A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.

A screen for regulators of survival of motor neuron protein levels.

The motor neuron disease spinal muscular atrophy (SMA) results from mutations that lead to low levels of the ubiquitously expressed protein survival of motor neuron (SMN). An ever-increasing collection of data suggests that therapeutics that elevate SMN may be effective in treating SMA. We executed an image-based screen of annotated chemical libraries and discovered several classes of compounds that were able to increase cellular SMN. Among the most important was the RTK-PI3K-AKT-GSK-3 signaling cascade. Chemical inhibitors of glycogen synthase kinase 3 (GSK-3) and short hairpin RNAs (shRNAs) directed against this target elevated SMN levels primarily by stabilizing the protein. It was particularly notable that GSK-3 chemical inhibitors were also effective in motor neurons, not only in elevating SMN levels, but also in blocking the death that was produced when SMN was acutely reduced by an SMN-specific shRNA. Thus, we have established a screen capable of detecting drug-like compounds that correct the main phenotypic change underlying SMA.

A Robust Pipeline for the Multi-Stage Accelerated Differentiation of Functional 3D Cortical Organoids from Human Pluripotent Stem Cells.

Disordered cellular development, abnormal neuroanatomical formations, and dysfunction of neuronal circuitry are among the pathological manifestations of cortical regions in the brain that are often implicated in complex neurodevelopmental disorders. With the advancement of stem cell methodologies such as cerebral organoid generation, it is possible to study these processes in vitro using 3D cellular platforms that mirror key developmental stages occurring throughout embryonic neurogenesis. Patterning-based stem cell models of directed neuronal development offer one approach to accomplish this, but these protocols often require protracted periods of cell culture to generate diverse cell types and current methods are plagued by a lack of specificity, reproducibility, and temporal control over cell derivation. Although ectopic expression of transcription factors offers another avenue to rapidly generate neurons, this process of direct lineage conversion bypasses critical junctures of neurodevelopment during which disease-relevant manifestations may occur. Here, we present a directed differentiation approach for generating human pluripotent stem cell (hPSC)-derived cortical organoids with accelerated lineage specification to generate functionally mature cortical neurons in a shorter timeline than previously established protocols. This novel protocol provides precise guidance for the specification of neuronal cell type identity as well as temporal control over the pace at which cortical lineage trajectories are established. Furthermore, we present assays that can be used as tools to interrogate stage-specific developmental signaling mechanisms. By recapitulating major components of embryonic neurogenesis, this protocol allows for improved in vitro modeling of cortical development while providing a platform that can be utilized to uncover disease-specific mechanisms of disordered development at various stages across the differentiation timeline. © 2023 Wiley Periodicals LLC. Basic Protocol 1: 3D hPSC neural induction Support Protocol 1: Neural rosette formation assay Support Protocol 2: Neurosphere generation Support Protocol 3: Enzymatic dissociation, NSC expansion, and cryopreservation Basic Protocol 2: 3D neural progenitor expansion Basic Protocol 3: 3D accelerated cortical lineage patterning and terminal differentiation.

16p13.11 deletion variants associated with neuropsychiatric disorders cause morphological and synaptic changes in induced pluripotent stem cell-derived neurons.

16p13.11 copy number variants (CNVs) have been associated with autism, schizophrenia, psychosis, intellectual disability, and epilepsy. The majority of 16p13.11 deletions or duplications occur within three well-defined intervals, and despite growing knowledge of the functions of individual genes within these intervals, the molecular mechanisms that underlie commonly observed clinical phenotypes remain largely unknown. Patient-derived, induced pluripotent stem cells (iPSCs) provide a platform for investigating the morphological, electrophysiological, and gene-expression changes that result from 16p13.11 CNVs in human-derived neurons. Patient derived iPSCs with varying sizes of 16p13.11 deletions and familial controls were differentiated into cortical neurons for phenotypic analysis. High-content imaging and morphological analysis of patient-derived neurons demonstrated an increase in neurite branching in patients compared with controls. Whole-transcriptome sequencing revealed expression level changes in neuron development and synaptic-related gene families, suggesting a defect in synapse formation. Subsequent quantification of synapse number demonstrated increased numbers of synapses on neurons derived from early-onset patients compared to controls. The identification of common phenotypes among neurons derived from patients with overlapping 16p13.11 deletions will further assist in ascertaining common pathways and targets that could be utilized for screening drug candidates. These studies can help to improve future treatment options and clinical outcomes for 16p13.11 deletion patients.

Systemic nature of spinal muscular atrophy revealed by studying insurance claims.

OBJECTIVE: We investigated the presence of non-neuromuscular phenotypes in patients affected by Spinal Muscular Atrophy (SMA), a disorder caused by a mutation in the Survival of Motor Neuron (SMN) gene, and whether these phenotypes may be clinically detectable prior to clinical signs of neuromuscular degeneration and therefore independent of muscle weakness. METHODS: We utilized a de-identified database of insurance claims to explore the health of 1,038 SMA patients compared to controls. Two analyses were performed: (1) claims from the entire insurance coverage window; and (2) for SMA patients, claims prior to diagnosis of any neuromuscular disease or evidence of major neuromuscular degeneration to increase the chance that phenotypes could be attributed directly to reduced SMN levels. Logistic regression was used to determine whether phenotypes were diagnosed at significantly different rates between SMA patients and controls and to obtain covariate-adjusted odds ratios. RESULTS: Results from the entire coverage window revealed a broad spectrum of phenotypes that are differentially diagnosed in SMA subjects compared to controls. Moreover, data from SMA patients prior to their first clinical signs of neuromuscular degeneration revealed numerous non-neuromuscular phenotypes including defects within the cardiovascular, gastrointestinal, metabolic, reproductive, and skeletal systems. Furthermore, our data provide evidence of a potential ordering of disease progression beginning with these non-neuromuscular phenotypes. CONCLUSIONS: Our data point to a direct relationship between early, detectable non-neuromuscular symptoms and SMN deficiency. Our findings are particularly important for evaluating the efficacy of SMN-increasing therapies for SMA, comparing the effectiveness of local versus systemically delivered therapeutics, and determining the optimal therapeutic treatment window prior to irreversible neuromuscular damage.

Applying Deep Neural Network Analysis to High-Content Image-Based Assays.

The etiological underpinnings of many CNS disorders are not well understood. This is likely due to the fact that individual diseases aggregate numerous pathological subtypes, each associated with a complex landscape of genetic risk factors. To overcome these challenges, researchers are integrating novel data types from numerous patients, including imaging studies capturing broadly applicable features from patient-derived materials. These datasets, when combined with machine learning, potentially hold the power to elucidate the subtle patterns that stratify patients by shared pathology. In this study, we interrogated whether high-content imaging of primary skin fibroblasts, using the Cell Painting method, could reveal disease-relevant information among patients. First, we showed that technical features such as batch/plate type, plate, and location within a plate lead to detectable nuisance signals, as revealed by a pre-trained deep neural network and analysis with deep image embeddings. Using a plate design and image acquisition strategy that accounts for these variables, we performed a pilot study with 12 healthy controls and 12 subjects affected by the severe genetic neurological disorder spinal muscular atrophy (SMA), and evaluated whether a convolutional neural network (CNN) generated using a subset of the cells could distinguish disease states on cells from the remaining unseen control-SMA pair. Our results indicate that these two populations could effectively be differentiated from one another and that model selectivity is insensitive to batch/plate type. One caveat is that the samples were also largely separated by source. These findings lay a foundation for how to conduct future studies exploring diseases with more complex genetic contributions and unknown subtypes.

Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease.

The mechanism underlying selective motor neuron (MN) death remains an essential question in the MN disease field. The MN disease spinal muscular atrophy (SMA) is attributable to reduced levels of the ubiquitous protein SMN. Here, we report that SMN levels are widely variable in MNs within a single genetic background and that this heterogeneity is seen not only in SMA MNs but also in MNs derived from controls and amyotrophic lateral sclerosis (ALS) patients. Furthermore, cells with low SMN are more susceptible to cell death. These findings raise the important clinical implication that some SMN-elevating therapeutics might be effective in MN diseases besides SMA. Supporting this, we found that increasing SMN across all MN populations using an Nedd8-activating enzyme inhibitor promotes survival in both SMA and ALS-derived MNs. Altogether, our work demonstrates that examination of human neurons at the single-cell level can reveal alternative strategies to be explored in the treatment of degenerative diseases.

Identification of a Maleimide-Based Glycogen Synthase Kinase-3 (GSK-3) Inhibitor, BIP-135, that Prolongs the Median Survival Time of Δ7 SMA KO Mouse Model of Spinal Muscular Atrophy.

The discovery of upregulated glycogen synthase kinase-3 (GSK-3) in various pathological conditions has led to the development of a host of chemically diverse small molecule GSK-3 inhibitors, such as BIP-135. GSK-3 inhibition emerged as an alternative therapeutic target for treating spinal muscular atrophy (SMA) when a number of GSK-3 inhibitors were shown to elevate survival motor neuron (SMN) levels in vitro and to rescue motor neurons when their intrinsic SMN level was diminished by SMN-specific short hairpin RNA (shRNA). Despite their cellular potency, the in vivo efficacy of GSK-3 inhibitors has yet to be evaluated in an animal model of SMA. Herein, we disclose that a potent and reasonably selective GSK-3 inhibitor, namely BIP-135, was tested in a transgenic Δ7 SMA KO mouse model of SMA, and found to prolong the median survival of these animals. In addition, this compound was shown to elevate the SMN protein level in SMA patient-derived fibroblast cells as determined by western blot, and was neuroprotective in a cell-based, SMA-related model of oxidative stress-induced neurodegeneration.

Neurokinin-1 enables measles virus trans-synaptic spread in neurons.

Measles virus (MV), a morbillivirus that remains a significant human pathogen, can infect the central nervous system, resulting in rare but often fatal diseases, such as subacute sclerosing panencephalitis. Previous work demonstrated that MV was transmitted trans-synaptically and that, while a cellular receptor for the hemagglutinin (H) protein was required for MV entry, it was dispensable for subsequent cell-to-cell spread. Here, we explored what role the other envelope protein, fusion (F), played in trans-synaptic transport. We made the following observations: (1) MV-F expression in infected neurons was similar to that seen in infected fibroblasts; (2) fusion inhibitory peptide (FIP), an inhibitor of MV fusion, prevented both infection and spread in primary neurons; (3) Substance P, a neurotransmitter with the same active site as FIP, also blocked neuronal MV spread; and (4) both genetic deletion and pharmacological inhibition of the Substance P receptor, neurokinin-1 (NK-1), reduced infection of susceptible mice. Together, these data implicate a role for NK-1 in MV CNS infection and spread, perhaps serving as an MV-F receptor or co-receptor on neurons.