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Collagen-heparan sulfate microcarriers support mesenchymal stem cell culture in microgravity and streamline post-flight recovery

ABSTRACT: The objective of this study was to validate a scalable, integrated workflow for Mesenchymal Stem Cell (MSC) culture in Low Earth Orbit (LEO) using pre-seeded, surface-engineered microcarriers (uCs). Cell thaw and expansion were conducted at ambient temperature (22 °C) in serum-free and DMSO-free media. Consequently, the primary objective of the miRNA sequencing was to verify the presence and stability of bioactive cargo within the extracellular vesicle (EV)-enriched fraction. This genomic analysis serves as a critical quality metric to ensure that the "pre-seed-freeze-thaw-and-expand" logistical framework preserves the integrity of the MSC secretome during orbital transit and post-flight. Small RNA sequencing was performed on EV-enriched fractions isolated from frozen conditioned media processed post-flight to assess the stability of bioactive miRNA signatures across different surface chemistries (engineered HS/COL vs. commercial COL uCs) and gravitational environments. MSCs in suspension (lacking microcarriers) served as the matrix-attachment control.

ORGANISM(S): Homo sapiens

PROVIDER: GSE328254 | GEO | 2026/08/02

REPOSITORIES: GEO

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Project description:Mesenchymal stromal cells (MSCs) are multipotent stromal cells with regenerative, immunomodulatory, and anti-inflammatory properties, making them a promising tool in cell-based therapies. Increasing evidence suggests the therapeutic effects of MSCs are largely mediated by secreted paracrine factors and extracellular vesicles (EVs), which facilitate extracellular communication by transferring bioactive molecules such as proteins, lipids, and nucleic acids to target cells. The lipid composition of EVs is critical for their stability, uptake, and functional activity. Sphingomyelinase (SMase), an enzyme that hydrolyzes sphingomyelin into ceramide, plays a key role in EV biogenesis by influencing membrane curvature allowing for invagination and budding of multivesicular bodies (MVBs). However, the impact of SMase treatment on the characteristics and therapeutic potential of MSC-derived EVs remains largely unexplored. In this study, human MSCs were cultured with SMase for 24 hours and EVs were isolated via ultracentrifugation. EV size distribution was analyzed using nanoparticle tracking analysis (NTA), microRNA (miRNA) profile was determined using RNA-sequencing, while lipidomic and proteomic profiling were assessed using mass spectrometry. Functional assays were conducted to assess changes in bioactivity. NTA revealed a shift in EV production, with a higher vesicle concentration in SMase induced MSCs compared to untreated cells. SMase treatment significantly altered MSC-EV composition, leading to increased ceramide and glycosphingolipids. In addition, the modifications in protein and miRNA EV cargo were found to be associated with pathways involving TNF-α signaling, vesicle trafficking, wound healing, and angiogenesis. Functional assays indicated greater suppression of TNF-α in activated macrophages and greater tubular network formation in HUVECS. These findings provide new insights into distinct biophysical and biochemical changes in MSC-derived EVs following SMase stimulation, potentially enhancing their therapeutic properties and may inform strategies for optimizing future EV-based therapies.

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Collagen-heparan sulfate microcarriers support mesenchymal stem cell culture in microgravity and streamline post-flight recovery

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