A team of researchers has outlined a new approach that uses mRNA to prevent senescence and strengthen mesenchymal stem cells (MSCs) against aging before they are transplanted into patients.
Stem cells go bad before they can be used
The researchers introduce this study by focusing on the translational problems with MSCs, specifically the propensity of these cells to become senescent during the replication process [1]. The researchers hold that oxidative stress is the key driver of this rapid aging, causing senescence pathways to trigger [2] and mitochondria to become dysfunctional [3], which causes even more oxidative stress.
We have previously reported that senolytics might be useful in reducing premature senescence in stem cells before they reach the patient. While this approach has value, particularly in stem cell types that rapidly become senescent in a few replications, it won’t protect those cells against the patient’s microenvironment once they are transplanted. While the researchers note that MSCs affect the microenvironment into which they are placed [4], they also note in this study that an oxidative microenvironment is a possible threat to MSCs on top of the existing issues with pre-implantation replication.
Therefore, this work focuses on protecting cells before they even begin replicating. Previously, this team had found out that transplanting healthy mitochondria into fibroblasts can prevent fibrosis [5]; here, the researchers encouraged mitochondrial growth by transfecting the stem cells with mRNA for nuclear respiratory factor 1 (NRF1).
Widespread benefits
First, the researchers made sure that their approach was actually increasing mitochondrial mass compared to a control group. Microscopic fluorescence examination and an analysis of the mitochondrial biomarker TFAM agreed that it did after 24 hours: MSCs that were exposed to this mRNA had roughly 50% more mitochondria than the control group, as measured by fluorescence, whether the cells were placed under peroxide-induced oxidative stress or not. Additionally, the mRNA transfection increased NRF1 production by roughly 30 times over controls, although oxidative stress itself also causes NRF1 to increase in response.
This NRF1 was found to be effective in blunting markers of oxidative stress under peroxide exposure. While it was not a perfect solution, cells that had been transfected with NRF1 had roughly 25% less oxidative stress as measured by the fluorescence of the MitoSOX reagent. Mitochondrial membrane depolarization, which occurs under oxidative stress, was also reduced in the treatment group. Most critically, these findings were replicated in cells undergoing replicative senescence.
NRF1 mRNA treatment also appeared to have benefits related to metabolism. An RNA sequencing analysis revealed that genes related to oxygen usage were significantly upregulated, while glycolysis, a form of anaerobic energy production, was downregulated, signifying a more efficient use of energy. The researchers believe that this primes cells to better handle an environment of increased oxidative stress.
This improvement of energy usage was even maintained after exposure to hydrogen peroxide. Under this intense stress, cells normally rely more on glycolysis and less on using oxygen productively. NRF1 transfection reversed nearly all of this change, restoring ATP production and encouraging more proper oxygen use.
At relatively high concentrations (400 micromoles), hydrogen peroxide even interferes with the fission and fusion of mitochondria. However, NRF1 protected against this as well, maintaining mitochondrial balance, which was also found to be true in aged, senescent MSCs.
A better treatment for senescence?
NRF1 mRNA had further benefits for senescent cells, reducing key markers of senescence, including the key marker SA-β-gal, and this held true whether the cells were driven senescent by exposure to hydrogen peroxide or by multiple replications. The researchers compared its effects on these biomarkers to the well-studied senolytic ABT263, although this is a senomorphic that changes senescent cells and not a senolytic that kills them.
While the researchers found that their mRNA begins to naturally degrade in 48 hours and the resulting increase in mitochondria starts to peter out after 72 hours, this initial time period is likely to be critical for replication and implantation. However, this is still just a cell study. Further work in animals will need to be done before this approach could be considered for use in human beings.
Additionally, this work suggests a close tie between cellular senescence and mitochondrial dysfunction. If directly benefiting mitochondria can indeed reduce senescence, this general approach may be useful for other cells, including ones already in the body. However, substantially more work must be done to determine if such an approach is indeed viable.
Literature
[1] McHugh, D., & Gil, J. (2018). Senescence and aging: Causes, consequences, and therapeutic avenues. Journal of Cell Biology, 217(1), 65-77.
[2] Weng, Z., Wang, Y., Ouchi, T., Liu, H., Qiao, X., Wu, C., … & Li, B. (2022). Mesenchymal stem/stromal cell senescence: hallmarks, mechanisms, and combating strategies. Stem Cells Translational Medicine, 11(4), 356-371.
[3] Miwa, S., Kashyap, S., Chini, E., & von Zglinicki, T. (2022). Mitochondrial dysfunction in cell senescence and aging. The Journal of clinical investigation, 132(13).
[4] Song, N., Scholtemeijer, M., & Shah, K. (2020). Mesenchymal stem cell immunomodulation: mechanisms and therapeutic potential. Trends in pharmacological sciences, 41(9), 653-664.
[5] Baudo, G., Wu, S., Massaro, M., Liu, H., Lee, H., Zhang, A., … & Blanco, E. (2023). Polymer-functionalized mitochondrial transplantation to fibroblasts counteracts a pro-fibrotic phenotype. International Journal of Molecular Sciences, 24(13), 10913.
