Ramalingam, R. | Search bioRxiv

bioRxiv2025

In vivo reprogramming of cytotoxic effector CD8+ T cells via fractalkine-conjugated mRNA-LNP

Selective in vivo reprogramming of cytotoxic effector CD8+ T (Teff) cells holds tremendous promise as a therapeutic tool but has not yet been accomplished. Here, we demonstrate that fractalkine-conjugated mRNA lipid nanoparticles (mRNA-LNP) can specifically target and deliver mRNA to CX3CR1+ Teff cells in vitro and in vivo. In mice, fractalkine-conjugated LNP target up to 90% of blood and splenic Teff cells, and delivery of IL-2-encoding mRNA to Teff cells enables robust exogenous IL-2 secretion. In rhesus macaques, fractalkine-conjugated mRNA-LNP target up to [~]100% of peripheral blood Teff cells and delivery of CD62L-mRNA enables transient CD62L expression. Collectively, these data demonstrate the potential of natural receptor ligand-based targeting of mRNA-LNP for effective and efficient transient in vivo modification of Teff cells.

immunology↗

bioRxiv2018

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

The long-term expansion of keratinocytes under serum- and feeder free conditions generally results in diminished proliferation and an increased commitment to terminal differentiation. Here we present a serum and xenogeneic feeder free culture system that retains the self-renewal capacity of primary human keratinocytes. In vivo, the tissue microenvironment is a major contributor to determining cell fate and a key component of the microenvironment is the extracellular matrix (ECM). Accordingly, acellular ECMs derived from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, were used as the basis for a xenogeneic-free keratinocyte expansion protocol. A phospholipase A2 decellularisation procedure produced matrices which, by proteomics analysis, resembled in composition the core matrix proteins of skin dermis. On these ECMs keratinocytes proliferated rapidly, retained their small size, expressed p63, did not express keratin 10 and rarely expressed keratin 16. Moreover, the colony forming efficiency of keratinocytes cultured on these acellular matrices was markedly enhanced. Collectively these data indicate that the dermal fibroblast-derived matrices support the in vitro expansion of keratinocytes that maintained stem-like characteristics under serum free conditions.

cell biology↗