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Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of cancer chemotherapy that can often limit treatment options for cancer patients or have life-long neurodegenerative consequences that reduce the patient’s quality of life. CIPN is caused by the detrimental actions of various chemotherapeutic agents on peripheral axons. Currently, there are no approved preventative measures or treatment options for CIPN, highlighting the need for the discovery of novel therapeutics and improving our understanding of disease mechanisms. In this study, we utilized human-induced pluripotent stem cell (hiPSC)-derived motor neurons as a platform to mimic axonal damage after treatment with vincristine, a chemotherapeutic used for the treatment of breast cancers, osteosarcomas, and leukemia. We screened a total of 1902 small molecules for neuroprotective properties in rescuing vincristine-induced axon growth deficits. From our primary screen, we identified 38 hit compounds that were subjected to secondary dose response screens. Six compounds showed favorable pharmacological profiles – AZD7762, A-674563, Blebbistatin, Glesatinib, KW-2449, and Pelitinib, all novel neuroprotectants against vincristine toxicity to neurons. In addition, four of these six compounds also showed efficacy against vincristine-induced growth arrest in human iPSC-derived sensory neurons. In this study, we utilized high-throughput screening of a large library of compounds in a therapeutically relevant assay. We identified several novel compounds that are efficacious in protecting different neuronal subtypes from the toxicity induced by a common chemotherapeutic agent, vincristine which could have therapeutic potential in the clinic. The online version contains supplementary material available at 10.1007/s00018-024-05340-x.

Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2, accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family, which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here, we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34 + -blood cells of a patient homozygous for the p.Cys408Gly mutation, the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain, perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency, including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation, ICF2-iPSCs showed decreased vitality and a lower percentage of CD34 + /CD43 + /CD45 + progenitors. Overall, the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype.

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality, inter-batch consistency, cryopreservation and scale remain, reducing experimental reproducibility and clinical translation. Here, we report a robust stirred suspension cardiac differentiation protocol, and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines, the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs, bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks, which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible, scalable, and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications, and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols. Subject terms: Cardiovascular biology, Induced pluripotent stem cells, Cardiovascular models

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines.

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 ( V606M ; R453C ), MYBPC3 ( G148R ) or digenic variants ( MYBPC3 P955fs , TNNI3 A157V ). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls, variant-positive CMs show sarcomere disorganization, higher contractility, calcium transients, and ATPase activity. However, only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM.

The delicate “Excitatory/Inhibitory balance” between neurons holds significance in neurodegenerative and neurodevelopmental diseases. With the ultimate goal of creating a faithful in vitro model of the human brain, in this study, we investigated the critical factor of heterogeneity, focusing on the interplay between excitatory glutamatergic (E) and inhibitory GABAergic (I) neurons in neural networks. We used high-density Micro-Electrode Arrays (MEA) with 2304 recording electrodes to investigate two neuronal culture configurations: 100% glutamatergic (100E) and 75% glutamatergic / 25% GABAergic (75E25I) neurons. This allowed us to comprehensively characterize the spontaneous electrophysiological activity exhibited by mature cultures at 56 Days in vitro , a time point in which the GABA shift has already occurred. We explored the impact of heterogeneity also through electrical stimulation, revealing that the 100E configuration responded reliably, while the 75E25I required more parameter tuning for improved responses. Chemical stimulation with BIC showed an increase in terms of firing and bursting activity only in the 75E25I condition, while APV and CNQX induced significant alterations on both dynamics and functional connectivity. Our findings advance understanding of diverse neuron interactions and their role in network activity, offering insights for potential therapeutic interventions in neurological conditions. Overall, this work contributes to the development of a valuable human-based in vitro system for studying physiological and pathological conditions, emphasizing the pivotal role of neuron diversity in neural network dynamics.

The transplantation of spinal cord progenitor cells (SCPCs) derived from human-induced pluripotent stem cells (iPSCs) has beneficial effects in treating spinal cord injury (SCI). However, the presence of residual undifferentiated iPSCs among their differentiated progeny poses a high risk as these cells can develop teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs, no specific surface markers can identify them for subsequent removal. By profiling the size of SCPCs after a 10-day differentiation process, we found that the large-sized group contains significantly more cells expressing pluripotent markers. In this study, we used a sized-based, label-free separation using an inertial microfluidic-based device to remove tumor-risk cells. The device can reduce the number of undifferentiated cells from an SCPC population with high throughput (ie, >3 million cells/minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining, flow cytometry analysis, and colony culture assay. We demonstrated the capabilities of our technology to reduce the percentage of OCT4-positive cells. Our technology has great potential for the “downstream processing” of cell manufacturing workflow, ensuring better quality and safety of transplanted cells.

Purpose: To investigate the therapeutic potential of inducible pluripotent stem cell (hiPSC)-based vascular repair, we evaluated two vascular reparative cell populations, CD34+ cells derived from hiPSC (hiPSC-CD34+) and endothelial colony forming cells (ECFCs) derived from hiPSC (iPS-ECFCs), alone and in combination, in a type 2 diabetic (db/db) mouse model of DR. Methods: hiPSC-CD34+ cells (1 × 104) or iPSC- ECFCs (1 × 105) alone or in combination (1.1 × 105) were injected into the vitreous of immunosuppressed db/db mice with six months of established diabetes. One month post-injection, mice underwent electroretinography (ERG) and optical coherence tomography (OCT) to evaluate functional and structural retinal recovery with iPSC administration. Immunohistochemistry (IHC) was used to assess recruitment and incorporation of cells into the retinal vasculature. Retinas from the experimental groups were analyzed using Functional Proteomics via Reverse Phase Protein Array (RPPA). Results: Functional assessment via ERG demonstrated significant improvements in retinal response in the diabetic cohorts treated with either hiPSC-derived CD34+ cells or hiPSC-ECFCs. Retinal thickness, assessed by OCT, was restored to near-nondiabetic levels in mice treated with hiPSC-CD34+ cells alone and the combination group, whereas hiPSC-ECFCs alone did not significantly affect retinal thickness. One month following intravitreal injection, hiPSC-CD34+ cells were localized to perivascular regions, whereas hiPSC-ECFCs were observed to integrate directly into the retinal vasculature. RPPA analysis revealed interaction-significant changes, and this was interpreted as a combination-specific, non-additive host responses (m6A, PI3K–AKT–mTOR, glycolysis, endothelial junction pathways). Conclusions: The studies support that injection of hiPSC-CD34+ cells and hiPSC-ECFCs, both individually and in combination, showed benefit; however, iPSC combination-specific effects were identified by measurement of retinal thickness and by RPPA.

Human induced pluripotent stem cell (hiPSC) derived neurons are powerful tools to model disease biology in the drug development space. Here we leveraged a spectrum of neurophysiological tools to characterize iPSC-derived NGN2 neurons. Specifically, we applied these technologies to detect phenotypes associated with presynaptic dysfunction and rescue in NGN2 neurons lacking a synaptic vesicle associated protein MUNC18-1, encoded by syntaxin binding protein 1 gene (STXBP1). STXBP1 homozygous knock out NGN2 neurons lacked miniature post synaptic currents and demonstrated disrupted network bursting as assayed with multielectrode array and calcium imaging. Furthermore, knock out neurons released less glutamate into culture media, consistent with a presynaptic deficit. These synaptic phenotypes were rescued by reconstitution of STXBP1 protein by AAV transduction in a dose-dependent manner. Our results identify a complementary suite of physiological methods suitable to examine the modulation of synaptic transmission in human neurons.

Type 1 interferons (IFN-Is) exhibit significant antiviral, antitumor, and immunoregulatory properties, demonstrating substantial therapeutic potential. However, IFN-Is are pleiotropic cytokines, and the available data on their effect under specific pathological conditions are inconclusive. Furthermore, the systemic administration of IFN-Is can result in side effects. Generating cells that can migrate to the pathological focus and provide regulated local production of IFN-Is could overcome this limitation and provide a model for an in-depth analysis of the biological and therapeutic effects of IFN-Is. Induced pluripotent stem cells (iPSCs) are a valuable source of various differentiated cell types, including human immune cells. In this study, we describe the generation of genetically modified human iPSCs with doxycycline-controlled overexpression of interferon β (IFNB1). Three IFNB1-overexpressing iPSC lines (IFNB-iPSCs) and one control line expressing the transactivator M2rtTA (TA-iPSCs) were generated using the CRISPR/Cas9 technology. The pluripotency of the generated cell lines has been confirmed by the following: (i) cell morphology; (ii) the expression of the pluripotency markers OCT4, SOX2, TRA 1-60, and NANOG; and (iii) the ability to spontaneously differentiate into the derivatives of the three germ layers. Upon the addition of doxycycline, all IFNB-iPSCs upregulated IFNB1 expression at RNA (depending on the iPSC line, 126-816-fold) and protein levels. The IFNB-iPSCs and TA-iPSCs generated here represent a valuable cellular model for studying the effects of IFN-β on the activity and differentiation trajectories of different cell types, as well as for generating different types of cells with controllable IFN-β expression.

Artificial skin models have emerged as valuable tools for evaluating cosmetic ingredients and developing treatments for skin regeneration. Among them, 3D skin equivalent models (SKEs) using human primary skin cells are widely utilized and supported by standardized testing guidelines. However, primary cells face limitations such as restricted donor availability and challenges in conducting genotype-specific studies. To overcome these issues, recent approaches have focused on differentiating skin cells from human-induced pluripotent stem cells (hiPSCs). In this study, we developed a protocol to differentiate high-purity skin cells, such as fibroblasts (hFIBROs) and keratinocytes (hKERAs), from hiPSCs. To construct the hiPSC-derived SKE (hiPSC-SKE), a dermis was first formed by culturing a collagen and hFIBROs mixture within an insert. Subsequently, hKERAs were seeded onto the dermis, and keratinization was induced under air-liquid culture conditions to establish an epidermis. Histological analysis with hematoxylin and eosin staining confirmed that the hiPSC-SKE recapitulated the layered architecture of native human skin and expressed appropriate epidermal and dermal markers. Moreover, exposure to Triton X-100, a known skin irritant, led to marked epidermal damage and significantly reduced cell viability, validating the model’s functional responsiveness. These findings indicate that the hiPSC-SKE model represents a promising alternative for various skin-related applications, including the replacement of animal testing.

BackgroundSevere peripheral nerve damage always requires surgical treatment. Autologous nerve transplantation is a standard treatment, but it is not sufficient due to length limitations and extended surgical time. Even with the available artificial nerves, there is still large room for improvement in their therapeutic effects. Novel treatments for peripheral nerve injury are greatly expected.MethodsUsing a specialized microfluidic device, we generated artificial neurite bundles from human iPSC-derived motor and sensory nerve organoids. We developed a new technology to isolate cell-free neurite bundles from spheroids. Transplantation therapy was carried out for large nerve defects in rat sciatic nerve with novel artificial nerve conduit filled with lineally assembled sets of human neurite bundles. Quantitative comparisons were performed over time to search for the artificial nerve with the therapeutic effect, evaluating the recovery of motor and sensory functions and histological regeneration. In addition, a multidimensional unbiased gene expression profiling was carried out by using next-generation sequencing.ResultAfter transplantation, the neurite bundle-derived artificial nerves exerted significant therapeutic effects, both functionally and histologically. Remarkably, therapeutic efficacy was achieved without immunosuppression, even in xenotransplantation. Transplanted neurite bundles fully dissolved after several weeks, with no tumor formation or cell proliferation, confirming their biosafety. Posttransplant gene expression analysis highlighted the immune system’s role in recovery.ConclusionThe combination of newly developed microfluidic devices and iPSC technology enables the preparation of artificial nerves from organoid-derived neurite bundles in advance for future treatment of peripheral nerve injury patients. A promising, safe, and effective peripheral nerve treatment is now ready for clinical application.Supplementary InformationThe online version contains supplementary material available at 10.1186/s41232-024-00319-4.