Xuan Zheng

Abstract Grafted mesenchymal stem cells (MSCs) yield neuroprotection in preclinical stroke models by secreting extracellular vesicles (EVs). The neuroprotective cargo of EVs, however, has not yet been identified. To investigate such cargo and its underlying mechanism, primary neurons were exposed to oxygen‐glucose‐deprivation (OGD) and cocultured with adipose‐derived MSCs (ADMSCs) or ADMSC‐secreted EVs. Under such conditions, both ADMSCs and ADMSC‐secreted EVs significantly reduced neuronal death. Screening for signalling cascades being involved in the interaction between ADMSCs and neurons revealed a decreased autophagic flux as well as a declined p53‐BNIP3 activity in neurons receiving either treatment paradigm. However, the aforementioned effects were reversed when ADMSCs were pretreated with the inhibitor of exosomal secretion GW4869 or when Hrs was knocked down. In light of miR‐25‐3p being the most highly expressed miRNA in ADMSC‐EVs interacting with the p53 pathway, further in vitro work focused on this pathway. Indeed, a miR‐25‐3p oligonucleotide mimic reduced cell death, whereas the anti‐oligonucleotide increased autophagic flux and cell death by modulating p53‐BNIP3 signalling in primary neurons exposed to OGD. Likewise, native ADMSC‐EVs but not EVs obtained from ADMSCs pretreated with the anti‐miR‐25‐3p oligonucleotide (ADMSC‐EVs anti‐miR‐25‐3p ) confirmed the aforementioned in vitro observations in C57BL/6 mice exposed to cerebral ischemia. The infarct size was reduced, and neurological recovery was increased in mice treated with native ADMSC‐EVs when compared to ADMSC‐EVs anti‐miR‐25‐3p . ADMSCs induce neuroprotection by improved autophagic flux through secreted EVs containing miR‐25‐3p. Hence, our work uncovers a novel key factor in naturally secreted ADMSC‐EVs for the regulation of autophagy and induction of neuroprotection in a preclinical stroke model.

Time-lapse microscopy is routinely used to follow cells within organoids, allowing direct study of division and differentiation patterns. There is an increasing interest in cell tracking in organoids, which makes it possible to study their growth and homeostasis at the single-cell level. As tracking these cells by hand is prohibitively time consuming, automation using a computer program is required. Unfortunately, organoids have a high cell density and fast cell movement, which makes automated cell tracking difficult. In this work, a semi-automated cell tracker has been developed. To detect the nuclei, we use a machine learning approach based on a convolutional neural network. To form cell trajectories, we link detections at different time points together using a min-cost flow solver. The tracker raises warnings for situations with likely errors. Rapid changes in nucleus volume and position are reported for manual review, as well as cases where nuclei divide, appear and disappear. When the warning system is adjusted such that virtually error-free lineage trees can be obtained, still less than 2% of all detected nuclei positions are marked for manual analysis. This provides an enormous speed boost over manual cell tracking, while still providing tracking data of the same quality as manual tracking.

Stem cells such as mesenchymal stem cells (MSCs) enhance neurological recovery in preclinical stroke models by secreting extracellular vesicles (EVs). Since previous reports have focused on the application of MSC-EVs only, the role of the most suitable host cell for EV enrichment and preclinical stroke treatment remains elusive. The present study aimed to evaluate the therapeutic potential of EVs derived from neural progenitor cells (NPCs) following experimental stroke. Using the PEG technique, EVs were enriched and characterized by electron microscopy, proteomics, rt-PCR, nanosight tracking analysis, and Western blotting. Different dosages of NPC-EVs displaying a characteristic profile in size, shape, cargo protein, and non-coding RNA contents were incubated in the presence of cerebral organoids exposed to oxygen-glucose deprivation (OGD), significantly reducing cell injury when compared with control organoids. Systemic administration of NPC-EVs in male C57BL6 mice following experimental ischemia enhanced neurological recovery and neuroregeneration for as long as 3 months. Interestingly, the therapeutic impact of such NPC-EVs was found to be not inferior to MSC-EVs. Flow cytometric analyses of blood and brain samples 7 days post-stroke demonstrated increased blood concentrations of B and T lymphocytes after NPC-EV delivery, without affecting cerebral cell counts. Likewise, a biodistribution analysis after systemic delivery of NPC-EVs revealed the majority of NPC-EVs to be found in extracranial organs such as the liver and the lung. This proof-of-concept study supports the idea of EVs being a general concept of stem cell-induced neuroprotection under stroke conditions, where EVs contribute to reverting the peripheral post-stroke immunosuppression.