Biology subjects

Keselman, P.

Publications and source records attributed to Keselman, P..

4 recordsLinked to original sources

bioRxiv2025

Orthogonal and Robust Analytics Enable Reproducible and Scalable Manufacturing of High Purity Extracellular Vesicles Derived from Mesenchymal Stromal Cells

The development of extracellular vesicle (EV) based therapeutics requires robust analytical assays and scalable downstream processing (DSP) strategies to ensure product quality and reproducibility. In this study, we established an analytical toolbox comprising scattering and fluorescence mode nanoparticle tracking analysis (NTA), fluorescence-based flow cytometry (Fl-FC), and multi-detector analytical size exclusion chromatography. Liposomes, selected for their physicochemical similarity to EVs, were used as reference materials to optimize assay parameters, fluorescence labeling conditions, and dynamic range, minimizing artifacts such as photobleaching and masking effects. Using these optimized analytical tools, we designed a scalable DSP workflow for human bone marrow mesenchymal stromal cell (hBM-MSC) derived EVs, incorporating clarification, tangential flow filtration (TFF), ion exchange chromatography (IEX), buffer exchange, and sterile filtration. IEX chromatography resulted in the elution of two cell-secreted populations, with similar scattering signals while eluate 1 showed approximately 30 times higher absorbance signal compared to eluate 2. Transmission electron microscopy revealed that eluate 1 contained non-vesicular extracellular particles (NVEPs), and eluate 2 was enriched in EVs and showed higher expression of positive markers such as CD81 and CD73 using Simple Western. Additionally, the two IEX eluates showed different proteomic and lipidomic profiles. Then the analytical toolbox was utilized to monitor the DSP process in terms of particle recovery and impurity removal throughout the process determining the high purity level of the final EV preparation. Together, these results demonstrate that orthogonal analytics coupled to a scalable DSP yield reproducible MSC-EV preparations while depleting commonly co-isolated NVEPs. This practical framework advances process analytics of MSC-EV manufacturing and supports reporting of identity, purity, and function aligned with existing guidelines in the field. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/672476v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@142ff8aorg.highwire.dtl.DTLVardef@196c0bforg.highwire.dtl.DTLVardef@485c36org.highwire.dtl.DTLVardef@9dff39_HPS_FORMAT_FIGEXP M_FIG C_FIG Liposomes were used as reference materials to optimize single particle analytics as well as multi detector analytical chromatography to assess particle recovery and impurity removal. This optimized analytical toolbox enabled accurate monitoring of downstream processing steps, establishing a robust workflow for reproducible and scalable mesenchymal stromal derived extracellular vesicles bioprocessing.

bioengineering↗

bioRxiv2025

A simplified co-culture reveals altered cardiotoxic responses to doxorubicin in hPSC-derived cardiomyocytes in the presence of endothelial cells

Cardiotoxicity is a significant challenge in cancer therapies, particularly with doxorubicin, a widely used anthracycline known for its broad anti-cancer spectrum but life-threatening cardiac side effects. There is a critical need for more predictive in vitro models to understand doxorubicin-induced cardiotoxicity and patient-specific drug responses. In this study, we used human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs), cardiac fibroblasts (hPSC-cFBs) and endothelial cells (hPSC-ECs) to investigate the cardiotoxic effects of doxorubicin in two-dimensional mono-and multi-cell type cultures. By mimicking the cumulative dose effect seen in patients through repeated doxorubicin treatments and using a machine learning-based in silico image analysis tool, we could precisely quantify caspase 3/7 activity as an early toxicity marker and identify hPSC-CMs in multi-cell type cultures. This innovative approach allowed continuous monitoring of apoptosis from phase-contrast images, revealing that hPSC-ECs showed higher sensitivity to doxorubicin than isogenic hPSC-CMs or hPSC-cFBs and significantly enhanced cardiomyocyte toxicity in co-culture. In contrast, dermal fibroblasts differentiated from the same hPSC line showed no toxic response under the same treatment regimen. These results challenge the conventional focus on cardiomyocytes as the target of drug-induced cardiac damage. Our findings not only highlight the complex interplay among different cardiac cell types in mediating the toxic effects of doxorubicin, but also demonstrate the potential of AI-enabled tools to advance personalized drug screening and safety assessments.

pharmacology and toxicology↗

bioRxiv2022

Novel insights into the fundamentals of palatal shelf elevation dynamics in normal mouse embryos

Embryonic palate development involves bilateral vertical growth of palatal shelves - extensions from the maxillary processes - next to the tongue until embryonic day (E) 13.5. Following vertical growth, palatal shelves elevate and adhere above the tongue by E14.5. Current models indicate that this process of elevation involves a complex vertical to horizontal reorienting of the palatal shelves. While earlier studies have implied that this is a rapid process, the precise timing has not been resolved. To understand the dynamics of palatal shelf elevation, we employed time-restricted pregnancies with a one-hour resolution and magnetic resonance imaging of intermediate stages. Our data showed that in almost all C57BL/6J embryos, palatal shelves have not yet elevated by E14.0. However, six hours later at E14.25, palatal shelves have completed elevation in 80% of embryos. Interestingly, all E14.25 embryos with unelevated palatal shelves (20%) were female, suggesting a delay in female embryos. In FVB/NJ embryos, the elevation window started earlier (E13.875-E14.25) without any noticeable sex differences. We frequently captured an intermediate stage with unilateral elevation of either right or left palatal shelf. Magnetic resonance imaging of various stages showed that palatal shelf elevation began with the formation of bilateral bulges in the posterior. These bulges progressed laterally and anteriorly over time. During elevation, we observed increased cell proliferation in the lingual region of the palatal shelf. Within the bulge, cell orientation was acutely tilted towards the tongue and actomyosin activity was increased, which together may participate in the projection of the bulge in the horizontal direction. Thus, our data reveal novel insights into the rapid dynamic changes in palatal shelf elevation that lay the foundation for future studies of normal and abnormal palatogenesis.

developmental biology↗

bioRxiv2021

Sex Differences in Cerebral Edema after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. Cerebral edema following TBI is known to play a critical role in injury severity and prognosis. In the current study we used multimodal magnetic resonance imaging (MRI) to assess cerebral edema 24 hours after unilateral contusive TBI in male and female rats. We then directly quantified brain water content in the same subjects ex vivo. We found that both males and females had similarly elevated T2 values after TBI compared with sham controls. Apparent diffusion coefficient (ADC) was more variable than T2 and did not show significant injury effects in males or females. Brain water was elevated in male TBI rats compared with sham controls, but there was no difference between female TBI and sham groups. Notably, MRI biomarkers of edema were more closely correlated with brain water in male rats; female rats did not show any relationship between brain water and T2 or ADC. These observations raise questions about the interpretation of radiological findings traditionally interpreted as edema in female TBI patients. A better understanding of sex differences and similarities in the pathophysiology of post-traumatic edema is needed to help improve patient management and the development of effective treatment strategies for men and women.

neuroscience↗