Native and engineered human megakaryocytic extracellular vesicles for targeted non-viral cargo delivery to blood stem cells

Das, Samik; Thompson, Will; Papoutsakis, E. T.

doi:10.1101/2023.04.11.536479

Cited by 1 publication

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“…We have already extensively characterized the large MkEVs produced from our specific cell culture and isolation protocols. 11,13,14,18,20,31 These findings, which fulfill the requirements outlined by the International Society for Extracellular Vesicles, 9 are outlined in Table S1. Our isolation protocol-illustrated in Figure S1-relies on differential centrifugation, a known "intermediate recovery, intermediate specificity" technique.…”

Section: Previous Characterization Of Large Mkevsmentioning

confidence: 62%

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Similar but distinct: The impact of biomechanical forces and culture age on the production, cargo loading, and biological efficacy of human megakaryocytic extracellular vesicles for applications in cell and gene therapies

Thompson

Papoutsakis

2023

Bioengineering & Transla Med

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Megakaryocytic extracellular vesicles (MkEVs) promote the growth and megakaryopoiesis of hematopoietic stem and progenitor cells (HSPCs) largely through endogenous miR‐486‐5p and miR‐22‐3p cargo. Here, we examine the impact of biomechanical force and culture age/differentiation on the formation, properties, and biological efficacy of MkEVs. We applied biomechanical force to Mks using two methods: shake flask cultures and a syringe pump system. Force increased MkEV production in a magnitude‐dependent manner, with similar trends emerging regardless of whether flow cytometry or nanoparticle tracking analysis was used for MkEV counting. Both methods produced MkEVs that were relatively depleted of miR‐486‐5p and miR‐22‐3p cargo. However, while the shake flask‐derived MkEVs were correspondingly less effective in promoting megakaryocytic differentiation of HSPCs, the syringe pump‐derived MkEVs were more effective in doing so, suggesting the presence of unique, unidentified miRNA cargo components. Higher numbers of MkEVs were also produced by “older” Mk cultures, though miRNA cargo levels and MkEV bioactivity were unaffected by culture age. A reduction in MkEV production by Mks derived from late‐differentiating HSPCs was also noted. Taken together, our results demonstrate that biomechanical force has an underappreciated and deeply influential role in MkEV biology, though that role may vary significantly depending on the nature of the force. Given the ubiquity of biomechanical force in vivo and in biomanufacturing, this phenomenon must be grappled with before MkEVs can attain clinical relevance.

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Section: Previous Characterization Of Large Mkevsmentioning

confidence: 62%

“…We have already extensively characterized the large MkEVs produced from our specific cell culture and isolation protocols 11,13,14,18,20,31 . These findings, which fulfill the requirements outlined by the International Society for Extracellular Vesicles, 9 are outlined in Table S1.…”

Section: Resultsmentioning

confidence: 95%