Generating Neuroimmune Assembloids Using Human Induced Pluripotent Stem Cell (iPSC)-Derived Cortical Organoids and Microglia.

The emergence of brain organoids has revolutionized our understanding of neurodevelopment and neurological diseases by providing an in vitro model system that recapitulates key aspects of human brain development. However, conventional organoid protocols often overlook the role of microglia, the resident immune cells of the central nervous system. Microglia dysfunction is implicated in various neurological disorders, highlighting the need for their inclusion in organoid models. Here, we present a novel method for generating neuroimmune assembloids using human-induced pluripotent stem cell (iPSC)-derived cortical organoids and microglia. Building upon our previous work generating myelinating cortical organoids, we extend our methodology to include the integration of microglia, ensuring their long-term survival and maturation within the organoids. We describe two integration methods: one involving direct addition of microglia progenitors to the organoids and an alternative approach where microglia and dissociated neuronal progenitors are aggregated together in a defined ratio. To facilitate downstream analysis, we also describe a dissociation protocol for single-cell RNA sequencing (scRNA-seq) and provide guidance on fixation, cryosectioning, and immunostaining of assembloid structures. Overall, our protocol provides a comprehensive framework for generating neuroimmune assembloids, offering researchers a valuable tool for studying the interactions between neural cell types and immune cells in the context of neurological diseases.

Establishing Neural Organoid Cultures for Investigating the Effects of Microgravity in Low-Earth Orbit (LEO).

Recent findings from studies involving astronauts and animal models indicate that microgravity increases immune cell activity and potentially alters the white and gray matter of the central nervous system (CNS). To further investigate the impact of microgravity on CNS cells, we established cultures of three-dimensional neural organoids containing isogenic microglia, the brain's resident immune cells, and sent them onboard the International Space Station. When using induced pluripotent stem cell (iPSC) lines from individuals affected by neuroinflammatory and neurodegenerative diseases such as multiple sclerosis (MS) and Parkinson's disease (PD), these cultures can provide novel insights into pathogenic pathways that may be exacerbated by microgravity. We have devised a cryovial culture strategy that enables organoids to be maintained through space travel and onboard the International Space Station (ISS) without the need for medium or carbon dioxide exchange. Here, we provide a comprehensive description of all the steps involved: generating various types of neural organoids, establishing long-term cultures, arranging plans for shipment to the Kennedy Space Center (KSC), and ultimately preparing organoids for launch into low-Earth orbit (LEO) and return to Earth for post-flight analyses.

Patient iPSC models reveal glia-intrinsic phenotypes in multiple sclerosis.

Multiple sclerosis (MS) is considered an inflammatory and neurodegenerative disease of the central nervous system, typically resulting in significant neurological disability that worsens over time. While considerable progress has been made in defining the immune system's role in MS pathophysiology, the contribution of intrinsic CNS-cell dysfunction remains unclear. Here, we generated the largest reported collection of iPSC lines from people with MS spanning diverse clinical subtypes and differentiated them into glia-enriched cultures. Using single-cell transcriptomic profiling, we observed several distinguishing characteristics of MS cultures pointing to glia-intrinsic disease mechanisms. We found that iPSC-derived cultures from people with primary progressive MS contained fewer oligodendrocytes. Moreover, iPSC-oligodendrocyte lineage cells and astrocytes from people with MS showed increased expression of immune and inflammatory genes that match those of glial cells from MS postmortem brains. Thus, iPSC-derived MS models provide a unique platform for dissecting glial contributions to disease phenotypes independent of the peripheral immune system and identify potential glia-specific targets for therapeutic intervention.

Molecular Mechanism behind the Safe Immunostimulatory Effect of Withania somnifera.

Withania somnifera (L.) Dunal (family Solanaceae) is a medicinal plant known for, among many pharmacological properties, an immune boosting effect. Our recent study revealed that its key immunostimulatory factor is lipopolysaccharide of plant-associated bacteria. This is peculiar, because, although LPS can elicit protective immunity, it is an extremely potent pro-inflammatory toxin (endotoxin). However, W. somnifera is not associated with such toxicity. In fact, despite the presence of LPS, it does not trigger massive inflammatory responses in macrophages. To gain insights into the safe immunostimulatory effect of W. somnifera, we conducted a mechanistic study on its major phytochemical constituent, withaferin A, which is known for anti-inflammatory activity. Endotoxin-triggered immunological responses in the presence and absence of withaferin A were characterized by both in vitro macrophage-based assay and in vivo cytokine profiling in mice. Collectively, our results demonstrate that withaferin A selectively attenuates the pro-inflammatory signaling triggered by endotoxin without impairing other immunological pathways. This finding provides a new conceptual framework to understand the safe immune-boosting effect of W. somnifera and possibly other medicinal plants. Furthermore, the finding opens a new opportunity to facilitate the development of safe immunotherapeutic agents, such as vaccine adjuvants.

Isolation of Human CD49f+ Astrocytes and In Vitro iPSC-Based Neurotoxicity Assays.

Given the critical roles of astrocytes in neuroinflammation and neurological diseases, models for studying human astrocyte biology are in increasing demand. Here, we present a protocol to isolate human astrocytes from induced pluripotent stem cell (iPSC)-based cultures, neural organoids, and primary tissue, using the surface marker CD49f. Moreover, we provide protocols for in vitro co-cultures of human iPSC-derived neurons and astrocytes, as well as for neurotoxicity assays that expose neurons to conditioned media from reactive astrocytes. For complete details on the use and execution of this protocol, please refer to Barbar et al. (2020).

CD49f Is a Novel Marker of Functional and Reactive Human iPSC-Derived Astrocytes.

New methods for investigating human astrocytes are urgently needed, given their critical role in the central nervous system. Here we show that CD49f is a novel marker for human astrocytes, expressed in fetal and adult brains from healthy and diseased individuals. CD49f can be used to purify fetal astrocytes and human induced pluripotent stem cell (hiPSC)-derived astrocytes. We provide single-cell and bulk transcriptome analyses of CD49f+ hiPSC-astrocytes and demonstrate that they perform key astrocytic functions in vitro, including trophic support of neurons, glutamate uptake, and phagocytosis. Notably, CD49f+ hiPSC-astrocytes respond to inflammatory stimuli, acquiring an A1-like reactive state, in which they display impaired phagocytosis and glutamate uptake and fail to support neuronal maturation. Most importantly, we show that conditioned medium from human reactive A1-like astrocytes is toxic to human and rodent neurons. CD49f+ hiPSC-astrocytes are thus a valuable resource for investigating human astrocyte function and dysfunction in health and disease.

Cell-line-based assay for the toxicity/benefit analysis of lipopolysaccharides in plants.

There are many immune-boosting medicinal plants that can potently activate innate immune cells. Recent studies indicate that the active factors of some immune-boosting plants are lipopolysaccharides (LPSs) of plant-associated bacteria. However, little is currently known about the potential risk and benefit of LPSs in medicinal plants. To facilitate their characterization, we established a simple cell-line-based assay that can be used to screen the toxicity and benefit of LPSs in medicinal plants. The assay can distinguish endotoxic diphosphoryl lipid A (DPL) from beneficial monophosphoryl lipid A (MPL), which is a clinically used immunological adjuvant for vaccines. The established assay was used to characterize commercial supplements of Ashwagandha, which was shown to contain immunostimulatory LPSs. The study revealed that Ashwagandha activates macrophages in a manner similar to MPL. The current finding underscores the importance of further studies to characterize the LPSs in immune-boosting medicinal plants.

Abundance of Plant-Associated Gammaproteobacteria Correlates with Immunostimulatory Activity of Angelica sinensis.

Background: Angelica sinensis is a medicinal plant known for a variety of biological effects, including its ability to stimulate innate immune cells in humans. Recent studies indicate that the immunostimulatory activity of A. sinensis arises from microbe-associated molecular patterns (MAMPs) of plant-associated bacteria. However, it is unknown which bacterial taxa in A. sinensis are responsible for the production of immunostimulatory MAMPs. Methods: Samples of A. sinensis were subjected to a cell-based assay to detect monocyte-stimulation and 16S ribosomal RNA amplicon sequencing, which revealed their immunostimulatory activity and microbial communities. The resulting data were analyzed by Linear discriminant analysis effect size (LEfSe), an online biostatistical tool for metagenomic biomarker discovery, to identify the bacterial taxonomical features correlated with the immunostimulatory activity. Results: A series of bacterial taxa under Gammaproteobacteria correlated positively with the immunostimulatory activity, whereas several Gram-positive taxa and Betaproteobacteria correlated negatively with the activity. Conclusions: The identified bacterial taxa set a new stage to characterize immunostimulatory MAMPs in plants.

Uncovering potential 'herbal probiotics' in Juzen-taiho-to through the study of associated bacterial populations.

Juzen-taiho-to (JTT) is an immune-boosting formulation of ten medicinal herbs. It is used clinically in East Asia to boost the human immune functions. The active factors in JTT have not been clarified. But, existing evidence suggests that lipopolysaccharide (LPS)-like factors contribute to the activity. To examine this possibility, JTT was subjected to a series of analyses, including high resolution mass spectrometry, which suggested the presence of structural variants of LPS. This finding opened a possibility that JTT contains immune-boosting bacteria. As the first step to characterize the bacteria in JTT, 16S ribosomal RNA sequencing was carried out for Angelica sinensis (dried root), one of the most potent immunostimulatory herbs in JTT. The sequencing revealed a total of 519 bacteria genera in A. sinensis. The most abundant genus was Rahnella, which is widely distributed in water and plants. The abundance of Rahnella appeared to correlate with the immunostimulatory activity of A. sinensis. In conclusion, the current study provided new pieces of evidence supporting the emerging theory of bacterial contribution in immune-boosting herbs.