Heterozygous knockout of Synaptotagmin13 phenocopies ALS features and TP53 activation in human motor neurons.

Spinal motor neurons (MNs) represent a highly vulnerable cellular population, which is affected in fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). In this study, we show that the heterozygous loss of SYT13 is sufficient to trigger a neurodegenerative phenotype resembling those observed in ALS and SMA. SYT13+/- hiPSC-derived MNs displayed a progressive manifestation of typical neurodegenerative hallmarks such as loss of synaptic contacts and accumulation of aberrant aggregates. Moreover, analysis of the SYT13+/- transcriptome revealed a significant impairment in biological mechanisms involved in motoneuron specification and spinal cord differentiation. This transcriptional portrait also strikingly correlated with ALS signatures, displaying a significant convergence toward the expression of pro-apoptotic and pro-inflammatory genes, which are controlled by the transcription factor TP53. Our data show for the first time that the heterozygous loss of a single member of the synaptotagmin family, SYT13, is sufficient to trigger a series of abnormal alterations leading to MN sufferance, thus revealing novel insights into the selective vulnerability of this cell population.

Mutation-induced LZTR1 polymerization provokes cardiac pathology in recessive Noonan syndrome.

Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. In this study, we investigate the mechanistic consequences of a homozygous variant LZTR1L580P by using patient-specific and CRISPR-Cas9-corrected induced pluripotent stem cell (iPSC) cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction identify an LZTR1L580P-specific disease mechanism provoking cardiac hypertrophy. The variant is predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymers. LZTR1 complex dysfunction results in the accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Furthermore, our data show that cardiomyocyte-specific MRAS degradation is mediated by LZTR1 via non-proteasomal pathways, whereas RIT1 degradation is mediated by both LZTR1-dependent and LZTR1-independent pathways. Uni- or biallelic genetic correction of the LZTR1L580P missense variant rescues the molecular and cellular disease phenotype, providing proof of concept for CRISPR-based therapies.

iPSC-Derived Astrocytes and Neurons Replicate Brain Gene Expression, Epigenetic, Cell Morphology and Connectivity Alterations Found in Autism.

Excessive inflammatory reactions and oxidative stress are well-recognized molecular findings in autism and these processes can affect or be affected by the epigenetic landscape. Nonetheless, adequate therapeutics are unavailable, as patient-specific brain molecular markers for individualized therapies remain challenging. METHODS: We used iPSC-derived neurons and astrocytes of patients with autism vs. controls (5/group) to examine whether they replicate the postmortem brain expression/epigenetic alterations of autism. Additionally, DNA methylation of 10 postmortem brain samples (5/group) was analyzed for genes affected in PSC-derived cells. RESULTS: We found hyperexpression of TGFB1, TGFB2, IL6 and IFI16 and decreased expression of HAP1, SIRT1, NURR1, RELN, GPX1, EN2, SLC1A2 and SLC1A3 in the astrocytes of patients with autism, along with DNA hypomethylation of TGFB2, IL6, TNFA and EN2 gene promoters and a decrease in HAP1 promoter 5-hydroxymethylation in the astrocytes of patients with autism. In neurons, HAP1 and IL6 expression trended alike. While HAP1 promoter was hypermethylated in neurons, IFI16 and SLC1A3 promoters were hypomethylated and TGFB2 exhibited increased promoter 5-hydroxymethlation. We also found a reduction in neuronal arborization, spine size, growth rate, and migration, but increased astrocyte size and a reduced growth rate in autism. In postmortem brain samples, we found DNA hypomethylation of TGFB2 and IFI16 promoter regions, but DNA hypermethylation of HAP1 and SLC1A2 promoters in autism. CONCLUSION: Autism-associated expression/epigenetic alterations in iPSC-derived cells replicated those reported in the literature, making them appropriate surrogates to study disease pathogenesis or patient-specific therapeutics.

Human organoid model of pontocerebellar hypoplasia 2a recapitulates brain region-specific size differences.

Pontocerebellar hypoplasia type 2a (PCH2a) is an ultra-rare, autosomal recessive pediatric disorder with limited treatment options. Its anatomical hallmark is hypoplasia of the cerebellum and pons accompanied by progressive microcephaly. A homozygous founder variant in TSEN54, which encodes a tRNA splicing endonuclease (TSEN) complex subunit, is causal. The pathological mechanism of PCH2a remains unknown due to the lack of a model system. Therefore, we developed human models of PCH2a using regionalized neural organoids. We generated induced pluripotent stem cell (iPSC) lines from three males with genetically confirmed PCH2a and subsequently differentiated cerebellar and neocortical organoids. Mirroring clinical neuroimaging findings, PCH2a cerebellar organoids were reduced in size compared to controls starting early in differentiation. Neocortical PCH2a organoids demonstrated milder growth deficits. Although PCH2a cerebellar organoids did not upregulate apoptosis, their stem cell zones showed altered proliferation kinetics, with increased proliferation at day 30 and reduced proliferation at day 50 compared to controls. In summary, we generated a human model of PCH2a, providing the foundation for deciphering brain region-specific disease mechanisms. Our first analyses suggest a neurodevelopmental aspect of PCH2a.

ß-Amyloid species production and tau phosphorylation in iPSC-neurons with reference to neuropathologically characterized matched donor brains.

A basic assumption underlying induced pluripotent stem cell (iPSC) models of neurodegeneration is that disease-relevant pathologies present in brain tissue are also represented in donor-matched cells differentiated from iPSCs. However, few studies have tested this hypothesis in matched iPSCs and neuropathologically characterized donated brain tissues. To address this, we assessed iPSC-neuron production of ß-amyloid (Aß) Aß40, Aß42, and Aß43 in 24 iPSC lines matched to donor brains with primary neuropathologic diagnoses of sporadic AD (sAD), familial AD (fAD), control, and other neurodegenerative disorders. Our results demonstrate a positive correlation between Aß43 production by fAD iPSC-neurons and Aß43 accumulation in matched brain tissues but do not reveal a substantial correlation in soluble Aß species between control or sAD iPSC-neurons and matched brains. However, we found that the ApoE4 genotype is associated with increased Aß production by AD iPSC-neurons. Pathologic tau phosphorylation was found to be increased in AD and fAD iPSC-neurons compared to controls and positively correlated with the relative abundance of longer-length Aß species produced by these cells. Taken together, our results demonstrate that sAD-predisposing genetic factors influence iPSC-neuron phenotypes and that these cells are capturing disease-relevant and patient-specific components of the amyloid cascade.

Inhibition of TBL1 cleavage alleviates doxorubicin-induced cardiomyocytes death by regulating the Wnt/ß-catenin signal pathway.

AIMS: Doxorubicin (DOX) is a widely used anthracycline anticancer agent; however, its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately, the pathophysiology of DICT has not yet been fully elucidated, and there are no effective strategies for its prevention or treatment. In this investigation, the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored. METHODS AND RESULTS: We observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites-D125, D136, and D215. Interestingly, overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis, effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with ß-catenin. As a result, Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/ß-catenin signalling, leading to DICT progression. Furthermore, the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However, these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally, in a DICT mouse model, TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation, ultimately protecting cardiomyocytes from cell death. CONCLUSION: Our findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function, even in the context of DOX administration. Consequently, this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT.

Use of iPSC-Derived Smooth Muscle Cells to Model Physiology and Pathology.

The implementation of human induced pluripotent stem cell (hiPSC) models has introduced an additional tool for identifying molecular mechanisms of disease that complement animal models. Patient-derived or CRISPR/Cas9-edited induced pluripotent stem cells differentiated into smooth muscle cells (SMCs) have been leveraged to discover novel mechanisms, screen potential therapeutic strategies, and model in vivo development. The field has evolved over almost 15 years of research using hiPSC-SMCs and has made significant strides toward overcoming initial challenges such as the lineage specificity of SMC phenotypes. However, challenges both specific (eg, the lack of specific markers to thoroughly validate hiPSC-SMCs) and general (eg, a lack of transparency and consensus around methodology in the field) remain. In this review, we highlight the recent successes and remaining challenges of the hiPSC-SMC model.

Neuroectoderm phenotypes in a human stem cell model of O-GlcNAc transferase associated with intellectual disability.

Pathogenic variants in the O-GlcNAc transferase gene (OGT) have been associated with a congenital disorder of glycosylation (OGT-CDG), presenting with intellectual disability which may be of neuroectodermal origin. To test the hypothesis that pathology is linked to defects in differentiation during early embryogenesis, we developed an OGT-CDG induced pluripotent stem cell line together with isogenic control generated by CRISPR/Cas9 gene-editing. Although the OGT-CDG variant leads to a significant decrease in OGT and O-GlcNAcase protein levels, there were no changes in differentiation potential or stemness. However, differentiation into ectoderm resulted in significant differences in O-GlcNAc homeostasis. Further differentiation to neuronal stem cells revealed differences in morphology between patient and control lines, accompanied by disruption of the O-GlcNAc pathway. This suggests a critical role for O-GlcNAcylation in early neuroectoderm architecture, with robust compensatory mechanisms in the earliest stages of stem cell differentiation.

Pathological analysis of Prader-Willi syndrome using adipocytes.

Prader-Willi syndrome (PWS) is a complex epigenetic disorder caused by the deficiency of paternally expressed genes in chromosome 15q11-q13. This syndrome also includes endocrine dysfunction, leading to short stature, hypogonadism, and obscure hyperphagia. Although recent progress has been made toward understanding the genetic basis for PWS, the molecular mechanisms underlying its pathology in obesity remain unclear. In this study, we examined the adipocytic characteristics of two PWS-induced pluripotent stem cell (iPSC) lines: those with the 15q11-q13 gene deletion (iPWS cells) and those with 15q11-q13 abnormal methylation (M-iPWS cells). The transcript levels of the lipid-binding protein aP2 were decreased in iPWS and M-iPWS adipocytes. Flow-cytometry analysis showed that PWS adipocytes accumulated more lipid droplets than did normal individual adipocytes. Furthermore, glucose uptake upon insulin stimulation was attenuated compared to that in normal adipocytes. Overall, our results suggest a significantly increased lipid content and defective in glucose metabolism in PWS adipocytes.

Generation of a human iPSC line CIPi004-A from a patient with neurofibromatosis type 1 and epilepsy harboring a heterozygous mutation in NF1 gene.

The NF1 gene is related to neurofibromatosis type 1 (NF1), which is an autosomal dominant disorder associated with multisystem involvement and epilepsy susceptibility. A human induced pluripotent stem cell (iPSC) line was derived from a pediatric patient with NF1 and epilepsy, harboring a heterozygous NF1 gene mutation. The iPSC line exhibits high levels of pluripotency markers, maintains the NF1 gene mutation, and demonstrates the capacity to undergo differentiation potential in vitro into three germ layers. The iPSC line will serve as a valuable resource for investigating the underlying mechanisms and conducting drug screening related to NF1 and NF1-associated epilepsy.

Integrated machine learning and multimodal data fusion for patho-phenotypic feature recognition in iPSC models of dilated cardiomyopathy.

Integration of multiple data sources presents a challenge for accurate prediction of molecular patho-phenotypic features in automated analysis of data from human model systems. Here, we applied a machine learning-based data integration to distinguish patho-phenotypic features at the subcellular level for dilated cardiomyopathy (DCM). We employed a human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of a DCM mutation in the sarcomere protein troponin T (TnT), TnT-R141W, compared to isogenic healthy (WT) control iPSC-CMs. We established a multimodal data fusion (MDF)-based analysis to integrate source datasets for Ca2+ transients, force measurements, and contractility recordings. Data were acquired for three additional layer types, single cells, cell monolayers, and 3D spheroid iPSC-CM models. For data analysis, numerical conversion as well as fusion of data from Ca2+ transients, force measurements, and contractility recordings, a non-negative blind deconvolution (NNBD)-based method was applied. Using an XGBoost algorithm, we found a high prediction accuracy for fused single cell, monolayer, and 3D spheroid iPSC-CM models (≥92 ± 0.08 %), as well as for fused Ca2+ transient, beating force, and contractility models (>96 ± 0.04 %). Integrating MDF and XGBoost provides a highly effective analysis tool for prediction of patho-phenotypic features in complex human disease models such as DCM iPSC-CMs.

Genetically engineered macrophages derived from iPSCs for self-regulating delivery of anti-inflammatory biologic drugs.

In rheumatoid arthritis, dysregulated cytokine signaling has been implicated as a primary factor in chronic inflammation. Many antirheumatic and biological therapies are used to suppress joint inflammation, but despite these advances, effectiveness is not universal, and delivery is often at high doses, which can predispose patients to significant off-target effects. During chronic inflammation, the inappropriate regulation of signaling factors by macrophages accelerates progression of disease by driving an imbalance of inflammatory cytokines, making macrophages an ideal cellular target. To develop a macrophage-based therapy to treat chronic inflammation, we engineered a novel induced pluripotent stem cell (iPSC)-derived macrophage capable of delivering soluble TNF receptor 1 (TNFR1), an anti-inflammatory biologic inhibitor of tumor necrosis factor alpha (TNF-α), in an auto-regulated manner in response to TNF-α. Murine iPSCs were differentiated into macrophages (iMACs) over a 17-day optimized protocol with continued successful differentiation confirmed at key timepoints. Varying inflammatory and immunomodulatory stimuli demonstrated traditional macrophage function and phenotypes. In response to TNF-α, therapeutic iMACs produced high levels of sTNFR1 in an autoregulated manner, which inhibited inflammatory signaling. This self-regulating iMAC system demonstrated the potential for macrophage-based drug delivery as a novel therapeutic approach for a variety of chronic inflammatory diseases.

LINC complex alterations are a key feature of sporadic and familial ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that primarily affects motor neurons, leading to progressive muscle weakness and loss of voluntary muscle control. While the exact cause of ALS is not fully understood, emerging research suggests that dysfunction of the nuclear envelope (NE) may contribute to disease pathogenesis and progression. The NE plays a role in ALS through several mechanisms, including nuclear pore defects, nucleocytoplasmic transport impairment, accumulation of mislocalized proteins, and nuclear morphology abnormalities. The LINC complex is the second biggest multi-protein complex in the NE and consists of the SUN1/2 proteins spanning the inner nuclear membrane and Nesprin proteins embedded in the outer membrane. The LINC complex, by interacting with both the nuclear lamina and the cytoskeleton, transmits mechanical forces to the nucleus regulating its morphology and functional homeostasis. In this study we show extensive alterations to the LINC complex in motor and cortical iPSC-derived neurons and spinal cord organoids carrying the ALS causative mutation in the C9ORF72 gene (C9). Importantly, we show that such alterations are present in vivo in a cohort of sporadic ALS and C9-ALS postmortem spinal cord and motor cortex specimens. We also found that LINC complex disruption strongly correlated with nuclear morphological alterations occurring in ALS neurons, independently of TDP43 mislocalization. Altogether, our data establish morphological and functional alterations to the LINC complex as important events in ALS pathogenic cascade, making this pathway a possible target for both biomarker and therapy development.

Dynamic molecular network analysis of iPSC-Purkinje cells differentiation delineates roles of ISG15 in SCA1 at the earliest stage.

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1), a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However, the regulatory network of SCA1 pathology, especially central regulators of the earliest developmental stages and inflammatory events, remains incompletely understood. Here, we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development, and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients.

Establishment of a human induced pluripotent stem cell line（SDQLCHi059-A）from a patient with congenital disorder of glycosylation carrying heterozygous mutation in MPI gene.

Congenital disorder of glycosylation (CDG) is inherited metabolicdiseasecaused by defects in the genes important for the process of protein and lipidglycosylation. We established an induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells of a 6-month-old boy with congenital disorder of glycosylation carrying heterozygous mutations c.1193 T > C (p.I398T) and c.376_384dup CCGCAGCAC (p.P126_H128 dupPQH) in MPI gene. This iPSC line was free of exogenous gene, expressed pluripotency markers, has normal karyotype, exhibited differentiation potential and harbored the same mutations found in the patient. This cell line will provide a reliable cell model for further studies on the potential therapeutic targets of CDG.

Generation of one induced pluripotent stem cell line JUCGRMi004-A from a Charcot-Marie-Tooth disease type 1A (CMT1A) patient with PMP22 duplication.

The CMT1A variant accounts for over 60% of cases of Charcot-Marie-Tooth disease (CMT), one of the most common human neuropathies. The cause of CMT1A has been identified as the duplication of PMP22, a myelin protein expressed in Schwann cells. Yet, the pathological mechanisms have not been elucidated, and no treatment is currently available. In our study, we established an iPS cell line from a CMT1A patient with PMP22 duplication. The generated iPSCs maintain pluripotency and in vitro differentiation potency.

The Role of Human Pluripotent Stem Cells in Amyotrophic Lateral Sclerosis: From Biological Mechanism to Practical Implications.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, characterized by progressive loss of both upper and lower motor neurons, resulting in clinical features such as muscle weakness, paralysis, and ultimately, respiratory failure. Nowadays, there is not effective treatment to reverse the progression of the disease, that leads to death within 3-5 years after the onset. Nevertheless, the induced pluripotent stem cells (iPS) technology could be the answer, providing disease modelling, drug testing, and cell-based therapies for this pathology. The aim of this work was to conduct a literature review of the past 5 years about the role of iPS in ALS, to better define the neurobiological mechanisms involved in the pathogenesis and the potential future therapies. The review also deals with advanced and currently available technologies used to reprogram cell lines and generate human motor neurons in vitro, which represent the source to study the pathological processes, the relationship between phenotype and genotype, the disease progression and the potential therapeutic targets of these group of disorders. Specific treatment options with stem cells involve Advance Gene Editing Technology, neuroprotective agents, and cells or exosomes transplantation, aimed to replace dead or damaged nerve cells. In summary, this review comprehensively addresses the role of human pluripotent stem cells (hPSCs) in motor neuron diseases (MND), with a focus on physiopathology, diagnostic and prognostic implications, specific and potential future treatment options. Understanding the biological mechanisms and practical implications of hPSCs in MND is crucial for advancing therapeutic strategies and improving outcomes for patients affected by these devastating diseases.

Generation of a human induced pluripotent stem cell line NTUHi004-A from a patient with Leigh syndrome harboring a homozygous missense mutation c.836 T > G (p.Met279Arg) in NDUFAF5 gene.

Leigh syndrome is a rare autosomal recessive disorder showcasing a diverse range of neurological symptoms. Classical Leigh syndrome is associated with mitochondrial complex I deficiency, primarily resulting from biallelic mutations in the NDUFAF5 gene, encoding the NADH:ubiquinone oxidoreductase complex assembly factor 5. Using the Sendai virus delivery system, we generated an induced pluripotent stem cell line from peripheral blood mononuclear cells of a 47-years-old female patient who carried a homozygous NDUFAF5 c.836 T > G (p.Met279Arg) mutation. This cellular model serves as a tool for investigating the underlying pathogenic mechanisms and for the development of potential treatments for Leigh syndrome.

Metabolic Rewiring and Altered Glial Differentiation in an iPSC-Derived Astrocyte Model Derived from a Nonketotic Hyperglycinemia Patient.

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine-glycine-one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.

Stem cell modeling of nervous system tumors.

Nervous system tumors, particularly brain tumors, represent the most common tumors in children and one of the most lethal tumors in adults. Despite decades of research, there are few effective therapies for these cancers. Although human nervous system tumor cells and genetically engineered mouse models have served as excellent platforms for drug discovery and preclinical testing, they have limitations with respect to accurately recapitulating important aspects of the pathobiology of spontaneously arising human tumors. For this reason, attention has turned to the deployment of human stem cell engineering involving human embryonic or induced pluripotent stem cells, in which genetic alterations associated with nervous system cancers can be introduced. These stem cells can be used to create self-assembling three-dimensional cerebral organoids that preserve key features of the developing human brain. Moreover, stem cell-engineered lines are amenable to xenotransplantation into mice as a platform to investigate the tumor cell of origin, discover cancer evolutionary trajectories and identify therapeutic vulnerabilities. In this article, we review the current state of human stem cell models of nervous system tumors, discuss their advantages and disadvantages, and provide consensus recommendations for future research.