How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a functional assessment.

BACKGROUND: Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. METHODS: Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. RESULTS: The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. CONCLUSIONS: We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

Parkinsonism Sac domain mutation in Synaptojanin-1 affects ciliary properties in iPSC-derived dopaminergic neurons.

Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4, 5)P2 4- and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis. A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1RQKI mice). Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function. Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1RQKI DA neurons and their isogenic controls. In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length. Further analysis of cilia of SJ1RQDA neurons revealed abnormal accumulation of the Ca2+ channel Cav1.3 and of ubiquitin chains, suggesting a defect in the clearing of ubiquitinated proteins at the ciliary base, where a focal concentration of SJ1 was observed. We suggest that SJ1 may contribute to the control of ciliary protein dynamics in DA neurons, with implications on cilia-mediated signaling.

Insights for disease modeling from single-cell transcriptomics of iPSC-derived Ngn2-induced neurons and astrocytes across differentiation time and co-culture.

BACKGROUND: Trans-differentiation of human-induced pluripotent stem cells into neurons via Ngn2-induction (hiPSC-N) has become an efficient system to quickly generate neurons a likely significant advance for disease modeling and in vitro assay development. Recent single-cell interrogation of Ngn2-induced neurons, however, has revealed some similarities to unexpected neuronal lineages. Similarly, a straightforward method to generate hiPSC-derived astrocytes (hiPSC-A) for the study of neuropsychiatric disorders has also been described. RESULTS: Here, we examine the homogeneity and similarity of hiPSC-N and hiPSC-A to their in vivo counterparts, the impact of different lengths of time post Ngn2 induction on hiPSC-N (15 or 21 days), and the impact of hiPSC-N/hiPSC-A co-culture. Leveraging the wealth of existing public single-cell RNA-seq (scRNA-seq) data in Ngn2-induced neurons and in vivo data from the developing brain, we provide perspectives on the lineage origins and maturation of hiPSC-N and hiPSC-A. While induction protocols in different labs produce consistent cell type profiles, both hiPSC-N and hiPSC-A show significant heterogeneity and similarity to multiple in vivo cell fates, and both more precisely approximate their in vivo counterparts when co-cultured. Gene expression data from the hiPSC-N show enrichment of genes linked to schizophrenia (SZ) and autism spectrum disorders (ASD) as has been previously shown for neural stem cells and neurons. These overrepresentations of disease genes are strongest in our system at early times (day 15) in Ngn2-induction/maturation of neurons, when we also observe the greatest similarity to early in vivo excitatory neurons. We have assembled this new scRNA-seq data along with the public data explored here as an integrated biologist-friendly web-resource for researchers seeking to understand this system more deeply: https://nemoanalytics.org/p?l=DasEtAlNGN2&g=NES . CONCLUSIONS: While overall we support the use of the investigated cellular models for the study of neuropsychiatric disease, we also identify important limitations. We hope that this work will contribute to understanding and optimizing cellular modeling for complex brain disorders.

Profiling the role of m6A effectors in the regulation of pluripotent reprogramming.

The N6-methyladenosine (m6A) RNA modification plays essential roles in multiple biological processes, including stem cell fate determination. To explore the role of the m6A modification in pluripotent reprogramming, we used RNA-seq to map m6A effectors in human iPSCs, fibroblasts, and H9 ESCs, as well as in mouse ESCs and fibroblasts. By integrating the human and mouse RNA-seq data, we found that 19 m6A effectors were significantly upregulated in reprogramming. Notably, IGF2BPs, particularly IGF2BP1, were among the most upregulated genes in pluripotent cells, while YTHDF3 had high levels of expression in fibroblasts. Using quantitative PCR and Western blot, we validated the pluripotency-associated elevation of IGF2BPs. Knockdown of IGF2BP1 induced the downregulation of stemness genes and exit from pluripotency. Proteome analysis of cells collected at both the beginning and terminal states of the reprogramming process revealed that the IGF2BP1 protein was positively correlated with stemness markers SOX2 and OCT4. The eCLIP-seq target analysis showed that IGF2BP1 interacted with the coding sequence (CDS) and 3'UTR regions of the SOX2 transcripts, in agreement with the location of m6A modifications. This study identifies IGF2BP1 as a vital pluripotency-associated m6A effector, providing new insight into the interplay between m6A epigenetic modifications and pluripotent reprogramming.

Cockayne Syndrome Patient iPSC-Derived Brain Organoids and Neurospheres Show Early Transcriptional Dysregulation of Biological Processes Associated with Brain Development and Metabolism.

Cockayne syndrome (CS) is a rare hereditary autosomal recessive disorder primarily caused by mutations in Cockayne syndrome protein A (CSA) or B (CSB). While many of the functions of CSB have been at least partially elucidated, little is known about the actual developmental dysregulation in this devasting disorder. Of particular interest is the regulation of cerebral development as the most debilitating symptoms are of neurological nature. We generated neurospheres and cerebral organoids utilizing Cockayne syndrome B protein (CSB)-deficient induced pluripotent stem cells derived from two patients with distinct severity levels of CS and healthy controls. The transcriptome of both developmental timepoints was explored using RNA-Seq and bioinformatic analysis to identify dysregulated biological processes common to both patients with CS in comparison to the control. CSB-deficient neurospheres displayed upregulation of the VEGFA-VEGFR2 signalling pathway, vesicle-mediated transport and head development. CSB-deficient cerebral organoids exhibited downregulation of brain development, neuron projection development and synaptic signalling. We further identified the upregulation of steroid biosynthesis as common to both timepoints, in particular the upregulation of the cholesterol biosynthesis branch. Our results provide insights into the neurodevelopmental dysregulation in patients with CS and strengthen the theory that CS is not only a neurodegenerative but also a neurodevelopmental disorder.

Challenges and Considerations of Preclinical Development for iPSC-Based Myogenic Cell Therapy.

Cell therapies derived from induced pluripotent stem cells (iPSCs) offer a promising avenue in the field of regenerative medicine due to iPSCs' expandability, immune compatibility, and pluripotent potential. An increasing number of preclinical and clinical trials have been carried out, exploring the application of iPSC-based therapies for challenging diseases, such as muscular dystrophies. The unique syncytial nature of skeletal muscle allows stem/progenitor cells to integrate, forming new myonuclei and restoring the expression of genes affected by myopathies. This characteristic makes genome-editing techniques especially attractive in these therapies. With genetic modification and iPSC lineage specification methodologies, immune-compatible healthy iPSC-derived muscle cells can be manufactured to reverse the progression of muscle diseases or facilitate tissue regeneration. Despite this exciting advancement, much of the development of iPSC-based therapies for muscle diseases and tissue regeneration is limited to academic settings, with no successful clinical translation reported. The unknown differentiation process in vivo, potential tumorigenicity, and epigenetic abnormality of transplanted cells are preventing their clinical application. In this review, we give an overview on preclinical development of iPSC-derived myogenic cell transplantation therapies including processes related to iPSC-derived myogenic cells such as differentiation, scaling-up, delivery, and cGMP compliance. And we discuss the potential challenges of each step of clinical translation. Additionally, preclinical model systems for testing myogenic cells intended for clinical applications are described.

Unlocking the Future: Pluripotent Stem Cell-Based Lung Repair.

The human respiratory system is susceptible to a variety of diseases, ranging from chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis to acute respiratory distress syndrome (ARDS). Today, lung diseases represent one of the major challenges to the health care sector and represent one of the leading causes of death worldwide. Current treatment options often focus on managing symptoms rather than addressing the underlying cause of the disease. The limitations of conventional therapies highlight the urgent clinical need for innovative solutions capable of repairing damaged lung tissue at a fundamental level. Pluripotent stem cell technologies have now reached clinical maturity and hold immense potential to revolutionize the landscape of lung repair and regenerative medicine. Meanwhile, human embryonic (HESCs) and human-induced pluripotent stem cells (hiPSCs) can be coaxed to differentiate into lung-specific cell types such as bronchial and alveolar epithelial cells, or pulmonary endothelial cells. This holds the promise of regenerating damaged lung tissue and restoring normal respiratory function. While methods for targeted genetic engineering of hPSCs and lung cell differentiation have substantially advanced, the required GMP-grade clinical-scale production technologies as well as the development of suitable preclinical animal models and cell application strategies are less advanced. This review provides an overview of current perspectives on PSC-based therapies for lung repair, explores key advances, and envisions future directions in this dynamic field.

Monoallelic KRAS (G13C) mutation triggers dysregulated expansion in induced pluripotent stem cell-derived hematopoietic progenitor cells.

BACKGROUND: Although oncogenic RAS mutants are thought to exert mutagenic effects upon blood cells, it remains uncertain how a single oncogenic RAS impacts non-transformed multipotent hematopoietic stem or progenitor cells (HPCs). Such potential pre-malignant status may characterize HPCs in patients with RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). This study sought to elucidate the biological and molecular alterations in human HPCs carrying monoallelic mutant KRAS (G13C) with no other oncogene mutations. METHODS: We utilized induced pluripotent stem cells (iPSCs) derived from two unrelated RALD patients. Isogenic HPC pairs harboring either wild-type KRAS or monoallelic KRAS (G13C) alone obtained following differentiation enabled reliable comparative analyses. The compound screening was conducted with an established platform using KRAS (G13C) iPSCs and differentiated HPCs. RESULTS: Cell culture assays revealed that monoallelic KRAS (G13C) impacted both myeloid differentiation and expansion characteristics of iPSC-derived HPCs. Comprehensive RNA-sequencing analysis depicted close clustering of HPC samples within the isogenic group, warranting that comparative studies should be performed within the same genetic background. When compared with no stimulation, iPSC-derived KRAS (G13C)-HPCs showed marked similarity with the wild-type isogenic control in transcriptomic profiles. After stimulation with cytokines, however, KRAS (G13C)-HPCs exhibited obvious aberrant cell-cycle and apoptosis responses, compatible with "dysregulated expansion," demonstrated by molecular and biological assessment. Increased BCL-xL expression was identified amongst other molecular changes unique to mutant HPCs. With screening platforms established for therapeutic intervention, we observed selective activity against KRAS (G13C)-HPC expansion in several candidate compounds, most notably in a MEK- and a BCL-2/BCL-xL-inhibitor. These two compounds demonstrated selective inhibitory effects on KRAS (G13C)-HPCs even with primary patient samples when combined. CONCLUSIONS: Our findings indicate that a monoallelic oncogenic KRAS can confer dysregulated expansion characteristics to non-transformed HPCs, which may constitute a pathological condition in RALD hematopoiesis. The use of iPSC-based screening platforms will lead to discovering treatments that enable selective inhibition of RAS-mutated HPC clones.

TAD boundary deletion causes PITX2-related cardiac electrical and structural defects.

While 3D chromatin organization in topologically associating domains (TADs) and loops mediating regulatory element-promoter interactions is crucial for tissue-specific gene regulation, the extent of their involvement in human Mendelian disease is largely unknown. Here, we identify 7 families presenting a new cardiac entity associated with a heterozygous deletion of 2 CTCF binding sites on 4q25, inducing TAD fusion and chromatin conformation remodeling. The CTCF binding sites are located in a gene desert at 1 Mb from the Paired-like homeodomain transcription factor 2 gene (PITX2). By introducing the ortholog of the human deletion in the mouse genome, we recapitulate the patient phenotype and characterize an opposite dysregulation of PITX2 expression in the sinoatrial node (ectopic activation) and ventricle (reduction), respectively. Chromatin conformation assay performed in human induced pluripotent stem cell-derived cardiomyocytes harboring the minimal deletion identified in family#1 reveals a conformation remodeling and fusion of TADs. We conclude that TAD remodeling mediated by deletion of CTCF binding sites causes a new autosomal dominant Mendelian cardiac disorder.

Exosomal miR-9-5p derived from iPSC-MSCs ameliorates doxorubicin-induced cardiomyopathy by inhibiting cardiomyocyte senescence.

Doxorubicin (DOX) is a chemotherapeutic agent widely used for tumor treatment. Nonetheless its clinical application is heavily limited by its cardiotoxicity. There is accumulated evidence that transplantation of mesenchymal stem cell-derived exosomes (MSC-EXOs) can protect against Dox-induced cardiomyopathy (DIC). This study aimed to examine the cardioprotective effects of EXOs isolated from human induced pluripotent stem cell-derived MSCs (iPSC-MSCs) against DIC and explore the potential mechanisms. EXOs were isolated from the cultural supernatant of human BM-MSCs (BM-MSC-EXOs) and iPSC-MSCs (iPSC-MSC-EXOs) by ultracentrifugation. A mouse model of DIC was induced by intraperitoneal injection of Dox followed by tail vein injection of PBS, BM-MSC-EXOs, or iPSC-MSC-EXOs. Cardiac function, cardiomyocyte senescence and mitochondrial dynamics in each group were assessed. In vitro, neonatal mouse cardiomyocytes (NMCMs) were subjected to Dox and treated with BM-MSC-EXOs or iPSC-MSC-EXOs. The mitochondrial morphology and cellular senescence of NMCMs were examined by Mitotracker staining and senescence-associated-ß-galactosidase assay, respectively. Compared with BM-MSC-EXOs, mice treated with iPSC-MSC-EXOs displayed improved cardiac function and decreased cardiomyocyte mitochondrial fragmentation and senescence. In vitro, iPSC-MSC-EXOs were superior to BM-MSC-EXOs in attenuation of cardiomyocyte mitochondrial fragmentation and senescence caused by DOX. MicroRNA sequencing revealed a higher level of miR-9-5p in iPSC-MSC-EXOs than BM-MSC-EXOs. Mechanistically, iPSC-MSC-EXOs transported miR-9-5p into DOX-treated cardiomyocytes, thereby suppressing cardiomyocyte mitochondrial fragmentation and senescence via regulation of the VPO1/ERK signal pathway. These protective effects and cardioprotection against DIC were largely reversed by knockdown of miR-9-5p in iPSC-MSC-EXOs. Our results showed that miR-9-5p transferred by iPSC-MSC-EXOs protected against DIC by alleviating cardiomyocyte senescence via inhibition of the VPO1/ERK pathway. This study offers new insight into the application of iPSC-MSC-EXOs as a novel therapeutic strategy for DIC treatment.

Telomerase is essential for cardiac differentiation and sustained metabolism of human cardiomyocytes.

Telomeres as the protective ends of linear chromosomes, are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes, telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study, we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently, the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect, highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies.

Clinical and molecular outcomes from the 5-Year natural history study of SSADH Deficiency, a model metabolic neurodevelopmental disorder.

BACKGROUND: Succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a model neurometabolic disease at the fulcrum of translational research within the Boston Children's Hospital Intellectual and Developmental Disabilities Research Centers (IDDRC), including the NIH-sponsored natural history study of clinical, neurophysiological, neuroimaging, and molecular markers, patient-derived induced pluripotent stem cells (iPSC) characterization, and development of a murine model for tightly regulated, cell-specific gene therapy. METHODS: SSADHD subjects underwent clinical evaluations, neuropsychological assessments, biochemical quantification of γ-aminobutyrate (GABA) and related metabolites, electroencephalography (standard and high density), magnetoencephalography, transcranial magnetic stimulation, magnetic resonance imaging and spectroscopy, and genetic tests. This was parallel to laboratory molecular investigations of in vitro GABAergic neurons derived from induced human pluripotent stem cells (hiPSCs) of SSADHD subjects and biochemical analyses performed on a versatile murine model that uses an inducible and reversible rescue strategy allowing on-demand and cell-specific gene therapy. RESULTS: The 62 SSADHD subjects [53% females, median (IQR) age of 9.6 (5.4-14.5) years] included in the study had a reported symptom onset at ∼ 6 months and were diagnosed at a median age of 4 years. Language developmental delays were more prominent than motor. Autism, epilepsy, movement disorders, sleep disturbances, and various psychiatric behaviors constituted the core of the disorder's clinical phenotype. Lower clinical severity scores, indicating worst severity, coincided with older age (R= -0.302, p = 0.03), as well as age-adjusted lower values of plasma γ-aminobutyrate (GABA) (R = 0.337, p = 0.02) and γ-hydroxybutyrate (GHB) (R = 0.360, p = 0.05). While epilepsy and psychiatric behaviors increase in severity with age, communication abilities and motor function tend to improve. iPSCs, which were differentiated into GABAergic neurons, represent the first in vitro neuronal model of SSADHD and express the neuronal marker microtubule-associated protein 2 (MAP2), as well as GABA. GABA-metabolism in induced GABAergic neurons could be reversed using CRISPR correction of the pathogenic variants or mRNA transfection and SSADHD iPSCs were associated with excessive glutamatergic activity and related synaptic excitation. CONCLUSIONS: Findings from the SSADHD Natural History Study converge with iPSC and animal model work focused on a common disorder within our IDDRC, deepening our knowledge of the pathophysiology and longitudinal clinical course of a complex neurodevelopmental disorder. This further enables the identification of biomarkers and changes throughout development that will be essential for upcoming targeted trials of enzyme replacement and gene therapy.

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies.

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

The human body contains around 20,000 different proteins that perform a myriad of essential roles. To understand how these proteins work in healthy individuals and during disease, we need to know their precise locations inside cells and how these locations may change in different situations. Genetic tools known as fluorescent proteins are often used as tags to study the location of specific proteins of interest within cells. When exposed to light, the fluorescent proteins emit specific colours of light that can be observed using microscopes. In a fluorescent protein system known as split mNeonGreen, researchers insert the DNA encoding two fragments of a fluorescent protein (one large, one small) separately into cells. The large fragment can be found throughout the cell, while the small fragment is attached to specific host proteins. When the two fragments meet, they assemble into the full mNeonGreen protein and can fluoresce. Researchers can use split mNeonGreen and other similar systems to generate large libraries of cells where the small fragment of a fluorescent protein is attached to thousands of different host proteins. However, so far these libraries are restricted to a handful of different types of cells. To address this challenge, Husser et al. inserted the DNA encoding the large fragment of mNeonGreen into human cells known as induced pluripotent stem cells, which are able to give rise to any other type of human cell. This then enabled the team to quickly and efficiently generate a library of stem cells that express the small fragment of mNeonGreen attached to different host proteins. Further experiments studied the locations of host proteins in the stem cells just before they divided into two cells. This suggested that there are differences between how induced pluripotent stem cells and other types of cells divide. In the future, the cells and the method developed by Husser et al. may be used by other researchers to create atlases showing where human proteins are located in many other types of cells.

A de novo dominant-negative variant is associated with OTULIN-related autoinflammatory syndrome.

OTULIN-related autoinflammatory syndrome (ORAS), a severe autoinflammatory disease, is caused by biallelic pathogenic variants of OTULIN, a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here, we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells, which confirms that the patient has ORAS. However, although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN, the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis.

Molecular insights into PCB neurotoxicity: Comparing transcriptomic responses across dopaminergic neurons, population blood cells, and Parkinson's disease pathology.

Parkinson's disease (PD) is a complex neurodegenerative disorder influenced by genetic factors and environmental exposures. Polychlorinated biphenyls (PCBs), a group of synthetic organic compounds, have been identified as potential environmental risk factors for neurodegenerative diseases, including PD. We explored PCB-induced neurotoxicity mechanisms using iPSC-derived dopaminergic neurons and assessed their transcriptomic responses to varying PCB concentrations (0.01 µM, 0.5 µM, and 10 µM). Specifically, we focused on PCB-180, a congener known for its accumulation in human brains. The exposure durations were 24 h and 74 h, allowing us to capture both short-term and more prolonged effects on gene expression patterns. We observed that PCB exposure led to the suppression of oxidative phosphorylation, synaptic function, and neurotransmitter release, implicating these pathways in PCB-induced neurotoxicity. In our comparative analysis, we noted similarities in PCB-induced changes with other PD-related compounds like MPP+ and rotenone. Our findings also aligned with gene expression changes in human blood derived from a population exposed to PCBs, highlighting broader inflammatory responses. Additionally, molecular patterns seen in iPSC-derived neurons were confirmed in postmortem PD brain tissues, validating our in vitro results. In conclusion, our study offers novel insights into the multifaceted impacts of PCB-induced perturbations on various cellular contexts relevant to PD. The use of iPSC-derived dopaminergic neurons allowed us to decipher intricate transcriptomic alterations, bridging the gap between in vitro and in vivo findings. This work underscores the potential role of PCB exposure in neurodegenerative diseases like PD, emphasizing the need to consider both systemic and cell specific effects.

An increase in ER stress and unfolded protein response in iPSCs-derived neuronal cells from neuronopathic Gaucher disease patients.

Gaucher disease (GD) is a lysosomal storage disorder caused by a mutation in the GBA1 gene, responsible for encoding the enzyme Glucocerebrosidase (GCase). Although neuronal death and neuroinflammation have been observed in the brains of individuals with neuronopathic Gaucher disease (nGD), the exact mechanism underlying neurodegeneration in nGD remains unclear. In this study, we used two induced pluripotent stem cells (iPSCs)-derived neuronal cell lines acquired from two type-3 GD patients (GD3-1 and GD3-2) to investigate the mechanisms underlying nGD by biochemical analyses. These iPSCs-derived neuronal cells from GD3-1 and GD3-2 exhibit an impairment in endoplasmic reticulum (ER) calcium homeostasis and an increase in unfolded protein response markers (BiP and CHOP), indicating the presence of ER stress in nGD. A significant increase in the BAX/BCL-2 ratio and an increase in Annexin V-positive cells demonstrate a notable increase in apoptotic cell death in GD iPSCs-derived neurons, suggesting downstream signaling after an increase in the unfolded protein response. Our study involves the establishment of iPSCs-derived neuronal models for GD and proposes a possible mechanism underlying nGD. This mechanism involves the activation of ER stress and the unfolded protein response, ultimately leading to apoptotic cell death in neurons.

Regulation of human microglial gene expression and function via RNAase-H active antisense oligonucleotides in vivo in Alzheimer's disease.

BACKGROUND: Microglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells, such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2), as the strongest risk factors for Alzheimer's disease (AD) highlights the importance of microglial biology in the brain. The sequence, structure and function of several microglial proteins are poorly conserved across species, which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs). METHODS: In this study, we identified, produced, and tested novel, selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models, as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo. RESULTS: We proved their efficacy in human iPSC microglia in vitro, as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-ß plaques in vivo in a model of AD. CONCLUSIONS: This study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.

Exosomes secreted from induced pluripotent stem cell ameliorate the lipopolysaccharide induced neuroinflammatory response via lncRNA-0949.

PURPOSE: To study the effect of exosomes derived from the induced pluripotent stem cells (iPSCs) in the neuroinflammatory response of microglia caused by lipopolysaccharide (LPS) and reveal the potential underlying mechanism. METHODS: A permanent microglia cell line HMO6 was activated by LPS. The features of exosomes were analyzed by nano flow cytometry, Western blot and transmission electron microscope. The RNA-seq was used to analyze the difference of noncoding RNA profiles between iPSC-Exos and HMO6 derived exosomes and proved that long no-coding RNA (lncRNA-0949) was highly expressed in the iPSC-Exos. Activated HMO6 cells were cocultured with iPSC-Exos in which lncRNA-0949 was overexpressed, knocked down or normally expressed. Quantitative real-time polymerase chain reaction (RT-qPCR), Enzyme-Linked Immunosorbent Assay and Western blot assay were adopted to analyze RNA and protein expression of inflammatory factors in HMO6 cells. RESULTS: The oxidative stress and inflammatory response of microglia were significantly attenuated with the iPSC derived exosomes treatment. LncRNA-0949 was effectively delivered into the HMO6 cells through the iPSC-Exos, which largely alleviated the production of malondialdehyde, IL-6, IL-1ß and TNF-α in HMO6 cells. Overexpression of lncRNA-0949 could enhance the anti-inflammatory effect of the iPSC-Exos, and knock-down of lncRNA-0949 impaired this availability. CONCLUSION: According to our results, lncRNA-0949 enriched exosomes from iPSC could potentially be used as a therapeutic strategy to prevent/treat neuroinflammatory diseases.

Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research.

Genome editing, notably CRISPR (cluster regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9), has revolutionized genetic engineering allowing for precise targeted modifications. This technique's combination with human induced pluripotent stem cells (hiPSCs) is a particularly valuable tool in cerebral organoid (CO) research. In this study, CRISPR/Cas9-generated fluorescently labeled hiPSCs exhibited no significant morphological or growth rate differences compared with unedited controls. However, genomic aberrations during gene editing necessitate efficient genome integrity assessment methods. Optical genome mapping, a high-resolution genome-wide technique, revealed genomic alterations, including chromosomal copy number gain and losses affecting numerous genes. Despite these genomic alterations, hiPSCs retain their pluripotency and capacity to generate COs without major phenotypic changes but one edited cell line showed potential neuroectodermal differentiation impairment. Thus, this study highlights optical genome mapping in assessing genome integrity in CRISPR/Cas9-edited hiPSCs emphasizing the need for comprehensive integration of genomic and morphological analysis to ensure the robustness of hiPSC-based models in cerebral organoid research.

Transgene-Free Cynomolgus Monkey iPSCs Generated under Chemically Defined Conditions.

Non-human primates (NHPs) are pivotal animal models for translating novel cell replacement therapies into clinical applications, including validating the safety and efficacy of induced pluripotent stem cell (iPSC)-derived products. Preclinical development and the testing of cell-based therapies ideally comprise xenogeneic (human stem cells into NHPs) and allogenic (NHP stem cells into NHPs) transplantation studies. For the allogeneic approach, it is necessary to generate NHP-iPSCs with generally equivalent quality to the human counterparts that will be used later on in patients. Here, we report the generation and characterization of transgene- and feeder-free cynomolgus monkey (Macaca fascicularis) iPSCs (Cyno-iPSCs). These novel cell lines have been generated according to a previously developed protocol for the generation of rhesus macaque, baboon, and human iPSC lines. Beyond their generation, we demonstrate the potential of the novel Cyno-iPSCs to differentiate into two clinically relevant cell types, i.e., cardiomyocytes and neurons. Overall, we provide a resource of novel iPSCs from the most frequently used NHP species in the regulatory testing of biologics and classical pharmaceutics to expand our panel of iPSC lines from NHP species with high relevance in preclinical testing and translational research.