(7):580.
2. Cheng L, Hill AF. Therapeutically harnessing extracellular vesicles. Nat Rev Drug Discov. 2022;21(5):379-399.
3. Koo D, et al. Optimizing cell therapy by sorting cells with high extracellular vesicle secretion. Nat Commun. 2024;15(1):4870.
4. Cheng RY-H, et al. SEC-seq: Association of molecular signatures with antibody secretion in thousands of single human plasma cells. Nat Commun. 2023;14(1):3567.
