Membrane repair following filtroporation-induced cell permeabilization.

Mechanical disruption of cell membranes to enable intracellular delivery has been gaining traction as a methodology. Here, we show that a mechanical disruption-based strategy, filtroporation (FP), can be applied to edit the beta globin gene efficiently in human hematopoietic stem and progenitor cells. Gene expression analyses from RNA-Seq datasets demonstrate that electroporation (EP) yields greater transcriptional changes compared to FP globally, and gene pathway enrichment analyses suggest EP promotes stem cell differentiation while FP promotes cell division. Membrane repair occurs within 30 s of disruption by FP, and calcium signaling plays a key role in membrane repair in this context. Studies with fluorescently tagged membrane repair proteins, GRAF1, SNAP23, and CHMP4B, implicate the involvement of three mechanisms to reconstitute the cell barrier following FP. This work supports the evidence that the choice of intracellular delivery method affects transcriptional stem cell profile, and that membrane repair after mechanical disruption is a fast, multi-pathway process.