Induced Pluripotent Stem Research Articles

Neural stem cells derived from α-synuclein-knockdown iPS cells alleviate Parkinson’s disease

Stem cells have the potential to replace damaged or defective cells and assist in the development of treatments for neurodegenerative diseases, including Parkinson’s disease (PD) and Alzheimer’s disease. iPS cells derived from patient-specific somatic cells are not only ethically acceptable, but they also avoid complications relating to immune rejection. Currently, researchers are developing stem cell-based therapies for PD using induced pluripotent stem (iPS) cells. iPS cells can differentiate into cells from any of the three germ layers, including neural stem cells (NSCs). Transplantation of neural stem cells (NSCs) is an emerging therapy for treating neurological disorders by restoring neuronal function. Nevertheless, there are still challenges associated with the quality and source of neural stem cells. This issue can be addressed by genetically edited iPS cells. In this study, shRNA was used to knock down the expression of mutant α-synuclein (SNCA) in iPS cells that were generated from SNCA A53T transgenic mice, and these iPS cells were differentiated to NSCs. After injecting these NSCs into SNCA A53T mice, the therapeutic effects of these cells were evaluated. We found that the transplantation of neural stem cells produced from SNCA A53T iPS cells with knocking down SNCA not only improved SNCA A53T mice coordination abilities, balance abilities, and locomotor activities but also significantly prolonged their lifespans. The results of this study suggest an innovative therapeutic approach that combines stem cell therapy and gene therapy for the treatment of Parkinson’s disease.

Ready-to-use iPSC-derived microglia progenitors for the treatment of CNS disease in mouse models of neuropathic mucopolysaccharidoses

Mucopolysaccharidoses are inherited metabolic disorders caused by the deficiency in lysosomal enzymes required to break down glycosaminoglycans. Accumulation of glycosaminoglycans leads to progressive, systemic degenerative disease. The central nervous system is particularly affected, resulting in developmental delays, neurological regression, and early mortality. Current treatments fail to adequately address neurological defects. Here we explore the potential of human induced pluripotent stem cell (hiPSC)-derived microglia progenitors as a one-time, allogeneic off-the-shelf cell therapy for several mucopolysaccharidoses (MPS). We show that hiPSC-derived microglia progenitors, possessing normal levels of lysosomal enzymes, can deliver functional enzymes into four subtypes of MPS knockout cell lines through mannose-6-phosphate receptor-mediated endocytosis in vitro. Additionally, our findings indicate that a single administration of hiPSC-derived microglia progenitors can reduce toxic glycosaminoglycan accumulation and prevent behavioral deficits in two different animal models of MPS. Durable efficacy is observed for eight months after transplantation. These results suggest a potential avenue for treating MPS with hiPSC-derived microglia progenitors.

Leiomodin 2 neonatal dilated cardiomyopathy mutation results in altered actin gene signatures and cardiomyocyte dysfunction

Neonatal dilated cardiomyopathy (DCM) is a poorly understood muscular disease of the heart. Several homozygous biallelic variants in LMOD2, the gene encoding the actin-binding protein Leiomodin 2, have been identified to result in severe DCM. Collectively, LMOD2-related cardiomyopathies present with cardiac dilation and decreased heart contractility, often resulting in neonatal death. Thus, it is evident that Lmod2 is essential to normal human cardiac muscle function. This study aimed to understand the underlying pathophysiology and signaling pathways related to the first reported LMOD2 variant (c.1193 G > A, p.Trp398*). Using patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and a mouse model harboring the homologous mutation to the patient, we discovered dysregulated actin-thin filament lengths, altered contractility and calcium handling properties, as well as alterations in the serum response factor (SRF)-dependent signaling pathway. These findings reveal that LMOD2 may be regulating SRF activity in an actin-dependent manner and provide a potential new strategy for the development of biologically active molecules to target LMOD2-related cardiomyopathies.

Epigenetic control and inheritance of rDNA arrays.

Ribosomal RNA (rRNA) genes exist in multiple copies arranged in tandem arrays known as ribosomal DNA (rDNA). The total number of gene copies is variable, and the mechanisms buffering this copy number variation remain unresolved. We surveyed the number, distribution, and activity of rDNA arrays at the level of individual chromosomes across multiple human and primate genomes. Each individual possessed a unique fingerprint of copy number distribution and activity of rDNA arrays. In some cases, entire rDNA arrays were transcriptionally silent. Silent rDNA arrays showed reduced association with the nucleolus and decreased interchromosomal interactions, indicating that the nucleolar organizer function of rDNA depends on transcriptional activity. Methyl-sequencing of flow-sorted chromosomes, combined with long read sequencing, showed epigenetic modification of rDNA promoter and coding region by DNA methylation. Silent arrays were in a closed chromatin state, as indicated by the accessibility profiles derived from Fiber-seq. Removing DNA methylation restored the transcriptional activity of silent arrays. Array activity status remained stable through the iPS cell re-programming. Family trio analysis demonstrated that the inactive rDNA haplotype can be traced to one of the parental genomes, suggesting that the epigenetic state of rDNA arrays may be heritable. We propose that the dosage of rRNA genes is epigenetically regulated by DNA methylation, and these methylation patterns specify nucleolar organizer function and can propagate transgenerationally.

An injectable decellularized extracellular matrix hydrogel with cortical neuron-derived exosomes enhances tissue repair following traumatic spinal cord injury

Traumatic spinal cord injury (SCI), known for its limited intrinsic regeneration capacity, often results in considerable neurological impairment. Studies suggest that therapeutic techniques utilizing exosomes (Exo) to promote tissue regeneration and modulate immune responses are promising for SCI treatment. However, combining exosome therapy with biomaterials for SCI treatment is not very effective. This study developed an adhesive hydrogel using exosomes secreted by cortical neurons derived from human induced pluripotent stem cells (iPSCs) and decellularized extracellular matrix (dECM) from human umbilical cord mesenchymal stem cells (hUCMSCs) to enhance motor function recovery post-SCI. In vitro assessments demonstrated the excellent cytocompatibility of the dECM hydrogel. Additionally, the Exo-dECM hydrogel facilitated the polarization of early M2 macrophages, reduced neuronal apoptosis, and established a pro-regenerative microenvironment in a rodent SCI model. Subsequent analyses revealed significant activation of endogenous neural stem cells and promotion of axon regeneration and remyelination at eight weeks post-surgery. The Exo-dECM hydrogel also promoted the functional recovery and preservation of urinary tissue in SCI-afflicted rats. These findings highlighted that the Exo-dECM hydrogel is a promising therapeutic strategy for treating SCI.

A scalable, spin-free approach to generate enhanced induced pluripotent stem cell-derived natural killer cells forcancer immunotherapy.

Natural killer (NK) cells play a vital role in innate immunity and show great promise in cancer immunotherapy. Traditional sources of NK cells, such as the peripheral blood, are limited by availability and donor variability. In addition, in vitro expansion can lead to functional exhaustion and gene editing challenges. This study aimed to harness induced pluripotent stem cell (iPSC) technology to provide a consistent and scalable source of NK cells, overcoming the limitations of traditional sources and enhancing the potential for cancer immunotherapy applications. We developed human placental-derived iPSC lines using reprogramming techniques. Subsequently, an optimized two-step differentiation protocol was introduced to generate high-purity NK cells. Initially, iPSCs were differentiated into hematopoietic-like stem cells using spin-free embryoid bodies (EBs). Subsequently, the EBs were transferred to ultra-low attachment plates to induce NK cell differentiation. iPSC-derived NK (iNK) cells expressed common NK cell markers (NKp46, NKp30, NKp44, CD16 and eomesodermin) at both RNA and protein levels. iNK cells demonstrated significant resilience to cryopreservation and exhibited enhanced cytotoxicity. The incorporation of a chimeric antigen receptor (CAR) construct further augmented their cytotoxic potential. This study exemplifies the feasibility of generating iNK cells with high purity and enhanced functional capabilities, their improved resilience to cryopreservation and the potential to have augmented cytotoxicity through CAR expression. Our findings offer a promising pathway for the development of potential cellular immunotherapies, highlighting the critical role of iPSC technology in overcoming challenges associated with traditional NK cell sources.

Modeling Lewy body disease with SNCA triplication iPSC-derived cortical organoids and identifying therapeutic drugs.

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)-derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration-approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.

Simultaneous electromechanical monitoring in engineered heart tissues using a mesoscale framework.

Engineered heart tissues (EHTs) generated from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent powerful platforms for human cardiac research, especially in drug testing and disease modeling. Here, we report a flexible, three-dimensional electronic framework that enables real-time, spatiotemporal analysis of electrophysiologic and mechanical signals in EHTs under physiological loading conditions for dynamic, noninvasive, longer-term assessments. These electromechanically monitored EHTs support multisite measurements throughout the tissue under baseline conditions and in response to stimuli. Demonstrations include uses in tracking physiological responses to pharmacologically active agents and in capturing electrophysiological characteristics of reentrant arrhythmias. This platform facilitates precise analysis of signal location and conduction velocity in human cardiomyocyte tissues, as the basis for a broad range of advanced cardiovascular studies.

An intrinsic mechanism of metabolic tuning promotes cardiac resilience to stress

Defining the molecular mechanisms underlying cardiac resilience is crucial to find effective approaches to protect the heart. A physiologic level of ROS is produced in the heart by fatty acid oxidation, but stressful events can boost ROS and cause mitochondrial dysfunction and cardiac functional impairment. Melusin is a muscle specific chaperone required for myocardial compensatory remodeling during stress. Here we report that Melusin localizes in mitochondria where it binds the mitochondrial trifunctional protein, a key enzyme in fatty acid oxidation, and decreases it activity. Studying both mice and human induced pluripotent stem cell-derived cardiomyocytes, we found that Melusin reduces lipid oxidation in the myocardium and limits ROS generation in steady state and during pressure overload and doxorubicin treatment, preventing mitochondrial dysfunction. Accordingly, the treatment with the lipid oxidation inhibitor Trimetazidine concomitantly with stressful stimuli limits ROS accumulation and prevents long-term heart dysfunction. These findings disclose a physiologic mechanism of metabolic regulation in the heart and demonstrate that a timely restriction of lipid metabolism represents a potential therapeutic strategy to improve cardiac resilience to stress.

CHIR99021 and Brdu Are Critical in Chicken iPSC Reprogramming via Small-Molecule Screening.

Background/Objectives: Induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells into cells with most of the ESC (embryonic stem cell) characteristics show promise toward solving ethical problems currently facing stem cell research and eventually yield clinical grade pluripotent stem cells for therapies and regenerative medicine. In recent years, an increasing body of research suggests that the chemical induction of pluripotency (CIP) method can yield iPSCs in vitro, yet its application in avian species remains unreported. Methods: Herein, we successfully obtained stably growing chicken embryonic fibroblasts (CEFs) using the tissue block adherence method and employed 12 small-molecule compounds to induce chicken iPSC formation. Results: The final optimized iPSC induction system was bFGF (10 ng/mL), CHIR99021 (3 μM), RepSox (5 μM), DZNep (0.05 μM), BrdU (10 μM), BMP4 (10 ng/mL), vitamin C (50 μg/mL), EPZ-5676 (5 μM), and VPA (0.1 mM). Optimization of the induction system revealed that the highest number of clones was induced with 8 × 104 cells per well and at 1.5 times the original concentration. Upon characterization, these clones exhibited iPSC characteristics, leading to the development of a stable compound combination for iPSC generation in chickens. Concurrently, employing a deletion strategy to investigate the functionality of small-molecule compounds during induction, we identified CHIR99021 and BrdU as critical factors for inducing chicken iPSC formation. Conclusions: In conclusion, this study provides a reference method for utilizing small-molecule combinations in avian species to reprogram cells and establish a network of cell fate determination mechanisms.

Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs

Translating genetic findings for neurodevelopmental and psychiatric disorders (NPDs) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop codons (iSTOP) that lead to mRNA nonsense-mediated decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 genes. Using RNA sequencing (RNA-seq), we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Despite high editing efficiency, three schizophrenia risk genes (SETD1A, TRIO, and CUL1) only had heterozygous LoF alleles, suggesting their essential roles for cell growth. We found that CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.

Adipocyte inflammation is the primary driver of hepatic insulin resistance in a human iPSC-based microphysiological system

Interactions between adipose tissue, liver and immune system are at the center of metabolic dysfunction-associated steatotic liver disease and type 2 diabetes. To address the need for an accurate in vitro model, we establish an interconnected microphysiological system (MPS) containing white adipocytes, hepatocytes and proinflammatory macrophages derived from isogenic human induced pluripotent stem cells. Using this MPS, we find that increasing the adipocyte-to-hepatocyte ratio moderately affects hepatocyte function, whereas macrophage-induced adipocyte inflammation causes lipid accumulation in hepatocytes and MPS-wide insulin resistance, corresponding to initiation of metabolic dysfunction-associated steatotic liver disease. We also use our MPS to identify and characterize pharmacological intervention strategies for hepatic steatosis and systemic insulin resistance and find that the glucagon-like peptide-1 receptor agonist semaglutide improves hepatocyte function by acting specifically on adipocytes. These results establish our MPS modeling the adipose tissue-liver axis as an alternative to animal models for mechanistic studies or drug discovery in metabolic diseases.

Generation of human induced pluripotent stem cell lines from a subject with UBAP1L-associated retinal dystrophy and CRISPR/cas9-corrected isogenic iPSC lines

A Human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a patient affected with an autosomal recessive retinal dystrophy carrying the homozygous c.910-7G>A variant in UBAP1L. Three isogenic control iPSC lines derived from this affected subject line were created using CRISPR/Cas9 engineering. All iPSC lines expressing the pluripotency markers, were able to differentiate into the three germ layers, and exhibit a normal karyotype. These cellular models will provide a powerful tool to study disease mechanisms associated with the recently reported UBAP1L- associated retinal dystrophy and better understand the role of the protein in retinal physiology.

Establishment of a non-integrated iPS cell line (SDQLCHi072-A) from a patient suffered from AUTS2 syndrome

In this study, PBMCs used in reprogramming were from a boy suffered from AUTS2 syndrome confirmed by clinical and genetic diagnosis. iPSCs were established by non-integrated method, which carried AUTS2 heterozygous mutation. The established iPSCs presented similar appearance and expressed pluripotent markers in mRNA and protein level. Additionally, the iPSCs possessed favorable differentiative capacity in vivo and normal karyotype. This is an effective potential model for the study about AUTS2 syndrome and the screen about drug therapy.

Characteristics and Transcriptomic Analysis of Cholinergic Neurons Derived from Induced Pluripotent Stem Cells with APP Mutation in Alzheimer's Disease.

The cholinergic hypothesis is one of the main theories that describe the pathogenesis of Alzheimer's disease (AD). Cholinergic neurons degenerate early and are severely damaged in AD. Despite extensive research, the causes of cholinergic neuron damage and the underlying molecular changes remain unclear. This study aimed to explore the characteristics and transcriptomic changes in cholinergic neurons derived from human induced pluripotent stem cells (iPSCs) with APP mutation. Peripheral blood mononuclear cells from patients with AD and healthy individuals were reprogrammed into iPSCs. The iPSCs were differentiated into cholinergic neurons. Cholinergic neurons were stained, neurotoxically tested, and electrophysiologically and transcriptomically analyzed. The iPSCs-derived cholinergic neurons from a patient with AD carrying a mutation in APP displayed enhanced susceptibility to Aβ1-42-induced neurotoxicity, characterized by severe neurotoxic effects, such as cell body coagulation and neurite fragmentation. Cholinergic neurons exhibited electrophysiological impairments and neuronal death after 21 days of culture in the AD group. Transcriptome analysis disclosed 883 differentially expressed genes (DEGs, 420 upregulated and 463 downregulated) participating in several signaling pathways implicated in AD pathogenesis. To assess the reliability of RNA sequencing, the expression of 16 target DEGs was validated using qPCR. Finally, the expression of the 8 core genes in different cell types of brain was analyzed by the AlzData database. In this study, iPSCs-derived cholinergic neurons from AD patients with APP mutations exhibit characteristics reminiscent of neurodegenerative disease. Transcriptome analysis revealed the corresponding DEGs and pathways, providing potential biomarkers and therapeutic targets for advancing AD research.

Generation and genetic repair of two human induced pluripotent stem cell lines from patients with Epidermolysis Bullosa simplex associated with a heterozygous mutation in the translation initiation codon of KLHL24

Fibroblasts from two patients carrying a distinct heterozygous mutation in the KLHL24 gene (c.1 A>G and c.2T>C) were reprogrammed to obtain hiPSC lines. Non-integrating Sendai virus and CRISPR-Cas9 editing were respectively used to deliver the reprogramming factors and repair the mutation in the patient-hiPSCs to obtain isogenic control pairs. No off-target nuclease activity was detected with the top-predicted sites. Patient and isogenic hiPSCs displayed typical morphology, expressed markers of the undifferentiated state, were able to differentiate into the three germ layers and had normal karyotypes. These isogenic pairs will expand the panel of hiPSC lines to model KLHL24-associated conditions.

Motion Sensing by a Highly Sensitive Nanogold Strain Sensor in a Biomimetic 3D Environment.

Recent advancements in flexible electronics have highlighted their potential in biomedical applications, primarily due to their human-friendly nature. This study introduces a new flexible electronic system designed for motion sensing in a biomimetic three-dimensional (3D) environment. The system features a self-healing gel matrix (chitosan-based hydrogel) that effectively mimics the dynamics of the extracellular matrix (ECM), and is integrated with a highly sensitive thin-film resistive strain sensor, which is fabricated by incorporating a cross-linked gold nanoparticle (GNP) thin film as the active conductive layer onto a biocompatible microphase-separated polyurethane (PU) substrate through a clean, rapid, and high-precision contact printing method. The GNP-PU strain sensor demonstrates high sensitivity (a gauge factor of ∼50), good stability, and waterproofing properties. The feasibility of detecting small motion was evaluated by sensing the beating of human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte spheroids embedded in the gel matrix. The integration of these components exemplifies a proof-of-concept for using flexible electronics comprising self-healing hydrogel and thin-film nanogold in cardiac sensing and offers promising insights into the development of next-generation biomimetic flexible electronic devices.

Induction of Pluripotent Stem Cells from Muscle Cells of Large Yellow Croaker (Larimichthys Crocea) Via Electrotransfection.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells reprogrammed from somatic cells back into an embryonic-like pluripotent state of stem cells to study development, disease and potential gene therapies. The induction and regulation mechanisms of iPSCs in fish are still unclear. By using the transfection technique, we investigated the crucial function of the OSKMNL factor co-expression for somatic reprogramming in the muscle cell line of large yellow croaker (Larimichthys crocea) (LYCMs) and successfully established a stable iPSCs line (Lc-OSNL-iPSCs). Stable culturing of iPSCs with high alkaline phosphatase activity and a stable karyotype was achieved. The qRT-PCR and immunofluorescence labeling results revealed that Lc-OSNL-iPSCs displayed a high expression level of pluripotent marker genes such as Nanog, Oct4, and Sox2. There were significant differences between Lc-OSNL-iPSCs, Lc-OSKMNL-iPSCs, and LYCMs, and the expression of several genes in maintaining cell pluripotency was up-regulated when the pluripotency signal pathway of stem cells was activated. The technical system for inducing iPSCs of Larimichthys crocea was constructed in this study. This system can serve as a basic model to understand germ cell differentiation mechanism, gender control, genetics, and breeding of large yellow croaker and a platform for studying iPSCs in fish. Interestingly, the acquired iPSCs serves as a useful material for the directional induction of muscle stem cells, thereby establishing the groundwork for obtaining "artificial fish" in the future.

Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells.

Acquisition of specific cell shapes and morphologies is a central component of cell fate transitions. Although signaling circuits and gene regulatory networks that regulate pluripotent stem cell differentiation have been intensely studied, how these networks are integrated in space and time with morphological transitions and mechanical deformations to control state transitions remains a fundamental open question. Here, we focus on two distinct models of pluripotency, primed pluripotent stem cells and pre-implantation inner cell mass cells of human embryos to discover that cell fate transitions associate with rapid changes in nuclear shape and volume which collectively alter the nuclear mechanophenotype. Mechanistic studies in human induced pluripotent stem cells further reveal that these phenotypical changes and the associated active fluctuations of the nuclear envelope arise from growth factor signaling-controlled changes in chromatin mechanics and cytoskeletal confinement. These collective mechano-osmotic changes trigger global transcriptional repression and a condensation-prone environment that primes chromatin for a cell fate transition by attenuating repression of differentiation genes. However, while this mechano-osmotic chromatin priming has the potential to accelerate fate transitions and differentiation, sustained biochemical signals are required for robust induction of specific lineages. Our findings uncover a critical mechanochemical feedback mechanism that integrates nuclear mechanics, shape and volume with biochemical signaling and chromatin state to control cell fate transition dynamics.

Specific cleavage of IGFBP-4 by papp-a in nervous tissue

Astrocytes are subtypes of glial cells involved in metabolic, structural, homeostatic, and neuroprotective processes that help neurons maintain viability. Insulin-like growth factors IGF-1 and IGF-2 are known to have neuroprotective effects on neurons and glial cells through interaction with specific receptors. IGF forms a complex with IGF-binding proteins (IGFBP) in nervous tissue and is released from the complex via IGFBP proteolysis by specific proteases. It has been reported that IGFBP-2, 5 and 6 are cleaved by specific proteases in the central nervous system (CNS), followed by IGF release; however, it was unknown whether IGFBP-4 was exposed to a particular proteolysis in nervous tissue. Using neurons and astrocytes derived from human induced pluripotent stem cell lines (hiPSC), as well as rat brain-sourced primary neuron-glia cultures, we demonstrated that IGFBP-4 is specifically cleaved in nervous tissue by the Pregnancy Associated Plasma Protein A (PAPP-A) protease and that this cleavage is IGF-dependent. Our results indicate that astrocyte rather than neuron PAPP-A cleaves IGFBP-4 in nervous tissue suggesting that this may be one of the fundamental mechanisms for IGF interchange between these two types of cells.