Injuries to peripheral nerves is a common health problem, often resulting in sensory and motor dysfunction in respective parts of the body. One of the most important facilitators of the regeneration process are Schwann cells (SCs), which basement membrane is chiefly comprised of laminin. Extracellular vesicles (EVs) are considered playing an important role in the intercellular communication and transfer of biological information. Especially, Mesenchymal stem cell-derived EVs (MSC-EVs) have been identified as a promising novel therapeutic option due to their potential function as target-directed drug delivery vehicles. However, the precise delivery of EVs upon administration is still a huge challenge due to the accumulation of EVs at off-target sites. To remedy this problem, this study focuses on the production of laminin-binding EVs derived from MSCs by modification of the large extracellular loop (LEL) of the overexpressed EV surface marker protein CD81, from the tetraspanin protein family, to increase their binding affinity to laminin.
Specific CD81-LEL sequences are cloned into lentiviral vectors encoding the expression cassette for full-length CD81 proteins fused with eGFP or Akaluciferase under the control of human cytomegalovirus promoter. Stable cell lines are obtained upon transformation of Wharton’s Jelly MSCs (WJ-MSCs). EVs derived by WJ-MSCs are further isolated and characterized by NTA. Their uptake by SCs is evaluated by detecting the emitted fluorescence with FACS.
Our results obtained from NTA demonstrate the successful cloning and production of laminin-binding EVs. The positive tracking of fluorescence with FACS verifies their binding affinity on rodent laminin and their increased uptake and pro-regenerative effect on SCs.
This study represents a substantial advance for further investigation on EVs regarding their influence on peripheral nerve regeneration processes.