Biology subjects

Marklein, R. A.

Publications and source records attributed to Marklein, R. A..

7 recordsLinked to original sources

bioRxiv2025

Development of a High-throughput Morphological Assay for Evaluating Mesenchymal Stromal Cell-derived Extracellular Vesicle Modulation of Brain Pericyte Secretory Phenotype

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are a promising therapeutic tool for treating many neurodegenerative diseases. Neuroinflammation plays a major role in many of these conditions through an orchestration of interdependent processes that lead to the breakdown of the blood-brain barrier (BBB), infiltration of immune cells and neuronal death. MSC-EVs have shown preliminary evidence of modulating neuroinflammation, but their mechanisms of action are still unknown. Therefore, we explored the potential of MSC-EVs in modulating brain pericytes, a cell type that plays a critical role in BBB maintenance but has not been investigated as a therapeutic target for MSC-EVs. Brain pericytes are multifaceted cells that can modulate neuroinflammation through their involvement in BBB homeostasis, as well as the innate and adaptive immune response. Pericyte morphology has been shown to change in response to inflammatory stimuli in vivo, hence, we used this behavior to develop a quantitative morphological profiling approach to assess the immunomodulatory function of MSC-EVs in a high-throughput, low-cost manner. Using this assay, we were able to demonstrate that MSC-EVs manufactured under various conditions (2D, 3D, and in response to cytokine priming) could induce distinct pericyte morphological responses indicative of changes in secretion of chemokines and cytokines relevant to neuroinflammation.

bioengineering↗

bioRxiv2024

High Throughput Morphological Screening Identifies Chemically Defined Media for Mesenchymal Stromal Cells that Enhances Proliferation and Supports Maintenance of Immunomodulatory Function

While mesenchymal stromal cell (MSC) therapies show promise for treating several indications due to their regenerative and immunomodulatory capacity, clinical translation has yet to be achieved due to a lack of robust, scalable manufacturing practices. Expansion using undefined fetal bovine serum (FBS) or human platelet lysate contributes to MSC functional heterogeneity and limits control of product quality. The need for tunable and consistent media has thus encouraged development of chemically defined media (CDM). However, CDM development strategies are rarely comprehensive nor considerate of a mediums impact on MSC therapeutic function. Standard practice often neglects high-level interactions of media components, such as growth factors, that are critical to MSC growth and function. Given that MSC morphology has been shown to predict their immunomodulatory function, we employed a high throughput screening (HTS) approach to elucidate effects of growth factor compositions on MSC phenotype and proliferation in a custom CDM. This approach led to the discovery and refinement of several formulations that enhanced MSC proliferation and demonstrated wide ranging impacts on MSC immunomodulation. Overall, this work reflects how our novel HTS approach serves as a generalizable tool for the comprehensive improvement of MSC manufacturing processes.

bioengineering↗

bioRxiv2024

Microglia Morphological Response to Mesenchymal Stromal Cell Extracellular Vesicles Demonstrates EV Therapeutic Potential for Modulating Neuroinflammation

BackgroundMesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation-relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs. ResultsUsing an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-{gamma}) and tumor necrosis factor-alpha (TNF-). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-{gamma}/IFN- stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism. ConclusionThis study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphological approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.

bioengineering↗

bioRxiv2023

High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia

Due to their immunomodulatory function, mesenchymal stromal cells (MSCs) are a promising therapeutic with the potential to treat neuroinflammation associated with neurodegenerative diseases. This function can be mediated by secreted extracellular vesicles (MSC-EVs). Despite established safety, MSC clinical translation has been unsuccessful due to inconsistent clinical outcomes resulting from functional heterogeneity. Current approaches to mitigate functional heterogeneity include priming MSCs with inflammatory signals to enhance function. However, comprehensive evaluation of priming and its effects on MSC-EV function has not been performed. Clinical translation of MSC-EV therapies requires significant manufacturing scale-up, yet few studies have investigated the effects of priming in bioreactors. As MSC morphology has been shown to predict their immunomodulatory function, we screened MSC morphological response to an array of priming signals and evaluated MSC-EV identity and potency in response to priming in flasks and bioreactors. We identified unique priming conditions corresponding to distinct morphologies. These conditions demonstrated a range of MSC-EV preparation quality and lipidome, allowing us to discover a novel MSC-EV manufacturing condition, as well as gain insight into potential mechanisms of MSC-EV microglia modulation. Our novel screening approach and application of priming to MSC-EV bioreactor manufacturing informs refinement of larger-scale manufacturing and enhancement of MSC-EV function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/567730v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1fca79forg.highwire.dtl.DTLVardef@60f543org.highwire.dtl.DTLVardef@186394borg.highwire.dtl.DTLVardef@11b94bd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMSCs morphologically respond to inflammatory priming conditions. C_LIO_LIPriming hits identified from morphological screen increase MSC-EV production. C_LIO_LIPriming MSCs in bioreactors enhances MSC-EV modulation of microglia. C_LIO_LIChanges in MSC-EV production and potency reflected by lipid content. C_LIO_LIFirst demonstration of effects of priming on MSC production of EVs in a bioreactor. C_LI

bioengineering↗

bioRxiv2021

Multi-omics characterization of mesenchymal stem/stromal cells for the identification of putative critical quality attributes

BackgroundMesenchymal stromal cells (MSCs) have shown great promise in the field of regenerative medicine as many studies have shown that MSCs possess immunomodulatory function. Despite this promise, no MSC therapies have been granted licensure from the FDA. This lack of successful clinical translation is due in part to MSC heterogeneity and a lack of critical quality attributes (CQAs). While MSC Indoleamine 2,3-dioxygnease (IDO) activity has been shown to correlate with MSC function, multiple CQAs may be needed to better predict MSC function. MethodsThree MSC lines (two bone marrow, one iPSC) were expanded to three passages. At the time of harvest for each passage, cell pellets were collected for nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography mass spectrometry (UPLC-MS), and media was collected for cytokine profiling. Harvested cells were also cryopreserved for assessing function using T cell proliferation and IDO activity assays. Linear regression was performed on functional and multiomics data to reduce the number of important features, and partial least squares regression (PLSR) was used to obtain putative CQAs based on variable importance in projection (VIP) scores. ResultsSignificant functional heterogeneity (in terms of T cell suppression and IDO activity) was observed between the three MSC lines, as well as donor-dependent differences based on passage. Omics characterization revealed distinct differences between cell lines using principal component analysis (PCA). Cell lines separated along principal component 1 based on tissue source (bone marrow vs. iPSC-derived) for NMR, MS, and cytokine profiles. PLSR modeling of important features predicts MSC functional capacity with NMR (R2=0.86), MS (R2=0.83), cytokines (R2=0.70), and a combination of all features (R2=0.88). DiscussionThe work described here provides a platform for identifying putative CQAs for predicting MSC functional capacity using PLSR modeling that could be used as release criteria and guide future manufacturing strategies for MSCs and other cell therapies.

bioengineering↗

bioRxiv2021

Morphological Landscapes from High Content Imaging Identify Optimal Priming Strategies that Enhance MSC Immunosuppression

Successful clinical translation of mesenchymal stromal cell (MSC) products has not been achieved in the United States and may be in large part due to MSC functional heterogeneity. Efforts have been made to identify priming conditions that produce MSCs with consistent immunomodulatory function; however, challenges remain with predicting and understanding how priming impacts MSC behavior. The purpose of this study was to develop a high throughput, image-based approach to assess MSC morphology in response to combinatorial priming treatments and establish morphological profiling as an effective approach to screen the effect of manufacturing changes (i.e. priming) on MSC immunomodulation. We characterized the morphological response of multiple MSC lines/passages to an array of Interferon-gamma (IFN-{gamma}) and Tumor Necrosis Factor alpha (TNF-) priming conditions, as well as the effects of priming on MSC modulation of activated T cells and MSC secretome. Although considerable functional heterogeneity, in terms of T cell suppression, was observed between different MSC lines and at different passages, this heterogeneity was significantly reduced with combined IFN-{gamma}/TNF- priming. The magnitude of this change correlated strongly with multiple morphological features and was also reflected by MSC secretion of immunomodulatory factors e.g. PGE2, ICAM-1, and CXCL16. Overall, this study further demonstrates the ability of priming to enhance MSC function, as well as the ability of morphology to better understand MSC heterogeneity and predict changes in function due to manufacturing.

bioengineering↗

bioRxiv2020

Priming of MSCs with inflammation-relevant signals affects extracellular vesicle biogenesis, surface markers, and modulation of T cell subsets

Although considerable evidence exists supporting the use of mesenchymal stromal cells (MSCs) for treating immune diseases, successful clinical translation has been challenging and has led researchers to investigate cell-free alternatives. MSC-derived extracellular vesicles (MSC-EVs) have been shown to mediate a significant portion of the observed therapeutic effect, including immunosuppression. MSCs have been shown to respond to different aspects of the injury microenvironment such as inflammatory cytokines and hypoxia, although acidosis has not been investigated and different conditions have not been assessed in terms of their effects on MSC-EV function. This study investigated the effects of acidosis, hypoxia, and inflammatory cytokine priming on MSCs and MSC-EVs. We cultured MSCs in the presence of acidosis, hypoxia, or inflammatory cytokines (Interferon-gamma and Tumor Necrosis Factor-alpha) and compared the characteristics of their EVs as well as their uptake by and suppression of different T cell subsets. MSCs showed a greater effect on suppressing activated CD4+ and CD8+ T cells than MSC-EVs. However, MSC-EVs from MSCs primed with acidosis increased CD4+ and CD8+ regulatory T cell frequency in vitro. This functional response was reflected by MSC-EV uptake. MSC-EVs from acidosis-primed MSCs were taken up by CD4+ and CD8+ regulatory T cells at a significantly higher level than MSC-EVs from control, hypoxic, and inflammatory cytokine groups. These data suggest that a simple low-cost alteration in MSC culture conditions, acidosis, can generate extracelluar vesicles that have a desirable influence on anti inflammatory T cell subtypes.

bioengineering↗