Biology subjects

Svenningsen, P.

Publications and source records attributed to Svenningsen, P..

4 recordsLinked to original sources

bioRxiv2026

Sustained 10% Oxygen Promotes Atrial Rather Than Ventricular Specification During Human iPSC-Cardiomyocyte Differentiation

Background: Induced pluripotent stem cell-derived ventricular cardiomyocytes (iPSC-vCMs) hold great promise for replacing ventricular cardiomyocytes lost after myocardial infarction. However, their immature phenotype limits successful engraftment by increasing the risk of post-transplant arrhythmias. In contrast to the atmospheric O2 used during most iPSC-vCM differentiations, O2 levels inside the developing heart remain low, but very little is known on the effect on O2 on iPSC-vCM differentiation. Methods: We used a GMP compliant Quad Physoxia glovebox platform providing continuous stable specified O2 tensions during all processes to simulate the in vivo O2 conditions more closely with the aim of improving iPSC-vCM maturation. Results: We demonstrate by single cell RNA sequencing, and data integration with datasets for human cardiomyocytes from the different heart chambers as well as cell morphology-, ploidy-, and functional studies, that sustained 10% O2 throughout iPSC-CM differentiation promotes atrial- instead of ventricular iPSC-CM subtype specification. Conclusions: While this rejects our original hypothesis and forces some concerns to iPSC-vCM manufacturing by sphere technology, these unexpected data may serve as an attractive and easy approach to refine atrial iPSC-CM specification to benefit their exponentially growing diagnostic-, cytotoxic-, and regenerative use.

cell biology↗

bioRxiv2024

Benchmarking transcriptional deconvolution methods for estimating tissue- and cell type-specific extracellular vesicle abundances

Extracellular vesicles (EVs) contain cell-derived lipids, proteins, and RNAs; however, the challenge to determine the tissue- and cell type-specific EV abundances in body fluids remains a significant hurdle for our understanding of EV biology. While tissue- and cell type-specific EV abundances can be estimated by matching the EVs transcriptome to a tissues/cell types expression signature using deconvolutional methods, a comparative assessment of deconvolution methods performance on EV transcriptome data is currently lacking. We benchmarked 11 deconvolution methods using data from 4 cell lines and their EVs, in silico mixtures, 118 human plasma, and 88 urine EVs. We identified deconvolution methods that estimated cell type-specific abundances of pure and in silico mixed cell line-derived EV samples with high accuracy. Using data from two urine EV cohorts with different EV isolation procedures, four deconvolution methods produced highly similar results. The four methods were also highly concordant in their tissue-specific plasma EV abundance estimates. We identified driving factors for deconvolution accuracy and highlight the importance of implementing biological knowledge in creating the tissue/cell type signature. Overall, our analyses demonstrate that the deconvolution algorithms DWLS and CIBERSORTx produce highly similar and accurate estimates of tissue- and cell type-specific EV abundances in biological fluids.

bioinformatics↗

bioRxiv2021

A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV- Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function.

molecular biology↗

bioRxiv2018

A bacterial display system for effective selection of protein-biotin ligase BirA variants with novel peptide specificity

Biotinylation creates a sensitive and specific tag for purification and detection of target proteins. The E. coli protein-biotin ligase BirA biotinylates a lysine within a synthetic biotin acceptor peptide (AP) and allow for specific tagging of proteins fused to the AP. The approach is not applicable to unmodified proteins, and we sought to develop an effective selection system that could form the basis for directed evolution of novel BirA variants with specificity towards unmodified proteins. The system was based on bacterial display of a target peptide sequence, which could be biotinylated by cytosolic BirA variants before being displayed on the surface. In a model selection, the bacterial display system accomplished >1.000.000 enrichment in a single selection step. A randomly mutated BirA library was used to identify novel variants. Bacteria displaying peptide sequences from 13 out of 14 tested proteins were strongly enriched after 3-5 selection rounds. Moreover, a clone selected for biotinylation of a C-terminal peptide from red-fluorescent protein TagRFP showed site-specific biotinylation. Thus, active BirA variants with novel specificity are effectively isolated with our bacterial display system and provides a basis for the development of BirA variants for site-selective biotinylation.

bioengineering↗