In vitro evolution of nucleic acids and proteins is a powerful strategy to optimize their biological and physical properties. To select proteins with the desired phenotype from large gene libraries, the proteins need to be linked to the gene they are encoded by. To facilitate selection of the desired phenotype and isolation of the encoding DNA, a novel bead display approach was developed, in which each member of a library of beads is first linked to multiple copies of a clonal gene variant by emulsion polymerase chain reaction. Beads are transferred to a second emulsion for an in vitro transcription–translation reaction, in which the protein encoded by each bead’s amplicon covalently binds to the bead present in the same picoliter reactor. The beads then contain multiple copies of a clonal gene variant and multiple molecules of the protein encoded by the bead’s gene variant and serve as the unit of selection. As a proof of concept, we screened a randomized library of the T7 promoter for high expression levels by flow cytometry and identified a T7 promoter variant with an ∼10-fold higher in vitro transcriptional activity, confirming that the multi-copy bead display approach can be efficiently applied to in vitro evolution.

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Neurodegeneration is a hallmark of many diseases and disorders of the central nervous system (CNS). High levels of neuroinflammation are often associated with irreparable damage to CNS cells due to the dysregulation of signaling cascades that are unable to restore a homeostatic balance. Due to the inherent complexity of the CNS, development of CNS-related therapeutics has met limited success. While stem cell therapy has been evaluated in the context of CNS repair, the mechanisms responsible for their functional properties have not been clearly defined. In recent years, there has been growing interest in the use of stem cell extracellular vesicles (EVs) for the treatment of various CNS pathologies as these vesicles are believed to mediate many of the functional effects associated with their donor stem cells. The potency of stem cell EVs is believed to be largely driven by their biological cargo which includes various types of RNAs, proteins, and cytokines. In this review, we describe the characteristic properties of stem cell EVs and summarize their reported neuroprotective and immunomodulatory functions. A special emphasis is placed on the identification of specific biological cargo, including proteins and non-coding RNA molecules, that have been found to be associated with stem cell EVs. Collectively, this review highlights the potential of stem cell EVs as an alternative to traditional stem cell therapy for the repair of cellular damage associated with diverse CNS pathologies.

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