Biology subjects

Voulgaris, D.

Publications and source records attributed to Voulgaris, D..

3 recordsLinked to original sources

bioRxiv2025

Human iPSC-based coculture model reveals neuroinflammatory crosstalk between microglia and astrocytes

BackgroundMicroglia and astrocytes have been implicated as central mediators of neuroinflammatory processes in several neurodegenerative diseases. However, their intricate crosstalk and contributions to pathogenesis remain elusive, highlighting the need for innovative in vitro approaches for investigating glial interactions in neuroinflammation. The aim of this study was to develop advanced human-based glial coculture models to explore the inflammatory roles and interactions of microglia and astrocytes in vitro. MethodsWe utilized human induced pluripotent stem cell (iPSC)-derived microglia and astrocytes cultured both in conventional culture dishes and in a compartmentalized microfluidic chip coculture platform. This novel platform features separate compartments for both cell types, enabling the creation of fluidically isolated microenvironments with spontaneous migration of microglia toward astrocytes through interconnecting microtunnels. To induce inflammatory activation, glial cultures were stimulated with lipopolysaccharide (LPS), a combination of tumor necrosis factor- (TNF-) and interleukin-1{beta} (IL-1{beta}), or interferon-{gamma} (IFN-{gamma}) for 24 hours. The glial activation and crosstalk were analyzed with immunocytochemistry, the secretion of inflammatory factors from the culture media was measured, and microglial migration was quantified. ResultsMicroglia-astrocyte cocultures were successfully generated in both conventional cultures and the microfluidic chip platform. Inflammatory stimulation with LPS and TNF-/IL-1{beta} elicited cell type-specific responses in microglia and astrocytes, respectively. Notably, the levels of secreted inflammatory mediators were altered under coculture conditions, revealing significant glial crosstalk. Utilization of our microfluidic coculture platform facilitated the study of microglial migration and glial activation within distinct inflammatory microenvironments. Microglia migrated efficiently toward the astrocyte compartment, and the chemoattractant adenosine diphosphate (ADP) notably increased microglial migration within this platform. Furthermore, inflammatory stimulation of the microfluidic chip cocultures successfully recapitulated glial crosstalk, revealing unique responses. This crosstalk was associated with elevated levels of complement component C3 in the cocultures, emphasizing the intricate interplay between microglia and astrocytes under inflammatory conditions. ConclusionsOur results depict an elaborate molecular crosstalk between inflammatory microglia and astrocytes, providing evidence of how glial cells orchestrate responses during neuroinflammation. Importantly, we demonstrate that the microfluidic coculture platform developed in this study for microglia and astrocytes provides a more functional and enhanced setup for investigating inflammatory glial interactions in vitro.

neuroscience↗

bioRxiv2021

Generation of star-shaped human induced astrocytes with a mature inflammatory phenotype for CNS disease modeling

Generating astrocytes from induced pluripotent stem cells has been hampered by either prolonged differentiation -spanning over two months -or by shorter protocols that generate immature astrocytes, devoid of salient inflammation-associated astrocytic traits pivotal for CNS neuropathological modeling. We directed human neural stem cells derived from induced pluripotent stem cells to astrocytic commitment and maturity by orchestrating an astrocytic-tuned culturing environment. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of AQP4 and, remarkably, the expression and functionality of glutamate transporter EAAT2. We also show that this protocol generates astrocytes that encompass traits critical in CNS disease modeling, such as glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile which is on par with primary astrocytes. This protocol generates a multifaceted astrocytic model suitable for CNS in vitro disease modeling and personalized medicine through brain-on-chip technologies.

neuroscience↗

bioRxiv2021

Continuous monitoring reveals protective effects of N-acetylcysteine amide on an isogenic microphysiological model of the neurovascular unit

Microphysiological systems mimic the in-vivo cellular ensemble and microenvironment with the goal of providing more human-like models for biopharmaceutical research. We report the first such model of the blood-brain barrier (BBB-on-chip) featuring both isogenic human induced pluripotent stem cell (hiPSC)-derived cells and continuous barrier integrity monitoring with <2-minute temporal resolution. We showcase its capabilities in the first microphysiological study of nitrosative stress and antioxidant prophylaxis. Relying on off-stoichiometry thiol-ene epoxy (OSTE+) for fabrication greatly facilitates assembly and sensor integration compared to the prevalent polydimethylsiloxane devices. The integrated cell-substrate endothelial resistance monitoring allows us to capture formation and breakdown of our blood-brain barrier model, consisting of co-cultured hiPSC-derived endothelial-like and astrocyte-like cells. We observe clear cellular disruption when exposing the BBB-on-chip to the nitrosative stressor linsidomine, and report on the barrier permeability and barrier-protective effects of the antioxidant N-acetylcysteine amide. Using metabolomic network analysis, we further find drug-induced changes consistent with prior literature regarding, e.g., cysteine and glutathione involvement. A model like ours opens new possibilities for drug screening studies and personalized medicine, relying solely on isogenic human-derived cells and providing high-resolution temporal readouts that can help in pharmacodynamic studies.

bioengineering↗