Julien Sirois

Human midbrain organoids (hMOs) are a powerful stem cell-derived model for studying human neurodevelopment and neurological disease in vitro; however, their broad adoption is limited by long culture times, high cost, and inter-batch variability. Cryopreservation offers a strategy to address these limitations by enabling long-term storage and reducing experimental heterogeneity, but its effectiveness depends on robust cryoprotective strategies. Here, we evaluated hMO cryopreservation using a standard 10% dimethyl sulfoxide (DMSO) protocol, alone and in combination with a panel of five ice recrystallization inhibitors (IRIs), including betaine-derived ammonium salts and N-aryl-d-gluconamides. Across all conditions, hMOs were successfully recovered following freeze–thawing, with preservation of overall structural integrity, including maintained morphology and reduced cellular debris. Importantly, post-thaw viability and cell recovery in the 10% DMSO condition were comparable to those of fresh controls, demonstrating that conventional DMSO-based cryopreservation is sufficient to maintain hMO survival and gross structural preservation. While supplementation with select IRIs, particularly N-ethyl betaine ethyl ester iodide (NEBEE-I), yielded modest improvements in viability relative to DMSO alone and a non-significant trend toward enhanced electrophysiological activity in multielectrode array recordings, these effects were secondary to the robust performance of the baseline DMSO protocol. Collectively, these findings establish standard 10% DMSO cryopreservation as an effective and broadly applicable method for hMO preservation, while suggesting that small-molecule IRIs may provide incremental benefits in selected outcome measures.

A central hallmark of Parkinson′s disease (PD) is the spread of α-Synuclein (α-Syn) aggregates, which is thought to contribute to its progressive nature. To better understand the mechanism of cellular internalization of α-Syn fibrils, we conducted a genome wide CRISPR activation (CRISPRa) screen to identify genetic modifiers of fibril uptake. We report here BRCA associated protein 1 (BAP1) as a regulator of fibril uptake into cells and validate its association with the genetic risk of PD. BAP1 regulates fibril entry into cells by acting as a master transcriptional regulator of the cell surface glycoproteome. BAP1 downregulates Heparan Sulfate Proteoglycan (HSPG) and upregulates O-linked glycoprotein expression, the net effect of which is to reduce fibril internalization. The effect of enhancing BAP1 expression is cell-type specific, as it reduces αSyn uptake in iPSC-derived dopaminergic neurons, but not in microglia. Using human midbrain organoids with integrated microglia, we find this effect is mediated through ASXL3 another PD risk gene and a non-obligatory component of the BAP1 Polycomb Repressive-Deubiquitinase Complex (PR-DUB). These findings are consistent with higher BAP1/ASXL3 co-expression in regions of the brain less vulnerable to PD pathology. Together, the work uncovers a novel role for BAP1/ASXL3 in regulating αSyn fibril internalization by remodelling the cell surface glycoproteome.

Microglia are the resident immune cells of the central nervous system and play key roles in the healthy brain during development and adulthood, as well as during neurodegenerative diseases - including Parkinson’s disease (PD). Yet the role of microglia in PD pathogenesis has not been fully elucidated. Limitations of 2D cell culture and animal models in simulating human microglia in the brain parenchyma have contributed to this knowledge gap. Human midbrain organoids (hMOs) provide a promising model that can recapitulate elements of PD pathology but lack microglial cells. Here we adapt protocols for the differentiation of hMOs and human iPSC-derived microglia (iMG) to generate iMG-hMO assembloids. Within assembloids, integrated iMG (intMG) express canonical microglia markers and induce the release of cytokines and chemokines. Transcriptomic profiling by single cell RNA sequencing reveals that intMG adopt a more mature and inflammation-responsive state compared to 2D iMG. The integration of microglia results in increased signaling through inflammatory and trophic pathways that drive altered transcriptional signatures of dopaminergic neurons and astrocytes within assembloids. Overall, iMG-hMO assembloids have the potential to more faithfully model the role of microglia and neuroinflammation in PD pathogenesis.

Summary: Post COVID-19 condition (Long COVID, LC) is frequently accompanied by persistent neurological symptoms, but the mechanisms linking intestinal dysbiosis to neuroinflammation remain unclear. Here we identify gut microbiota-derived extracellular vesicles (GMEVs) as functional mediators linking LC-associated dysbiosis to systemic and neuroimmune inflammation. In a longitudinally characterized cohort, individuals with LC and neurological symptoms exhibit a persistent intestinal microbiome signature. Transplantation of LC-associated microbiota into germ-free mice induces intestinal barrier disruption and neuroinflammatory phenotypes. GMEVs from individuals with LC activate inflammasome-associated programs and impair epithelial barrier function, promote inflammatory responses in macrophages, and induce coordinated pro-inflammatory transcriptional programs in human induced pluripotent stem cell (iPSC)-derived microglia. Chronic oral administration of LC-derived GMEVs remodels the microbiota and induces intestinal and systemic inflammation with glial activation in vivo . Together, these findings support a vesicle-centered framework in which microbiota-derived extracellular vesicles translate dysbiosis into sustained immune and neuroimmune activation in a post-viral inflammatory state.

Summary: Cellular heterogeneity is a common issue in differentiation protocols of oligodendrocytes (OLs) from human induced pluripotent stem cells. Our previous work described a novel method to generate OLs and highlighted the presence of glial progenitors. Here, we unravel the glial heterogeneity and characterize the response of isolated subpopulations to differentiation. This study provides a novel tool for studying the dynamics of glial development in vitro and on a transcriptomic level.

Background and Objectives: Oligodendrocytes (OL) and their myelin-forming processes are targeted and lost during the disease course of Multiple Sclerosis (MS), targeted by infiltrating leukocytes and their effector cytokines. Myelin repair is considered to be dependent on recruitment and differentiation of oligodendrocyte precursor cells (OPCs). The basis of failure of re-myelination during the disease course of MS remains to be defined. The aim of this study is to determine the impact of pro-inflammatory molecules tumor necrosis factor ⍰ (TNF⍰) and interferon gamma (IFN _γ ) on the differentiation of human OPCs. Methods: We generated human OPCs from induced pluripotent stem cells with a reporter gene under the OL-specific transcription factor SOX10. We treated the cells in vitro with TNF⍰ or IFN _γ and evaluated effects in terms of cell viability, expression of OL-lineage markers, and co- expression of astrocyte markers. To relate our findings to the molecular properties of OPCs as found in the MS brain we re-analyzed publicly available single nuclear RNAseq datasets. Results: Our analysis indicated that both TNF⍰ and IFN _γ decreased the proportion of cells differentiating into the OL-lineage; consistent with previous reports. We now observe the TNF⍰ effect is linked to aberrant OPC differentiation. A subset of O4+, reporter-positive cells co- expressed the astrocytic marker Aquaporin-4 (AQP4). On the transcriptomic level, the cells acquire an astrocyte-like signature alongside a conserved reactive phenotype. Analysis of single- nuclear RNAseq datasets from human MS brain revealed a subset of OPCs expressing an astrocytic signature. Discussion: In the context of MS, these results imply that OPCs are present but inhibited from differentiating along the OL-lineage, with a subset acquiring a reactive and stem-cell like phenotype, reducing their capacity to contribute towards repair. These findings help define a potential basis for the impaired myelin repair in MS and provide a prospective route for regenerative treatment.

Fibrinogen is a blood-derived protein involved in coagulation, and can make its way into the central nervous system (CNS) following breakdown of the blood-brain barrier. This molecule has been implicated in multiple sclerosis (MS), a disease marked by inflammation and demyelination in the CNS, as well as other neurological disorders. However, the effect of this molecule has not been studied on human myelinating cells. This study examines how fibrinogen influences human oligodendrocyte (OL) lineage cells at various stages of development. Using induced pluripotent stem cell-derived (iPSC) OL precursors and human primary OLs, we examined the effects of fibrinogen on cell differentiation, viability and myelination-related function. Here we show that fibrinogen induces an aberrant differentiation of early lineage OLs, by inhibiting their maturation and inducing an astrocytic phenotype, as seen in previous studies. On mature OLs, fibrinogen was found to promote myelination capacity as shown by ensheathment assays as well as on the RNA level. These effects were associated with the activation of BMP signalling, both in early and mature OLs. Transcriptomic analysis of human MS brain tissue shows similar pro-myelination changes in a subset of OLs, suggesting in vivo relevance. These findings indicate that fibrinogen has a lineage-dependent effect, where it may be inhibitory earlier in the lineage while promoting OL function in later stages. Understanding this dual role will provide insight into remyelination failure in MS and highlights the importance of timing and target in future therapeutic strategies. Significance Statement: In multiple sclerosis (MS), the blood protein fibrinogen leaks into the brain and has been shown to interfere with myelin repair. This study demonstrates that fibrinogen has opposite effects on human oligodendrocyte-lineage cells depending on their stage of maturation. While it blocks the differentiation of early-stage cells, it enhances the functional capacity of mature oligodendrocytes. These findings help explain why remyelination may fail in MS and suggest that fibrinogen could both hinder and support repair, depending on the cell context. This dual role has important implications for developing stage-specific therapies for MS.

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor‐based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10‐P2A‐mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells. Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, toward enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but also holds promise to accelerate research and translational work with iPSC‐derived OLs.

The fatal motor neuron (MN) disease Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive MN degeneration. Phrenic MNs (phMNs) controlling the activity of the diaphragm are prone to degeneration in ALS, leading to death by respiratory failure. Understanding of the mechanisms of phMN degeneration in ALS is limited, mainly because human experimental models to study phMNs are lacking. Here we describe a method enabling the derivation of phrenic-like MNs from human iPSCs (hiPSC-phMNs) within 30 days. This protocol uses an optimized combination of small molecules followed by cell-sorting based on a cell-surface protein enriched in hiPSC-phMNs, and is highly reproducible using several hiPSC lines. We show further that hiPSC-phMNs harbouring ALS-associated amplification of the C9orf72 gene progressively lose their electrophysiological activity and undergo increased death compared to isogenic controls. These studies establish a previously unavailable protocol to generate human phMNs offering a disease-relevant system to study mechanisms of respiratory MN dysfunction.