Biology subjects

Chotteau, V.

Publications and source records attributed to Chotteau, V..

4 recordsLinked to original sources

bioRxiv2025

Single-Cell Transcriptomics Reveals Dynamics of NK Cell Expansion in a Feeder Cell-Free Culture of PBMCs - Implications for Immunotherapy

Natural killer (NK) cell therapies hold great promise for cancer treatment; however, donor-to-donor heterogeneity in the ex vivo expansion process remains a critical bottleneck in their supply. This study aimed to identify factors influencing donor variability in a two-week long ex vivo NK cell expansion from PBMCs, analyzed across three donors. Single-cell transcriptomics was applied to investigate the distribution of cell types and phenotypes, as well as trajectory inference and differential gene expression. Our results identified that several factors were associated with the variability in the final NK cell fraction and expansion, and that their influence was prevalent between culture day 3 and 8. Compared to high final NK cell fraction, a culture with low final NK cell fraction exhibited an upregulation of some stress and inflammatory genes and an increase in one specific subcluster of the NK cells already on culture day 3. It showed a low score of CD56Bright CD16- phenotype and high score of CD56Dim CD16+ phenotype. It had also an increased presence of cytotoxic CD8+ Tm cells. Among the observed subclusters of CD8+ Tm cells, it exhibited a higher presence of a subcluster associated with a less differentiated and less cytotoxic phenotype as well as a lower prevalence of a subcluster associated with chemokine and cytotoxic genes. Finally, it had a major expansion of one of the CD8+ Tm cells subclusters annotated as NK-like T cell and characterized by a high CCR5 mRNA expression while the levels of CCL3, CCL4, and CCL5 mRNA were downregulated. The present findings point towards a potential link between CCL signaling and improved NK cell expansion performance, including possible markers for further investigations, and suggest future strategies to increase the final NK cell fraction and expansion based on donor-specific markers.

bioengineering↗

bioRxiv2021

Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.

systems biology↗

bioRxiv2020

Transcriptome analysis of EPO and GFP HEK293 Cell-lines Reveal Shifts in Energy and ER Capacity Support Improved Erythropoietin Production in HEK293F Cells

Higher eukaryotic cell lines like HEK293 are the preferred hosts for production of therapeutic proteins requiring human post translational processing. However, recombinant protein production can result in severe stress on the cellular machinery, resulting in limited titre and product quality. To investigate the cellular and metabolic characteristics associated with these limitations, we compared erythropoietin (secretory) and GFP (non-secretory) protein producer HEK293 cell-lines using transcriptomics analysis. Despite the high demand for ATP in all protein producer clones, a significantly higher capacity for ATP production was observed with erythropoietin producers as evidenced by the enrichment of upregulated genes in the oxidative phosphorylation pathway. In addition, ribosomal genes exhibited specific patterns of expression depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased erythropoietin secretion, we detected higher ER stress, including upregulation of the ATF6B gene. Our results are significant in recognizing key pathways for recombinant protein production and identifying potential target genes for further development of secretory power in mammalian cell factories. In BriefAlthough the protein secretion process has been widely studied, the complexity of it leaves many questions with regards to defining bottlenecks for successful protein secretion to be answered. By investigating the transcriptomic profiles of different HEK293 clones with varying translational rates producing either the secreted protein erythropoietin or the intracellular GFP, we reveal that high ATP production and improved capacity of specific post-translational pathways are key factors associated with boosting erythropoietin production. HighlightsO_LITranscriptomics analysis of a panel of HEK293 stable cell lines expressing GFP or erythropoietin (EPO) at varying translational rates C_LIO_LIExpression of mitochondrial ribosomal genes is positively correlated with EPO secretion C_LIO_LIExpression of different cytosolic ribosomal genes are correlated with productivity in a recombinant-protein specific manner C_LIO_LIHigh EPO producing clones have significant upregulation of ATF6B, potentially enabling a beneficial ER stress response to cope with high protein secretion C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/299966v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1e35efforg.highwire.dtl.DTLVardef@71cee2org.highwire.dtl.DTLVardef@1c56d33org.highwire.dtl.DTLVardef@843a6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2020

Evolution from adherent to suspension - systems biology of HEK293 cell line development

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production and is today used for manufacturing of several therapeutic proteins and viral vectors. Despite this increasing popularity there is still limited knowledge of the detailed genetic and composition of derivatives of this strain. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

systems biology↗