Biology subjects

Belliveau, J.

Publications and source records attributed to Belliveau, J..

3 recordsLinked to original sources

bioRxiv2023

Kinetic and functional analysis of abundant microRNAs in extracellular vesicles from normal and stressed cultures of Chinese Hamster Ovary (CHO) cells

Chinese hamster ovary (CHO) cells release and exchange large quantities of extracellular vesicles (EVs). EVs are highly enriched in microRNAs (miRs, or miRNAs), which are responsible for most of their biological effects. We have recently shown that the miR content of CHO EVs varies significantly under culture stress conditions. Here, we provide a novel stoichiometric ("per-EV") quantification of miR and protein levels in large CHO EVs produced under ammonia, lactate, osmotic, and age-related stress. Each stress resulted in distinct EV miR levels, with selective miR loading by parent cells. Our data provide a proof of concept for the use of CHO EV cargo as a diagnostic tool for identifying culture stress. We also tested the impact of three select miRs (let-7a, miR-21, and miR-92a) on CHO cell growth and viability. Let-7a--abundant in CHO EVs from stressed cultures--reduced CHO cell viability, while miR-92a--abundant in CHO EVs from unstressed cultures--promoted cell survival. Overexpression of miR-21 had a slight detrimental impact on CHO cell growth and viability during late exponential-phase culture, an unexpected result based on the reported anti-apoptotic role of miR-21 in other mammalian cell lines. These findings provide novel relationships between CHO EV cargo and cell phenotype, suggesting that CHO EVs may exert both pro- and anti-apoptotic effects on target cells, depending on the conditions under which they were produced. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/545902v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f85cd7org.highwire.dtl.DTLVardef@973599org.highwire.dtl.DTLVardef@13ff81eorg.highwire.dtl.DTLVardef@ba8eba_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

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

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) 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.

cell biology↗

bioRxiv2021

Extracellular Vesicles Facilitate Large-Scale, Homogenizing Dynamic Exchange of Proteins and RNA Among Cultured Chinese Hamster Ovary (CHO) and Human Cells

Cells in culture are viewed as unique individuals in a large population communicating through extracellular molecules and, more recently extracellular vesicles (EVs). Our data here paints a different picture: the homogenizing effect of large-scale exchange of cellular material through EVs. We show that Chinese Hamster Ovary (CHO) cells dynamically produce and uptake EVs, to exchange proteins and RNAs at large-scale. To visualize the dynamic production and cellular uptake of EVs, we used correlative confocal microscopy and scanning electron microscopy, as well as flow cytometry to interrogate labeled cells. We employed cells expressing fluorescent proteins (GFP, miRFP703) and tagged cells with protein and RNA dyes. Flow cytometry was used to quantify the exchange of cellular RNA between cells through EVs. This EV-mediated dynamic exchange observed in CHO cultures was also observed in cultures of the human CHRF-288-11 cell line and of primary human hematopoietic stem and progenitor cells. This study demonstrates an underappreciated native cell communication and protein/RNA exchange mechanism mediated by EVs spanning cell type and lines, suggesting the proximity of cells in normal and tumor tissues may also result in prolific cellular exchange. This exchange would be expected to homogenize the cell-population cytoplasm and dynamically regulate cell proliferation and cellular state.

bioengineering↗