Modulation of mesenchymal stromal cell characteristics by microcarrier culture in bioreactors.

Mesenchymal stromal cells (MSCs) are promising candidates for cell therapy. Their therapeutic use requires extensive expansion to obtain a sufficiently high number of cells for clinical applications. State-of-the-art expansion systems, that is, primarily culture flask-based systems, are limited regarding scale-up, automation, and reproducibility. To overcome this bottleneck, microcarrier (MC)-based expansion processes have been developed. For the first time, MSCs from the perinatal sources umbilical cord (UC) and amniotic membrane (AM) were expanded on MCs. This study focuses on the comparison of flask- and Cytodex 1 MC-expanded MSCs by evaluating the influence of the expansion process on biological MSC characteristics. Furthermore, we tested the hypothesis to obtain more homogeneous MSC preparations by expanding cells on MCs in controlled large-scale bioreactors. MSCs were extensively characterized determining morphology, cell growth, surface marker expression, and functional properties such as differentiation capacity, secretion of paracrine factors, and gene expression. Based on their gene expression profile MSCs from different donors and sources clearly clustered in distinct groups solely depending on the expansion process-MC or flask culture. MC- and flask-expanded MSCs significantly differed from each other regarding surface markers and both paracrine factors and gene expression profiles. Furthermore, based on gene expression analysis, MC cultivation of MSCs in controlled bioreactor systems resulted in less heterogeneity between cells from different donors. In conclusion, MC-based MSC expansion in controlled bioreactors has the potential to reliably produce MSCs with altered characteristics and functions as compared to flask-expanded MSCs. These findings may be useful for the generation of MSCs with tailored properties for clinical applications.

Multicenter evaluation of blood-based biomarkers for the detection of endometriosis and adenomyosis: A prospective non-interventional study.

OBJECTIVE: To evaluate blood-based biomarkers to detect endometriosis and/or adenomyosis across nine European centers (June 2014-April 2018). METHODS: This prospective, non-interventional study assessed the diagnostic accuracy of 54 blood-based biomarker immunoassays in samples from 919 women (aged 18-45 years) with suspicion of endometriosis and/or adenomyosis versus symptomatic controls. Endometriosis was stratified by revised American Society for Reproductive Medicine stage. Symptomatic controls were "pathologic symptomatic controls" or "pathology-free symptomatic controls". The main outcome measure was receiver operating characteristic-area under the curve (ROC-AUC) and Wilcoxon P values corrected for multiple testing (q values). RESULTS: CA-125 performed best in "all endometriosis cases" versus "all symptomatic controls" (AUC 0.645, 95% confidence interval [CI] 0.600-0.690, q < 0.001) and increased (P < 0.001) with disease stage. In "all endometriosis cases" versus "pathology-free symptomatic controls", S100-A12 performed best (AUC 0.692, 95% CI 0.614-0.769, q = 0.001) followed by CA-125 (AUC 0.649, 95% CI 0.569-0.729, q = 0.021). In "adenomyosis only cases" versus "symptomatic controls" or "pathology-free symptomatic controls", respectively, the top-performing biomarkers were sFRP-4 (AUC 0.615, 95% CI 0.551-0.678, q = 0.045) and S100-A12 (AUC 0.701, 95% CI 0.611-0.792, q = 0.004). CONCLUSION: This study concluded that no biomarkers tested could diagnose or rule out endometriosis/adenomyosis with high certainty.

EphA4-dependent axon guidance is mediated by the RacGAP alpha2-chimaerin.

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. We show that the Rac-specific GTPase-activating protein alpha2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. alpha-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. Our results reveal an alpha-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo.

Structurally and functionally unique complexins at retinal ribbon synapses.

Ribbon synapses in retinal sensory neurons maintain large pools of readily releasable synaptic vesicles. This allows them to release several hundreds of vesicles per second at every presynaptic release site. The molecular components that cause this high transmitter release efficiency of ribbon synapses are unknown. In the present study, we identified and characterized two novel vertebrate complexins (CPXs), CPXs III and IV, that are the only CPX isoforms present in retinal ribbon synapses. CPXs III and IV are COOH-terminally farnesylated, and, like CPXs I and II, bind to SNAP receptor complexes. CPXs III and IV can functionally replace CPXs I and II, and their COOH-terminal farnesylation regulates their synaptic targeting and modulatory function in transmitter release. The novel CPXs III and IV may contribute to the unique release efficacy of retinal sensory neurons.

Divergent and convergent signaling by the diacylglycerol second messenger pathway in mammals.

Diacylglycerol is an essential second messenger in mammalian cells. The most prominent intracellular targets of diacylglycerol and the functionally analogous phorbol esters belong to the protein kinase C family, but at least five alternative types of high affinity diacylglycerol/phorbol ester receptors are known: protein kinase D, diacylglycerol kinases alpha, beta, and gamma, RasGRPs, chimaerins, and Munc13s. These function independently of protein kinase C isozymes, and form a network of signaling pathways in the diacylglycerol second messenger system that regulates processes as diverse as gene transcription, lipid signaling, cytoskeletal dynamics, intracellular membrane trafficking, or neurotransmitter release.

Mesenchymal stromal cell characteristics vary depending on their origin.

Mesenchymal stromal cells (MSCs) are rare progenitor cells that can be isolated from various tissues. They exhibit multilineage differentiation potential, support regenerative processes, and interact with various immune cells. Therefore, MSCs represent a promising tool for regenerative medicine. However, source-dependent and donor-dependent differences of MSC properties, including implications on their clinical application are still largely unknown. We evaluated MSCs derived from perinatal tissues umbilical cord (UC) and amniotic membrane (AM) in comparison to adult MSCs from bone marrow (BM), which were used as gold standard. We found genetic background-independent differences between MSCs from UC and AM. While AM- and UC-MSCs were closer to each other than to BM-MSCs, they also exhibited differences between each other. AM-MSCs from different donors but not UC-MSCs displayed high interdonor variability. In addition, we show that although all MSCs expressed similar surface markers, MSC populations from UC and AM showed differential profiles of gene expression and paracrine factor secretion to BM-derived MSCs. Notably, pathway analysis of gene expression data revealed intriguing differences between MSCs suggesting that MSCs from UC and AM possess in general a higher potential of immunomodulatory capacity, whereas BM-MSCs showed a higher potential of supporting regenerative processes as exemplified by neuronal differentiation and development. These differences between perinatal and BM-derived MSCs may be relevant for clinical applications.