The microRNomes of Chinese hamster ovary (CHO) cells and their extracellular vesicles, and how they respond to osmotic and ammonia stress

Abstract

A new area of focus in Chinese hamster ovary (CHO) biotechnology is the role of small (exosomes) and large (microvesicles or microparticles) extracellular vesicles (EVs). CHO cells in culture exchange large quantities of proteins and RNA through these EVs, yet the content and role of these EVs remain elusive. MicroRNAs (miRs or miRNA) are central to adaptive responses to stress and more broadly to changes in culture conditions. Given that EVs are highly enriched in miRs, and that EVs release large quantities of miRs both in vivo and in vitro, EVs and their miR content likely play an important role in adaptive responses. Here we report the miRNA landscape of CHO cells and their EVs under normal culture conditions and under ammonia and osmotic stress. We show that both cells and EVs are highly enriched in five miRs (among over 600 miRs) that make up about half of their total miR content, and that these highly enriched miRs differ significantly between normal and stress culture conditions. Notable is the high enrichment in miR‐92a and miR‐23a under normal culture conditions, in contrast to the high enrichment in let‐7 family miRs (let‐7c, let‐7b, and let‐7a) under both stress conditions. The latter suggests a preserved stress‐responsive function of the let‐7 miR family, one of the most highly preserved miR families across species, where among other functions, let‐7 miRs regulate core oncogenes, which, depending on the biological context, may tip the balance between cell cycle arrest and apoptosis. While the expected—based on their profound enrichment—important role of these highly enriched miRs remains to be dissected, our data and analysis constitute an important resource for exploring the role of miRs in cell adaptation as well as for synthetic applications.

Kinetic and functional analysis of abundant microRNAs in extracellular vesicles from normal and stressed cultures of Chinese hamster ovary cells

“…This finding suggests that the bulk of the total miR content in CHO EVs from stressed cultures are not composed of solely the five miRs identified in standard cultures during the exponential phase and investigated individually here (i.e., miR‐92a, miR‐21, miR‐25, let‐7c, miR‐23a). Indeed, previous RNA sequencing of the osmotic and ammonia‐stressed cultures supports this, as the identities of the most abundant miRs in CHO EVs differ when parent cells are exposed to stress (Belliveau & Papoutsakis, 2023). We would expect a similar finding following the application of lactate stress.…”

“…CHO EVs were isolated and characterized as described (Belliveau & Papoutsakis, 2022, 2023). As noted, only large CHO EVs (i.e., “CHO MPs”) were collected and analyzed for this study.…”

“…In our previous publication (Belliveau & Papoutsakis, 2023) reporting on the microRNome of CHO cells and their EVs, the larger EVs were called MPs, and the smaller EVs were called exosomes. Our previous publications (Belliveau & Papoutsakis, 2022, 2023) have provided detailed CHO EV characterization that meets or exceeds the MISEV2018 guidelines from the International Society for Extracellular Vesicles (Théry et al, 2018). Here, we use the same isolation methods as before, expanding our CHO EV characterization by focusing primarily on various miR cargoes and their potential functions in CHO cell culture.…”

Advanced cell technologies: Making protein, cell, and gene therapies a reality

“…Cholesterol biosynthesis was one of those pathways, results showing that the addition of statins to cell culture increased vesicle productivity up to ninefold. In addition, Belliveau and Papoutsakis (2023) investigated the impact of osmotic and ammonia stress on the microRNA (miRs) within EVs of CHO cells. While normal culture conditions included higher levels of mir‐92a and mir‐23a in EVs, stress conditions resulted in the enrichment of let‐7a, let‐7b, and let‐7c miRs, which regulate core oncogenes and may alter the balance between cell cycle and apoptosis.…”

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

“…[5][6][7] We have previously demonstrated that EVs contain a variety of different endogenous cargo, such as micro RNAs (miRNAs), that are dynamically exchanged between cells and are critical for determining the fate of the target cell following uptake. [1,[8][9][10] As an initial aim, we wanted to determine if unmodified huMkEVs could localize to and affect the phenotype of HSPCs and lay the groundwork for huMkEV-based HSPC-specific delivery of therapeutics. Thus, the ultimate goal of this study was to examine if loaded huMkEVs can functionally deliver with specificity/tropism for HSPCs, plasmid DNA, both in vitro and in vivo and demonstrate their potential for targeted therapy of genetic hematological disorders.…”