Adipose stem cells (ASCs) stand out as promising therapeutic resources in regenerative medicine due to their exceptional self-renewal capabilities^1,2. When transplanted at diseased sites, their ability to modulate the pathological microenvironment plays a crucial role in various therapeutic areas³. Despite their therapeutic potential, ASCs face challenges in eliciting long-term and sufficient efficacy^4,5. To address the issue, researchers have delved into various engineering strategies in order to endow ASCs with extended and reinforced protein generation capacity, thereby facilitating the release of therapeutic agents at diseased sites. Notably, recent studies have highlighted the effectiveness of lipid nanoparticles (LNPs) in delivering RNA cargos, including messenger RNAs (mRNAs) and self-amplifying RNAs (saRNAs), to different types of primary cells^6,7. Such a delivery platform enables the reprogramming of cells to exhibit desired functions while maintaining a favorable safety profile.

Building upon this concept, our hypothesis posited that LNPs may serve as effective carriers for delivering mRNAs and saRNAs encoding therapeutic proteins, thereby enhancing the therapeutic abilities of ASCs (Fig. 1). In the exploration of this strategy, first, Dr. Yuebao Zhang developed a series of sugar alcohol-derived ionizable lipids with unique properties of chirality, rigidity, and low toxicity, laying the groundwork for subsequent systematic screening and optimization processes to identify the optimal LNP formulation for RNA cargo delivery. Concurrently, we selected saRNAs for achieving sustained protein expression in engineered ASCs.

However, we observed that the emergence of double-stranded RNA intermediates during replicative translation triggered the activation of intracellular RNA sensors, initiating translational blockade through the PKR-eIF2α pathway. After extensive reviews of the literature and group discussion, we investigated delivering saRNAs into ASCs concomitant with mRNAs encoding an immune evasion protein E3. This approach substantially improved delivery efficiency and prolonged protein expression from the saRNA translation for up to 9 days post-treatment.

We then proceeded to conduct impaired wound healing experiments simulating diabetic foot ulcers (DFU) in a db/db mouse model. Specifically, we co-delivered CXCL12-saRNAs and E3 mRNAs to engineer ASCs, which were further resuspended in hydrogels and embedded on top of the wounds. The treatment resulted in an expedited healing process, achieving complete wound closure by Day 15. Sustained production of CXCL12 chemokines effectively shifted the inflammatory wound microenvironment to an anti-inflammatory state, as evidenced by the upregulation of anti-inflammatory cytokines, thereby facilitating the prolonged persistence of ASCs. Notably, the enhanced viabilities of the engineered ASCs around the wound areas contributed to the development of thicker epidermal layers with enhanced angiogenesis and muscle regeneration capabilities.

In summary, the engineering of ASCs leveraging the LNP-RNA platform enables potent therapeutic effects through sufficient and durable protein productions, all while minimizing toxicity owing to the 'hit and run' nature of ASCs. Our work provides a promising strategy for the treatment of diabetic wounds and broadens the applicability of LNP-RNA-engineered ASCs to tackle various regenerative medical challenges.

Fig. 1 | Illustration of LNP-engineered ASCs with enhanced protein-generating ability for diabetic wound healing. This illustration was created with BioRender.com.

 Our paper: Xue, Y., Zhang, Y., Zhong, Y. et al. LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing. Nat Commun 15, 739 (2024). https://doi.org/10.1038/s41467-024-45094-5

References:

1               Xue, Y., Baig, R. & Dong, Y. Recent advances of biomaterials in stem cell therapies. Nanotechnology 33 (2022). https://doi.org/10.1088/1361-6528/ac4520

2               Ankrum, J. A., Ong, J. F. & Karp, J. M. Mesenchymal stem cells: immune evasive, not immune privileged. Nat Biotechnol 32, 252-260 (2014). https://doi.org/10.1038/nbt.2816

3               Kimbrel, E. A. & Lanza, R. Next-generation stem cells - ushering in a new era of cell-based therapies. Nat Rev Drug Discov 19, 463-479 (2020). https://doi.org/10.1038/s41573-020-0064-x

4               Sanchez-Diaz, M. et al. Biodistribution of Mesenchymal Stromal Cells after Administration in Animal Models and Humans: A Systematic Review. J Clin Med 10 (2021). https://doi.org/10.3390/jcm10132925

5               von Bahr, L. et al. Analysis of Tissues Following Mesenchymal Stromal Cell Therapy in Humans Indicates Limited Long-Term Engraftment and No Ectopic Tissue Formation. Stem Cells 30, 1575-1578 (2012). https://doi.org/10.1002/stem.1118

6               Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nature Reviews Materials 6, 1078-1094 (2021). https://doi.org/10.1038/s41578-021-00358-0

7               Zhao, X. et al. mRNA Delivery Using Bioreducible Lipidoid Nanoparticles Facilitates Neural Differentiation of Human Mesenchymal Stem Cells. Adv Healthc Mater 10, e2000938 (2021). https://doi.org/10.1002/adhm.202000938