Search Results (35)

Organoids allow to model healthy and diseased human tissues. and have applications in developmental biology, drug discovery, and cell therapy. Traditionally cultured in immersion/suspension, organoids face issues like lack of standardization, fusion, hypoxia-induced necrosis, continuous agitation, and high media volume requirements. To address these issues, we developed an air–liquid interface (ALi) technology for culturing organoids, termed AirLiwell. It uses non-adhesive microwells for generating and maintaining individualized organoids on an air–liquid interface. This method ensures high standardization, prevents organoid fusion, eliminates the need for agitation, simplifies media changes, reduces media volume, and is compatible with Good Manufacturing Practices. We compared the ALi method to standard immersion culture for midbrain organoids, detailing the process from human pluripotent stem cell (hPSC) culture to organoid maturation and analysis. Air–liquid interface organoids (3D-ALi) showed optimized size and shape standardization. RNA sequencing and immunostaining confirmed neural/dopaminergic specification. Single-cell RNA sequencing revealed that immersion organoids (3D-i) contained 16% fibroblast-like, 23% myeloid-like, and 61% neural cells (49% neurons), whereas 3D-ALi organoids comprised 99% neural cells (86% neurons). Functionally, 3D-ALi organoids showed a striking electrophysiological synchronization, unlike the heterogeneous activity of 3D-i organoids. This standardized organoid platform improves reproducibility and scalability, demonstrated here with midbrain organoids. The use of midbrain organoids is particularly relevant for neuroscience and neurodegenerative diseases, such as Parkinson’s disease, due to their high incidence, opening new perspectives in disease modeling and cell therapy. In addition to hPSC-derived organoids, the method’s versatility extends to cancer organoids and 3D cultures from primary human cells. Full article

The 2016 Zika virus (ZIKV) epidemic has largely subsided, but a key question remains. How did ZIKV evolve to become a virulent human pathogen compared to the virus of its original discovery? What specific virologic and pathologic changes contributed to increased pathogenicity in humans? Phylogenetic studies have identified two genetically distinct ZIKV, the African and Asian lineages, which differ in their pathogenicity. Previous studies including ours suggest that the envelope (E) protein plays a key role in viral entry, immune activation, and neuropathogenesis. This study aimed to further elucidate virologic and pathogenic differences between these lineages by assessing their ability to bind and replicate in host cells, induce apoptotic cell death, trigger inflammatory responses, and influence human neural progenitor cell (hNPC)-derived neurosphere formation. We compared a historic African ZIKV strain (MR766) with an epidemic Brazilian strain (BR15) and evaluated the effects of the E protein inhibitor quercetin-3-β-O-D-glucoside (Q3G) and an E protein-neutralizing antibody (AbII). Our results revealed distinct virologic properties and that MR766 exhibited stronger inhibition of neurosphere formation due to enhanced viral binding to neuronal SH-SY5Y cells, while BR15 infection triggered a heightened pro-inflammatory cytokine response with reduced viral binding. Chimeric virus studies suggested that the E protein likely influences viral binding, replication efficiency, immune activation, and neuropathogenesis. Notably, Q3G exhibited antiviral activities against both MR766 and BR15, whereas AbII preferentially inhibited MR766. These findings highlight the virological differences between ancestral and epidemic viral strains, as well as the critical role of E protein in viral permissiveness, immune response, and neuropathogenesis, providing insights for developing targeted antiviral strategies. Full article

Bisphenol AF (BPAF) is widely utilized as an analog of bisphenol A (BPA) in the plastics industry. However, there is limited evidence on its neurodevelopmental toxicity. Existing studies suggest that BPAF has greater accumulation in vivo than other bisphenol analogs, and could pass through the placental barrier and the blood–brain barrier. In this study, we used the human neural progenitor cells line ReNcell CX, which was derived from 14-week human cortical brain tissue, as an in vitro model to investigate the neurodevelopmental toxicity effects of BPAF and BPA on ReNcell CX cells, and explored the possible mechanism by which BPAF induced neurodevelopmental toxicity on ReNcell CX cells. The results showed that BPAF reduced the proliferation of neural progenitor cells and changed the differentiation towards neurons after exposure for 24 h. Compared with BPA, ReNcell CX cells are more susceptible to BPAF exposure. In a 3D neurospheres model, BPAF affected the distance that neurons migrated outwards at the concentration of 2 μM. Furthermore, BPAF increased ROS levels in cells and reduced the expression of key proteins in the Nrf2/HO-1 pathway and its downstream molecules, such as SOD, GSH, and CAT. In conclusion, BPAF induces damage to critical nodes in neural progenitor cell development through the Nrf2/HO-1 pathway. Therefore, clarifying its neurodevelopmental toxicity and elaborating on the neurodevelopmental toxicity effects and mechanisms of bisphenol AF will help identify intervention targets for neurodevelopmental toxicity, and will have important public health significance for the safety assessment and risk prediction of bisphenol-related chemicals. Full article

Improved in vitro models are needed to reduce costs and delays in central nervous system (CNS) drug discovery. The FDA Modernization Acts 2.0 and 3.0 require human-centered alternative testing methods to mitigate animal-based experiments and discovery delays, and to ensure human safety. Developing cost-efficient, flexible microfluidic chips is essential to advance organ-on-chip (OoC) technology for drug discovery and disease modeling. While CNC micromilling shows promise for fabricating microfluidic devices, it remains underutilized due to limited accessibility. We present a simple CNC-milled flexible microfluidic chip fabricated from thermoplastic poly (methyl methacrylate) (PMMA). The structure of the microplate included drilled openings for connecting the wells. The chip’s biocompatibility was evaluated with isolated primary neuronal cultures from postnatal Wistar rat pups (p1). Primary cells cultured in the device showed high viability, differentiation, and 3D neurosphere formation, similar to conventional well-plate cultures. Neuronal cultures showed neurite growth and functional markers. Although cleanroom-based methods provide higher accuracy, the chip effectively promotes cell viability, differentiation, and alignment, offering an ideal platform for tissue modeling and OoC applications. It allows cell biologists to quickly create prototypes at lower cost and in less time than required for soft lithography and is a viable alternative to the current manufacturing methods. Full article

The degeneration of spiral ganglion neurons (SGNs), which convey auditory signals from hair cells to the brain, can be a primary cause of sensorineural hearing loss (SNHL) or can occur secondary to hair cell loss. Emerging therapies for SNHL include the replacement of damaged SGNs using stem cell-derived otic neuronal progenitors (ONPs). However, the availability of renewable, accessible, and patient-matched sources of human stem cells is a prerequisite for successful replacement of the auditory nerve. In this study, we derived ONP and SGN-like cells by a reliable and reproducible stepwise guidance differentiation procedure of self-renewing human dental pulp stem cells (hDPSCs). This in vitro differentiation protocol relies on the modulation of BMP and TGFβ pathways using a free-floating 3D neurosphere method, followed by differentiation on a Geltrex-coated surface using two culture paradigms to modulate the major factors and pathways involved in early otic neurogenesis. Gene and protein expression analyses revealed efficient induction of a comprehensive panel of known ONP and SGN-like cell markers during the time course of hDPSCs differentiation. Atomic force microscopy revealed that hDPSC-derived SGN-like cells exhibit similar nanomechanical properties as their in vivo SGN counterparts. Furthermore, spiral ganglion neurons from newborn rats come in close contact with hDPSC-derived ONPs 5 days after co-culturing. Our data demonstrate the capability of hDPSCs to generate SGN-like neurons with specific lineage marker expression, bipolar morphology, and the nanomechanical characteristics of SGNs, suggesting that the neurons could be used for next-generation cochlear implants and/or inner ear cell-based strategies for SNHL. Full article

This review addresses the need for innovative co-culture systems integrating the enteric nervous system (ENS) with intestinal organoids. The breakthroughs achieved through these techniques will pave the way for a transformative era in gastrointestinal (GI) disease modeling and treatment strategies. This review serves as an introduction to the companion protocol paper featured in this journal. The protocol outlines the isolation and co-culture of myenteric and submucosal neurons with small intestinal organoids. This review provides an overview of the intestinal organoid culture field to establish a solid foundation for effective protocol application. Remarkably, the ENS surpasses the number of neurons in the spinal cord. Referred to as the “second brain”, the ENS orchestrates pivotal roles in GI functions, including motility, blood flow, and secretion. The ENS is organized into myenteric and submucosal plexuses. These plexuses house diverse subtypes of neurons. Due to its proximity to the gut musculature and its cell type complexity, there are methodological intricacies in studying the ENS. Diverse approaches such as primary cell cultures, three-dimensional (3D) neurospheres, and induced ENS cells offer diverse insights into the multifaceted functionality of the ENS. The ENS exhibits dynamic interactions with the intestinal epithelium, the muscle layer, and the immune system, influencing epithelial physiology, motility, immune responses, and the microbiome. Neurotransmitters, including acetylcholine (ACh), serotonin (5-HT), and vasoactive intestinal peptide (VIP), play pivotal roles in these intricate interactions. Understanding these dynamics is imperative, as the ENS is implicated in various diseases, ranging from neuropathies to GI disorders and neurodegenerative diseases. The emergence of organoid technology presents an unprecedented opportunity to study ENS interactions within the complex milieu of the small and large intestines. This manuscript underscores the urgent need for standardized protocols and advanced techniques to unravel the complexities of the ENS and its dynamic relationship with the gut ecosystem. The insights gleaned from such endeavors hold the potential to revolutionize GI disease modeling and treatment paradigms. Full article

Hyaluronic acid (HA), a major glycosaminoglycan of the brain extracellular matrix, modulates cell behaviors through binding its receptor, Cd44. In this study, we assessed the influence of HA on high-grade brain tumors in vitro. The model comprised cell cultures derived from six rodent carcinogen-induced brain tumors, forming 3D spheroids prone to spontaneous fusion. Supplementation of the standard culture medium with 0.25% HA significantly inhibited the fusion rates, preserving the shape and size uniformity of spheroids. The 3D cultures were assigned to two groups; a Cd44lo group had a tenfold decreased relative expression of Cd44 than another (Cd44hi) group. In addition, these two groups differed by expression levels of Sox2 transcription factor; the correlation analysis revealed a tight negative association for Cd44 and Sox2. Transcriptomic responses of spheroids to HA exposure also depended on Cd44 expression levels, from subtle in Cd44lo to more pronounced and specific in Cd44hi, involving cell cycle progression, PI3K/AKT/mTOR pathway activation, and multidrug resistance genes. The potential HA-induced increase in brain tumor 3D models’ resistance to anticancer drug therapy should be taken into account when designing preclinical studies using HA scaffold-based models. The property of HA to prevent the fusion of brain-derived spheroids can be employed in CNS regenerative medicine and experimental oncology to ensure the production of uniform, controllably fusing neurospheres when creating more accurate in vitro brain models. Full article

Neuroblastoma (NB) is an embryonal tumor arising from the sympathetic central nervous system. The epidermal growth factor (EGF) plays a role in NB growth and metastatic behavior. Recently, we have demonstrated that cathepsin D (CD) contrasts EGF-induced NB cell growth in 2D by downregulating EGFR/MAPK signaling. Aggressive NB is highly metastatic to the bone and the brain. In the metastatic process, adherent cells detach to form clusters of suspended cells that adhere once they reach the metastatic site and form secondary colonies. Whether CD is involved in the survival of metastatic NB clones is not known. Therefore, in this study, we addressed how CD differentially affects cell growth in suspension versus the adherent condition. To mimic tumor heterogeneity, we co-cultured transgenic clones silenced for or overexpressing CD. We compared the growth kinetics of such mixed clones in 2D and 3D models in response to EGF, and we found that the Over CD clone had an advantage for growth in suspension, while the CD knocked-down clone was favored for the adherent growth in 2D. Interestingly, on switching from 3D to 2D culture conditions, the expression of E-cadherin and of N-cadherin increased in the KD-CD and Over CD clones, respectively. The fact that CD plays a dual role in cancer cell growth in 2D and 3D conditions indicates that during clonal evolution, subclones expressing different level of CD may arise, which confers survival and growth advantages depending on the metastatic step. By searching the TCGA database, we found up to 38 miRNAs capable of downregulating CD. Interestingly, these miRNAs are associated with biological processes controlling cell adhesion and cell migration. The present findings support the view that during NB growth on a substrate or when spreading as floating neurospheres, CD expression is epigenetically modulated to confer survival advantage. Thus, epigenetic targeting of CD could represent an additional strategy to prevent NB metastases. Full article

Within the last decade, a wide variety of protocols have emerged for the generation of retinal organoids. A subset of studies have compared protocols based on stem cell source, the physical features of the microenvironment, and both internal and external signals, all features that influence embryoid body and retinal organoid formation. Most of these comparisons have focused on the effect of signaling pathways on retinal organoid development. In this study, our aim is to understand whether starting cell conditions, specifically those involved in embryoid body formation, affect the development of retinal organoids in terms of differentiation capacity and reproducibility. To investigate this, we used the popular 3D floating culture method to generate retinal organoids from stem cells. This method starts with either small clumps of stem cells generated from larger clones (clumps protocol, CP) or with an aggregation of single cells (single cells protocol, SCP). Using histological analysis and gene-expression comparison, we found a retention of the pluripotency capacity on embryoid bodies generated through the SCP compared to the CP. Nonetheless, these early developmental differences seem not to impact the final retinal organoid formation, suggesting a potential compensatory mechanism during the neurosphere stage. This study not only facilitates an in-depth exploration of embryoid body development but also provides valuable insights for the selection of the most suitable protocol in order to study retinal development and to model inherited retinal disorders in vitro. Full article

A major goal of regenerative medicine of the central nervous system is to accelerate the regeneration of nerve tissue, where astrocytes, despite their positive and negative roles, play a critical role. Thus, scaffolds capable of producing astrocytes from neural precursor cells (NPCs) are most desirable. Our study shows that NPCs are converted into reactive astrocytes upon cultivation on coralline-derived calcium carbonate coated with poly-D-lysine (PDL-CS). As shown via nuclei staining, the adhesion of neurospheres containing hundreds of hippocampal neural cells to PDL-CS resulted in disaggregation of the cell cluster as well as the radial migration of dozens of cells away from the neurosphere core. Migrating cells per neurosphere averaged 100 on PDL-CS, significantly higher than on uncoated CS (28), PDL-coated glass (65), or uncoated glass (20). After 3 days of culture on PDL-CS, cell migration plateaued and remained stable for four more days. In addition, NPCs expressing nestin underwent continuous morphological changes from round to spiky, extending and elongating their processes, resembling activated astrocytes. The extension of the process increased continuously during the maturation of the culture and doubled after 7 days compared to day 1, whereas bifurcation increased by twofold during the first 3 days before plateauing. In addition, nestin positive cells’ shape, measured through the opposite circularity level correlation, decreased approximately twofold after three days, indicating spiky transformation. Moreover, nestin-positive cells co-expressing GFAP increased by 2.2 from day 1 to 7, reaching 40% of the NPC population on day 7. In this way, PDL-CS promotes NPC differentiation into reactive astrocytes, which could accelerate the repair of neural tissue. Full article

Radiation therapy (RT) is a crucial treatment modality for central nervous system (CNS) tumors but toxicity to healthy CNS tissues remains a challenge. Additionally, environmental exposure to radiation during nuclear catastrophes or space travel presents a risk of CNS toxicity. However, the underlying mechanisms of radiation-induced CNS toxicity are not fully understood. Neural progenitor cells (NPCs) are highly radiosensitive, resulting in decreased neurogenesis in the hippocampus. This study aimed to characterize a novel platform utilizing rat NPCs cultured as 3D neurospheres (NSps) to screen the safety and efficacy of experimental drugs with and without radiation exposure. The effect of radiation on NSp growth and differentiation was assessed by measuring sphere volume and the expression of neuronal differentiation markers Nestin and GFAP and proliferation marker Ki67. Radiation exposure inhibited NSp growth, decreased proliferation, and increased GFAP expression, indicating astrocytic differentiation. RNA sequencing analysis supported these findings, showing upregulation of Notch, BMP2/4, S100b, and GFAP gene expression during astrogenesis. By recapitulating radiation-induced toxicity and astrocytic differentiation, this single-NSp culture system provides a high-throughput preclinical model for assessing the effects of various radiation modalities and evaluates the safety and efficacy of potential therapeutic interventions in combination with radiation. Full article

Neural injuries disrupt the normal functions of the nervous system, whose complexities limit current treatment options. Because of their enhanced therapeutic effects, neurospheres have the potential to advance the field of regenerative medicine and neural tissue engineering. Methodological steps can pose challenges for implementing neurosphere assemblies; for example, conventional static cultures hinder yield and throughput, while the presence of the necrotic core, time-consuming methodology, and high variability can slow their progression to clinical application. Here we demonstrate the optimization of primary neural cell-derived neurospheres, developed using a high-throughput, stress-free, 3D bioreactor. This process provides a necessary baseline for future studies that could develop co-cultured assemblies of stem cells combined with endothelial cells, and/or biomaterials and nanomaterials for clinical therapeutic use. Neurosphere size and neurite spreading were evaluated under various conditions using Image J software. Primary neural cells obtained from the hippocampi of three-day-old rat pups, when incubated for 24 h in a reactor coated with 2% Pluronic and seeded on Poly-D-Lysine-coated plates establish neurospheres suitable for therapeutic use within five days. Most notably, neurospheres maintained high cell viability of ≥84% and expressed the neural marker MAP2, neural marker β-Tubulin III, and glial marker GFAP at all time points when evaluated over seven days. Establishing these factors reduces the variability in developing neurospheres, while increasing the ease and output of the culture process and maintaining viable cellular constructs. Full article

Currently, there is no cure for human immunodeficiency virus type 1 (HIV-1) infection. However, combined antiretroviral therapy (cART) aids in viral latency and prevents the progression of HIV-1 infection into acquired immunodeficiency syndrome (AIDS). cART has extended many lives, but people living with HIV-1 (PLWH) face lifelong ailments such as HIV-associated neurocognitive disorders (HAND) that range from asymptomatic HAND to HIV-1-associated dementia. HAND has been attributed to chronic inflammation and low-level infection within the central nervous system (CNS) caused by proinflammatory cytokines and viral products. These molecules are shuttled into the CNS within extracellular vesicles (EVs), lipid bound nanoparticles, and are released from cells as a form of intercellular communication. This study investigates the impact of cannabidiol (CBD), as a promising and potential therapeutic for HAND patients, and a similar synthetic molecule, HU308, on the EVs released from HIV-1-infected myeloid cells as well as HIV-1-infected 3D neurospheres. The data shows that both CBD and HU308 decrease non-coding and coding viral RNA (TAR and env) as well as proinflammatory cytokines as IL-1β and TNF-α mRNA. This decrease in viral RNA occurs in in vitro differentiated primary macrophages, in EVs released from HIV-1-infected cells monocytes, and infected neurospheres. Furthermore, a 3D neurosphere model shows an overall decrease in proinflammatory mRNA with HU308. Finally, using a humanized mouse model of HIV-1 infection, plasma viral RNA was shown to significantly decrease with HU308 alone and was most effective in combination with cART, even when compared to the typical cART treatment. Overall, CBD or HU308 may be a viable option to decrease EV release and associated cytokines which would dampen the virus spread and may be used in effective treatment of HAND in combination with cART. Full article

Glioblastoma multiforme (GBM) is one of the most aggressive types of cancer characterized by poor patient outcomes. To date, it is believed that the major cause of its recurrence and chemoresistance is represented by the enrichment of GBM stem cells (GSCs) sustained by the abnormal activation of a number of signaling pathways. In this study, we found that in GBM cells, treatment with low toxicity doses of the γ-secretase inhibitor RO4929097 (GSI), blocking the Notch pathway activity, in combination with resveratrol (RSV) was able to reverse the basal mesenchymal phenotype to an epithelial-like phenotype, affecting invasion and stemness interplay. The mechanism was dependent on cyclin D1 and cyclin-dependent kinase (CDK4), leading to a reduction of paxillin (Pxn) phosphorylation. Consequently, we discovered the reduced interaction of Pxn with vinculin (Vcl), which, during cell migration, transmits the intracellular forces to the extracellular matrix. The exogenous expression of a constitutively active Cdk4 mutant prevented the RSV + GSI inhibitory effects in GBM cell motility/invasion and augmented the expression of stemness-specific markers, as well as the neurosphere sizes/forming abilities in untreated cells. In conclusion, we propose that Cdk4 is an important regulator of GBM stem-like phenotypes and invasive capacity, highlighting how the combined treatment of Notch inhibitors and RSV could be prospectively implemented in the novel therapeutic strategies to target Cdk4 for these aggressive brain tumors. Full article

The in vitro derivation of Schwann cells from human bone marrow stromal cells (hBMSCs) opens avenues for autologous transplantation to achieve remyelination therapy for post-traumatic neural regeneration. Towards this end, we exploited human induced pluripotent stem-cell-derived sensory neurons to direct Schwann-cell-like cells derived from among the hBMSC-neurosphere cells into lineage-committed Schwann cells (hBMSC-dSCs). These cells were seeded into synthetic conduits for bridging critical gaps in a rat model of sciatic nerve injury. With improvement in gait by 12-week post-bridging, evoked signals were also detectable across the bridged nerve. Confocal microscopy revealed axially aligned axons in association with MBP-positive myelin layers across the bridge in contrast to null in non-seeded controls. Myelinating hBMSC-dSCs within the conduit were positive for both MBP and human nucleus marker HuN. We then implanted hBMSC-dSCs into the contused thoracic cord of rats. By 12-week post-implantation, significant improvement in hindlimb motor function was detectable if chondroitinase ABC was co-delivered to the injured site; such cord segments showed axons myelinated by hBMSC-dSCs. Results support translation into a protocol by which lineage-committed hBMSC-dSCs become available for motor function recovery after traumatic injury to both peripheral and central nervous systems. Full article