Generation of three isogenic gene-edited Huntington's disease human embryonic stem cell lines with DOX-inducible NGN2 expression cassette in the AAVS1 safe locus.

Neurogenin 2 (NGN2), a neuronal transcription factor, can expedite differentiation of stem cells into mature glutamatergic neurons. We have utilized an allelic series of previously published and characterized isogenic Huntington's disease (IsoHD) human embryonic stem cell lines (Ooi et al., 2019), carrying different CAG repeat lengths in the first exon of the huntingtin gene. These IsoHDs were modified using CRISPR/Cas9 to insert NGN2 under the TET-ON doxycycline inducible promoter. The resulting IsoHD-NGN2 cell lines retained pluripotency in the absence of doxycycline (DOX), and via addition of DOX to the culturing media differentiation to neurons was achieved within 14 days.

Generation of two patient specific GABRD variants and their isogenic controls for modeling epilepsy.

Developmental and epileptic encephalopathies (DEEs) are early-onset conditions that cause intractable seizures and developmental delays. Missense variants in Gamma-aminobutyric acid type A receptor (GABAAR) subunits commonly cause DEEs. Ahring et al. (2022) showed a variant in the gene that encodes the delta subunit (GABRD) is strongly associated with the gain-of-function of extrasynaptic GABAAR. Here, we report the generation of two patient-specific human induced pluripotent stem cells (hiPSC) lines with (i) a de novo variant and (ii) a maternal variant, both for the pathogenic GABRD c.872 C>T, (p.T291I). The variants in the generated cell line were corrected using the CRISPR-Cas9 gene editing technique (respective isogenic control lines).

Generation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Astrocytes for Amyotrophic Lateral Sclerosis and Other Neurodegenerative Disease Studies.

Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest. Key features • This protocol requires preexisting experience in hiPSC culturing for a successful outcome. • The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points. • The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.

Rapid Point-of-Care Identification of Aspergillus Species in Microbial Keratitis.

Importance: Microbial keratitis (MK) is a common cause of unilateral visual impairment, blindness, and eye loss in low-income and middle-income countries. There is an urgent need to develop and implement rapid and simple point-of-care diagnostics for MK to increase the likelihood of good outcomes. Objective: To evaluate the diagnostic performance of the Aspergillus-specific lateral-flow device (AspLFD) to identify Aspergillus species causing MK in corneal scrape and corneal swab samples of patients presenting with microbial keratitis. Design, Setting, and Participants: This diagnostic study was conducted between May 2022 and January 2023 at the corneal clinic of Aravind Eye Hospital in Madurai, Tamil Nadu, India. All study participants were recruited during their first presentation to the clinic. Patients aged 15 years or older met the eligibility criteria if they were attending their first appointment, had a corneal ulcer that was suggestive of a bacterial or fungal infection, and were about to undergo diagnostic scrape and culture. Main Outcomes and Measures: Sensitivity and specificity of the AspLFD with corneal samples collected from patients with MK. During routine diagnostic scraping, a minimally invasive corneal swab and an additional corneal scrape were collected and transferred to aliquots of sample buffer and analyzed by lateral-flow device (LFD) if the patient met the inclusion criteria. Photographs of devices were taken with a smartphone and analyzed using a ratiometric approach, which was developed for this study. The AspLFD results were compared with culture reports. Results: The 198 participants who met the inclusion criteria had a mean (range) age of 51 (15-85) years and included 126 males (63.6%). Overall, 35 of 198 participants with corneal scrape (17.7%) and 17 of 40 participants with swab samples (42.5%) had positive culture results for Aspergillus species. Ratiometric analysis results for the scrape samples found that the AspLFD achieved high sensitivity (0.89; 95% CI, 0.74-0.95), high negative predictive value (0.97; 95% CI, 0.94-0.99), low negative likelihood ratio (0.12; 95% CI, 0.05-0.30), and an accuracy of 0.94 (95% CI, 0.90-0.97). Ratiometric analysis results for the swab samples showed that the AspLFD had high sensitivity (0.94; 95% CI, 0.73-1.00), high negative predictive value (0.95; 95% CI, 0.76-1.00), low negative likelihood ratio (0.07; 95% CI, 0.01-0.48), and an accuracy of 0.88 (95% CI, 0.73-0.96). Conclusions and Relevance: Results of this diagnostic study suggest that AspLFD along with the ratiometric analysis of LFDs developed for this study has high diagnostic accuracy in identifying Aspergillus species from corneal scrapes and swabs. This technology is an important step toward the provision of point-of-care diagnostics for MK and could inform the clinical management strategy.

Alteration of microglial metabolism and inflammatory profile contributes to neurotoxicity in a hiPSC-derived microglia model of frontotemporal dementia 3.

Frontotemporal dementia (FTD) is a common cause of early-onset dementia, with no current treatment options. FTD linked to chromosome 3 (FTD3) is a rare sub-form of the disease, caused by a point mutation in the Charged Multivesicular Body Protein 2B (CHMP2B). This mutation causes neuronal phenotypes, such as mitochondrial deficiencies, accompanied by metabolic changes and interrupted endosomal-lysosomal fusion. However, the contribution of glial cells to FTD3 pathogenesis has, until recently, been largely unexplored. Glial cells play an important role in most neurodegenerative disorders as drivers and facilitators of neuroinflammation. Microglia are at the center of current investigations as potential pro-inflammatory drivers. While gliosis has been observed in FTD3 patient brains, it has not yet been systematically analyzed. In the light of this, we investigated the role of microglia in FTD3 by implementing human induced pluripotent stem cells (hiPSC) with either a heterozygous or homozygous CHMP2B mutation, introduced into a healthy control hiPSC line via CRISPR-Cas9 precision gene editing. These hiPSC were differentiated into microglia to evaluate the pro-inflammatory profile and metabolic state. Moreover, hiPSC-derived neurons were cultured with conditioned microglia media to investigate disease specific interactions between the two cell populations. Interestingly, we identified two divergent inflammatory microglial phenotypes resulting from the underlying mutations: a severe pro-inflammatory profile in CHMP2B homozygous FTD3 microglia, and an "unresponsive" CHMP2B heterozygous FTD3 microglial state. These findings correlate with our observations of increased phagocytic activity in CHMP2B homozygous, and impaired protein degradation in CHMP2B heterozygous FTD3 microglia. Metabolic mapping confirmed these differences, revealing a metabolic reprogramming of the CHMP2B FTD3 microglia, displayed as a compensatory up-regulation of glutamine metabolism in the CHMP2B homozygous FTD3 microglia. Intriguingly, conditioned CHMP2B homozygous FTD3 microglia media caused neurotoxic effects, which was not evident for the heterozygous microglia. Strikingly, IFN-γ treatment initiated an immune boost of the CHMP2B heterozygous FTD3 microglia, and conditioned microglia media exposure promoted neural outgrowth. Our findings indicate that the microglial profile, activity, and behavior is highly dependent on the status of the CHMP2B mutation. Our results suggest that the heterozygous state of the mutation in FTD3 patients could potentially be exploited in form of immune-boosting intervention strategies to counteract neurodegeneration.

Generation of eight hiPSCs lines from two pathogenic variants in CACNA1A using the CRISPR-Cas9 gene editing technology.

Developmental and epileptic encephalopathies (DEEs) are rare severe neurodevelopmental disorders with a cumulative incidence of 1:6.000 live births. Many epileptic conditions arise from single nucleotide variants in CACNA1A (calcium voltage-gated channel subunit alpha1 A), encoding the CaV2.1 calcium channel subunit. Human induced pluripotent stem cells (hiPSCs) are an optimal choice for modeling DEEs, as they can be differentiated in vitro into diverse neuronal subpopulations. Here, we report the generation of hiPSC lines with two pathogenic CACNA1A variants c.1767C > T, p. (Arg589Cys), referred to as R589C and c. 2139G > A, p.(Ala713Thr), referred to as A713T, previously associated with epilepsy. The variants were introduced into a hiPSC line from a healthy individual via CRISPR-Cas9 gene editing technology.

Golgi fragmentation - One of the earliest organelle phenotypes in Alzheimer's disease neurons.

Alzheimer's disease (AD) is the most common cause of dementia, with no current cure. Consequently, alternative approaches focusing on early pathological events in specific neuronal populations, besides targeting the well-studied amyloid beta (Aß) accumulations and Tau tangles, are needed. In this study, we have investigated disease phenotypes specific to glutamatergic forebrain neurons and mapped the timeline of their occurrence, by implementing familial and sporadic human induced pluripotent stem cell models as well as the 5xFAD mouse model. We recapitulated characteristic late AD phenotypes, such as increased Aß secretion and Tau hyperphosphorylation, as well as previously well documented mitochondrial and synaptic deficits. Intriguingly, we identified Golgi fragmentation as one of the earliest AD phenotypes, indicating potential impairments in protein processing and post-translational modifications. Computational analysis of RNA sequencing data revealed differentially expressed genes involved in glycosylation and glycan patterns, whilst total glycan profiling revealed minor glycosylation differences. This indicates general robustness of glycosylation besides the observed fragmented morphology. Importantly, we identified that genetic variants in Sortilin-related receptor 1 (SORL1) associated with AD could aggravate the Golgi fragmentation and subsequent glycosylation changes. In summary, we identified Golgi fragmentation as one of the earliest disease phenotypes in AD neurons in various in vivo and in vitro complementary disease models, which can be exacerbated via additional risk variants in SORL1.

Neural Derivates of Canine Induced Pluripotent Stem Cells-Like Cells From a Mild Cognitive Impairment Dog.

Domestic dogs are superior models for translational medicine due to greater anatomical and physiological similarities with humans than rodents, including hereditary diseases with human equivalents. Particularly with respect to neurodegenerative medicine, dogs can serve as a natural, more relevant model of human disease compared to transgenic rodents. Herein we report attempts to develop a canine-derived in vitro model for neurodegenerative diseases through the generation of induced pluripotent stem cells from a 14-year, 9-month-old female West Highland white terrier with mild cognitive impairment (MCI). Canine induced pluripotent stem cells-like cells (ciPSCLC) were generated using human OSKM and characterized by positive expression of pluripotency markers. Due to inefficient viral vector silencing we refer to them as ciPSCLCs. Subsequently, the ciPSCLC were subjected to neural induction according to two protocols both yielding canine neural progenitor cells (cNPCs), which expressed typical NPC markers. The cNPCs were cultured in neuron differentiation media for 3 weeks, resulting in the derivation of morphologically impaired neurons as compared to iPSC-derived human counterparts generated in parallel. The apparent differences encountered in this study regarding the neural differentiation potential of ciPSCLC reveals challenges and new perspectives to consider before using the canine model in translational neurological studies.

Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3.

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration.

A protein-centric view of in vitro biological model systems for schizophrenia.

Schizophrenia (SCZ) is a severe brain disorder, characterized by psychotic, negative, and cognitive symptoms, affecting 1% of the population worldwide. The precise etiology of SCZ is still unknown; however, SCZ has a high heritability and is associated with genetic, environmental, and social risk factors. Even though the genetic contribution is indisputable, the discrepancies between transcriptomics and proteomics in brain tissues are consistently challenging the field to decipher the disease pathology. Here we provide an overview of the state of the art of neuronal two-dimensional and three-dimensional model systems that can be combined with proteomics analyses to decipher specific brain pathology and detection of alternative entry points for drug development.

Glutamate-glutamine homeostasis is perturbed in neurons and astrocytes derived from patient iPSC models of frontotemporal dementia.

Frontotemporal dementia (FTD) is amongst the most prevalent early onset dementias and even though it is clinically, pathologically and genetically heterogeneous, a crucial involvement of metabolic perturbations in FTD pathology is being recognized. However, changes in metabolism at the cellular level, implicated in FTD and in neurodegeneration in general, are still poorly understood. Here we generate induced human pluripotent stem cells (hiPSCs) from patients carrying mutations in CHMP2B (FTD3) and isogenic controls generated via CRISPR/Cas9 gene editing with subsequent neuronal and glial differentiation and characterization. FTD3 neurons show a dysregulation of glutamate-glutamine related metabolic pathways mapped by 13C-labelling coupled to mass spectrometry. FTD3 astrocytes show increased uptake of glutamate whilst glutamate metabolism is largely maintained. Using quantitative proteomics and live-cell metabolic analyses, we elucidate molecular determinants and functional alterations of neuronal and glial energy metabolism in FTD3. Importantly, correction of the mutations rescues such pathological phenotypes. Notably, these findings implicate dysregulation of key enzymes crucial for glutamate-glutamine homeostasis in FTD3 pathogenesis which may underlie vulnerability to neurodegeneration. Neurons derived from human induced pluripotent stem cells (hiPSCs) of patients carrying mutations in CHMP2B (FTD3) display major metabolic alterations compared to CRISPR/Cas9 generated isogenic controls. Using quantitative proteomics, 13C-labelling coupled to mass spectrometry metabolic mapping and seahorse analyses, molecular determinants and functional alterations of neuronal and astrocytic energy metabolism in FTD3 were characterized. Our findings implicate dysregulation of glutamate-glutamine homeostasis in FTD3 pathogenesis. In addition, FTD3 neurons recapitulate glucose hypometabolism observed in FTD patient brains. The impaired mitochondria function found here is concordant with disturbed TCA cycle activity and decreased glycolysis in FTD3 neurons. FTD3 neuronal glutamine hypermetabolism is associated with up-regulation of PAG expression and, possibly, ROS production. Distinct compartments of glutamate metabolism can be suggested for the FTD3 neurons. Endogenous glutamate generated from glutamine via PAG may enter the TCA cycle via AAT (left side of neuron) while exogenous glutamate taken up from the extracellular space may be incorporated into the TCA cycle via GDH (right side of the neuron) FTD3 astrocytic glutamate uptake is upregulated whilst glutamate metabolism is largely maintained. Finally, pharmacological reversal of glutamate hypometabolism manifesting from decreased GDH expression should be explored as a novel therapeutic intervention for treating FTD3.

Neurons derived from sporadic Alzheimer's disease iPSCs reveal elevated TAU hyperphosphorylation, increased amyloid levels, and GSK3B activation.

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia, affecting one in eight adults over 65 years of age. The majority of AD cases are sporadic, with unknown etiology, and only 5% of all patients with AD present the familial monogenic form of the disease. In the present study, our aim was to establish an in vitro cell model based on patient-specific human neurons to study the pathomechanism of sporadic AD. METHODS: We compared neurons derived from induced pluripotent stem cell (iPSC) lines of patients with early-onset familial Alzheimer's disease (fAD), all caused by mutations in the PSEN1 gene; patients with late-onset sporadic Alzheimer's disease (sAD); and three control individuals without dementia. The iPSC lines were differentiated toward mature cortical neurons, and AD pathological hallmarks were analyzed by RT-qPCR, enzyme-linked immunosorbent assay, and Western blotting methods. RESULTS: Neurons from patients with fAD and patients with sAD showed increased phosphorylation of TAU protein at all investigated phosphorylation sites. Relative to the control neurons, neurons derived from patients with fAD and patients with sAD exhibited higher levels of extracellular amyloid-ß 1-40 (Aß1-40) and amyloid-ß 1-42 (Aß1-42). However, significantly increased Aß1-42/Aß1-40 ratios, which is one of the pathological markers of fAD, were observed only in samples of patients with fAD. Additionally, we detected increased levels of active glycogen synthase kinase 3 ß, a physiological kinase of TAU, in neurons derived from AD iPSCs, as well as significant upregulation of amyloid precursor protein (APP) synthesis and APP carboxy-terminal fragment cleavage. Moreover, elevated sensitivity to oxidative stress, as induced by amyloid oligomers or peroxide, was detected in both fAD- and sAD-derived neurons. CONCLUSIONS: On the basis of the experiments we performed, we can conclude there is no evident difference except secreted Aß1-40 levels in phenotype between fAD and sAD samples. To our knowledge, this is the first study in which the hyperphosphorylation of TAU protein has been compared in fAD and sAD iPSC-derived neurons. Our findings demonstrate that iPSC technology is suitable to model both fAD and sAD and may provide a platform for developing new treatment strategies for these conditions.

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6+/NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background.

Modeling neurodegenerative diseases with patient-derived induced pluripotent cells: Possibilities and challenges.

The rising prevalence of progressive neurodegenerative diseases coupled with increasing longevity poses an economic burden at individual and societal levels. There is currently no effective cure for the majority of neurodegenerative diseases and disease-affected tissues from patients have been difficult to obtain for research and drug discovery in pre-clinical settings. While the use of animal models has contributed invaluable mechanistic insights and potential therapeutic targets, the translational value of animal models could be further enhanced when combined with in vitro models derived from patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls generated using CRISPR-Cas9 mediated genome editing. The iPSCs are self-renewable and capable of being differentiated into the cell types affected by the diseases. These in vitro models based on patient-derived iPSCs provide the opportunity to model disease development, uncover novel mechanisms and test potential therapeutics. Here we review findings from iPSC-based modeling of selected neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia and spinocerebellar ataxia. Furthermore, we discuss the possibilities of generating three-dimensional (3D) models using the iPSCs-derived cells and compare their advantages and disadvantages to conventional two-dimensional (2D) models.

Astrocyte Differentiation of Human Pluripotent Stem Cells: New Tools for Neurological Disorder Research.

Astrocytes have a central role in brain development and function, and so have gained increasing attention over the past two decades. Consequently, our knowledge about their origin, differentiation and function has increased significantly, with new research showing that astrocytes cultured alone or co-cultured with neurons have the potential to improve our understanding of various central nervous system diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, or Alexander disease. The generation of astrocytes derived from pluripotent stem cells (PSCs) opens up a new area for studying neurologic diseases in vitro; these models could be exploited to identify and validate potential drugs by detecting adverse effects in the early stages of drug development. However, as it is now known that a range of astrocyte populations exist in the brain, it will be important in vitro to develop standardized protocols for the in vitro generation of astrocyte subsets with defined maturity status and phenotypic properties. This will then open new possibilities for co-cultures with neurons and the generation of neural organoids for research purposes. The aim of this review article is to compare and summarize the currently available protocols and their strategies to generate human astrocytes from PSCs. Furthermore, we discuss the potential role of human-induced PSCs derived astrocytes in disease modeling.

Establishment of induced pluripotent stem cell (iPSC) line from a 63-year old patient with late onset Alzheimer's disease (LOAD).

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterised 63-year old woman with late onset Alzheimer's disease (LOAD). The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The transgene-free iPSC showed pluripotency verified by immunocytochemistry for pluripotency markers and differentiated spontaneously towards the 3 germ layers in vitro. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to further study the pathomechanism of sporadic AD, to identify early biomarkers and also for drug testing and gene therapy studies.

Neurosphere Based Differentiation of Human iPSC Improves Astrocyte Differentiation.

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.