A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism.

Memory T cells demonstrate superior in vivo persistence and antitumor efficacy. However, methods for manufacturing less differentiated T cells are not yet well-established. Here, we show that producing chimeric antigen receptor (CAR)-T cells using berbamine (BBM), a natural compound found in the Chinese herbal medicine Berberis amurensis, enhances the antitumor efficacy of CAR-T cells. BBM is identified through cell-based screening of chemical compounds using induced pluripotent stem cell-derived T cells, leading to improved viability with a memory T cell phenotype. Transcriptomics and metabolomics using stem cell memory T cells reveal that BBM broadly enhances lipid metabolism. Furthermore, the addition of BBM downregulates the phosphorylation of p38 mitogen-activated protein kinase and enhanced mitochondrial respiration. CD19-CAR-T cells cultured with BBM also extend the survival of leukaemia mouse models due to their superior in vivo persistence. This technology offers a straightforward approach to enhancing the antitumor efficacy of CAR-T cells.

Characterization of Novel Cell-Adhesive Peptides for Biomaterial Development.

In previous studies, my group developed cell-adhesive peptide-polysaccharide complexes as biomaterials for tissue engineering. Having a wide variety of cell-adhesive peptides is important as the biological functions of peptide-polysaccharide complexes are highly dependent on the biological activity of peptides. This paper reviews the biological activities of two types of recently characterized cell-adhesive peptides. The first is peptides rich in basic amino acids originating from octaarginine. We analyzed the relationships between the amino acid composition of basic peptides and cell adhesion, elongation, and proliferation and identified the most suitable peptide for cell culture. The second was arginine-glycine-aspartic acid (RGD)-containing peptides that promote the adhesion of induced pluripotent stem cells (iPSCs). We identified the RGD-surrounding sequences necessary for iPSC adhesion, clarified the underlying mechanism, and improved cell adhesion by modifying the structure-activity relationships. The novel cell-adhesive peptides identified in our previous studies may aid in the development of novel peptide-based biomaterials.

OliTag-seq enhances in cellulo detection of CRISPR-Cas9 off-targets.

The potential for off-target mutations is a critical concern for the therapeutic application of CRISPR-Cas9 gene editing. Current detection methodologies, such as GUIDE-seq, exhibit limitations in oligonucleotide integration efficiency and sensitivity, which could hinder their utility in clinical settings. To address these issues, we introduce OliTag-seq, an in-cellulo assay specifically engineered to enhance the detection of off-target events. OliTag-seq employs a stable oligonucleotide for precise break tagging and an innovative triple-priming amplification strategy, significantly improving the scope and accuracy of off-target site identification. This method surpasses traditional assays by providing comprehensive coverage across various sgRNAs and genomic targets. Our research particularly highlights the superior sensitivity of induced pluripotent stem cells (iPSCs) in detecting off-target mutations, advocating for using patient-derived iPSCs for refined off-target analysis in therapeutic gene editing. Furthermore, we provide evidence that prolonged Cas9 expression and transient HDAC inhibitor treatments enhance the assay's ability to uncover off-target events. OliTag-seq merges the high sensitivity typical of in vitro assays with the practical application of cellular contexts. This approach significantly improves the safety and efficacy profiles of CRISPR-Cas9 interventions in research and clinical environments, positioning it as an essential tool for the precise assessment and refinement of genome editing applications.

An ultrathin membrane mediates tissue-specific morphogenesis and barrier function in a human kidney chip.

Organ-on-chip (OOC) systems are revolutionizing tissue engineering by providing dynamic models of tissue structure, organ-level function, and disease phenotypes using human cells. However, nonbiological components of OOC devices often limit the recapitulation of in vivo-like tissue-tissue cross-talk and morphogenesis. Here, we engineered a kidney glomerulus-on-a-chip that recapitulates glomerular morphogenesis and barrier function using a biomimetic ultrathin membrane and human-induced pluripotent stem cells. The resulting chip comprised a proximate epithelial-endothelial tissue interface, which reconstituted the selective molecular filtration function of healthy and diseased kidneys. In addition, fenestrated endothelium was successfully induced from human pluripotent stem cells in an OOC device, through in vivo-like paracrine signaling across the ultrathin membrane. Thus, this device provides a dynamic tissue engineering platform for modeling human kidney-specific morphogenesis and function, enabling mechanistic studies of stem cell differentiation, organ physiology, and pathophysiology.

Maturation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) on polycaprolactone and polyurethane nanofibrous mats.

Investigating the potential of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) in in vitro heart models is essential to develop cardiac regenerative medicine. iPSC-CMs are immature with a fetal-like phenotype relative to cardiomyocytes in vivo. Literature indicates methods for enhancing the structural maturity of iPSC-CMs. Among these strategies, nanofibrous scaffolds offer more accurate mimicry of the functioning of cardiac tissue structures in the human body. However, further research is needed on the use of nanofibrous mats to understand their effects on iPSC-CMs. Our research aimed to evaluate the suitability of poly(ε-caprolactone) (PCL) and polyurethane (PU) nanofibrous mats with different elasticities as materials for the maturation of iPSC-CMs. Analysis of cell morphology and orientation and the expression levels of selected genes and proteins were performed to determine the effect of the type of nanofibrous mats on the maturation of iPSC-CMs after long-term (10-day) culture. Understanding the impact of 3D structural properties in in vitro cardiac models on induced pluripotent stem cell-derived cardiomyocyte maturation is crucial for advancing cardiac tissue engineering and regenerative medicine because it can help optimize conditions for obtaining more mature and functional human cardiomyocytes.

Biomass-derived carbon dots as fluorescent quantum probes to visualize and modulate inflammation.

Quantum dots, which won the Nobel Prize in Chemistry, have recently gained significant attention in precision medicine due to their unique properties, such as size-tunable emission, high photostability, efficient light absorption, and vibrant luminescence. Consequently, there is a growing demand to identify new types of quantum dots from various sources and explore their potential applications as stimuli-responsive biosensors, biomolecular imaging probes, and targeted drug delivery agents. Biomass-waste-derived carbon quantum dots (CQDs) are an attractive alternative to conventional QDs, which often require expensive and toxic precursors, as they offer several merits in eco-friendly synthesis, preparation from renewable sources, and cost-effective production. In this study, we evaluated three CQDs derived from biomass waste for their potential application as non-toxic bioimaging agents in various cell lines, including human dermal fibroblasts, HeLa, cardiomyocytes, induced pluripotent stem cells, and an in-vivo medaka fish (Oryzias latipes) model. Confocal microscopic studies revealed that CQDs could assist in visualizing inflammatory processes in the cells, as they were taken up more by cells treated with tumor necrosis factor-α than untreated cells. In addition, our quantitative real-time PCR gene expression analysis has revealed that citric acid-based CQDs can potentially reduce inflammatory markers such as Interleukin-6. Our studies suggest that CQDs have potential as theragnostic agents, which can simultaneously identify and modulate inflammatory markers and may lead to targeted therapy for immune system-associated diseases.

Repression of developmental transcription factor networks triggers aging-associated gene expression in human glial progenitor cells.

Human glial progenitor cells (hGPCs) exhibit diminished expansion competence with age, as well as after recurrent demyelination. Using RNA-sequencing to compare the gene expression of fetal and adult hGPCs, we identify age-related changes in transcription consistent with the repression of genes enabling mitotic expansion, concurrent with the onset of aging-associated transcriptional programs. Adult hGPCs develop a repressive transcription factor network centered on MYC, and regulated by ZNF274, MAX, IKZF3, and E2F6. Individual over-expression of these factors in iPSC-derived hGPCs lead to a loss of proliferative gene expression and an induction of mitotic senescence, replicating the transcriptional changes incurred during glial aging. miRNA profiling identifies the appearance of an adult-selective miRNA signature, imposing further constraints on the expansion competence of aged GPCs. hGPC aging is thus associated with acquisition of a MYC-repressive environment, suggesting that suppression of these repressors of glial expansion may permit the rejuvenation of aged hGPCs.

Interactions of the Immune System with Human Kidney Organoids.

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids.

BACKGROUND: Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin. METHODS: Differentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy. RESULTS: We observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles. CONCLUSION: Our data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

Graft cell expansion from hiPSC-RPE strip after transplantation in primate eyes with or without RPE damage.

Clinical studies using suspensions or sheets of human pluripotent cell-derived retinal pigment epithelial cells (hiPSC-RPE) have been conducted globally for diseases such as age-related macular degeneration. Despite being minimally invasive, cell suspension transplantation faces challenges in targeted cell delivery and frequent cell leakage. Conversely, although the RPE sheet ensures targeted delivery with correct cell polarity, it requires invasive surgery, and graft preparation is time-consuming. We previously reported hiPSC-RPE strips as a form of quick cell aggregate that allows for reliable cell delivery to the target area with minimal invasiveness. In this study, we used a microsecond pulse laser to create a local RPE ablation model in cynomolgus monkey eyes. The hiPSC-RPE strips were transplanted into the RPE-ablated and intact sites. The hiPSC-RPE strip stably survived in all transplanted monkey eyes. The expansion area of the RPE from the engrafted strip was larger at the RPE injury site than at the intact site with no tumorigenic growth. Histological observation showed a monolayer expansion of the transplanted RPE cells with the expression of MERTK apically and collagen type 4 basally. The hiPSC-RPE strip is considered a beneficial transplantation option for RPE cell therapy.

Establishment of Transgene-Free Porcine Induced Pluripotent Stem Cells.

Although protocols to generate authentic transgene-free mouse and human induced pluripotent stem cells (iPSCs) are now well established, standard methods for reprogramming porcine somatic cells still suffer from low efficiency and transgene retention. The Basic Protocol describes reprogramming procedures to establish transgene-free porcine iPSCs (PiPSCs) from porcine fibroblasts. This method uses episomal plasmids encoding POU5F1, SOX2, NANOG, KLF4, SV40LT, c-MYC, LIN28A, and microRNA-302/367, combined with an optimized medium, to establish PiPSC lines. Support protocols describe the establishment and characterization of clonal PiPSC lines, as well as the preparation of feeder cells and EBNA1 mRNA. This optimized, step-by-step approach tailored to this species enables the efficient derivation of PiPSCs in ∼4 weeks. The establishment of transgene-free PiPSCs provides a new and valuable model for studies of larger mammalian species' development, disease, and regenerative biology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Reprogramming of porcine fibroblasts with episomal plasmids Support Protocol 1: Preparation of mouse embryonic fibroblasts for feeder layer Support Protocol 2: Preparation of in vitro-transcribed EBNA1 mRNA Support Protocol 3: Establishment of clonal porcine induced pluripotent stem cell (PiPSC) lines Support Protocol 4: PiPSC characterization: Genomic DNA PCR and RT-PCR Support Protocol 5: PiPSC characterization: Immunostaining.

Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays.

The neuromodulatory effects of focused ultrasound (FUS) have been demonstrated in animal models, and FUS has been used successfully to treat movement and psychiatric disorders in humans. However, despite the success of FUS, the mechanism underlying its effects on neurons remains poorly understood, making treatment optimization by tuning FUS parameters difficult. To address this gap in knowledge, we studied human neurons in vitro using neurons cultured from human-induced pluripotent stem cells (HiPSCs). Using HiPSCs allows for the study of human-specific neuronal behaviors in both physiologic and pathologic states. This report presents a protocol for using a high-throughput system that enables the monitoring and quantification of the neuromodulatory effects of FUS on HiPSC neurons. By varying the FUS parameters and manipulating the HiPSC neurons through pharmaceutical and genetic modifications, researchers can evaluate the neural responses and elucidate the neuro-modulatory effects of FUS on HiPSC neurons. This research could have significant implications for the development of safe and effective FUS-based therapies for a range of neurological and psychiatric disorders.

Low-adhesion culture selection for human iPS cell-derived cardiomyocytes.

Despite progress in generating cardiomyocytes from pluripotent stem cells, these populations often include non-contractile cells, necessitating cardiomyocyte selection for experimental purpose. This study explores a novel cardiomyocyte enrichment mechanism: low-adhesion culture selection. The cardiac cells derived from human induced pluripotent stem cells were subjected to a coating-free low-adhesion culture using bovine serum albumin and high molecular weight dextran sulfate. This approach effectively increased the population of cardiac troponin T-positive cardiomyocytes. Similar results were obtained with commercially available low-adhesion culture dishes. Subsequently, we accessed the practicality of selection of cardiomyocytes using this phenomenon by comparing it with established methods such as glucose-free culture and selection based on puromycin resistance genes. The cardiomyocytes enriched through low-adhesion culture selection maintained autonomous pulsation and responsiveness to beta-stimuli. Moreover, no significant differences were observed in the expression of genes related to subtype commitment and maturation when compared to other selection methods. In conclusion, cardiomyocytes derived from pluripotent stem cells were more low-adhesion culture resistant than their accompanying non-contractile cells, and low-adhesion culture is an alternative method for selection of pluripotent stem cell-derived cardiomyocytes.

Three-dimensional liquid metal-based neuro-interfaces for human hippocampal organoids.

Human hippocampal organoids (hHOs) derived from human induced pluripotent stem cells (hiPSCs) have emerged as promising models for investigating neurodegenerative disorders, such as schizophrenia and Alzheimer's disease. However, obtaining the electrical information of these free-floating organoids in a noninvasive manner remains a challenge using commercial multi-electrode arrays (MEAs). The three-dimensional (3D) MEAs developed recently acquired only a few neural signals due to limited channel numbers. Here, we report a hippocampal cyborg organoid (cyb-organoid) platform coupling a liquid metal-polymer conductor (MPC)-based mesh neuro-interface with hHOs. The mesh MPC (mMPC) integrates 128-channel multielectrode arrays distributed on a small surface area (~2*2 mm). Stretchability (up to 500%) and flexibility of the mMPC enable its attachment to hHOs. Furthermore, we show that under Wnt3a and SHH activator induction, hHOs produce HOPX+ and PAX6+ progenitors and ZBTB20+PROX1+ dentate gyrus (DG) granule neurons. The transcriptomic signatures of hHOs reveal high similarity to the developing human hippocampus. We successfully detect neural activities from hHOs via the mMPC from this cyb-organoid. Compared with traditional planar devices, our non-invasive coupling offers an adaptor for recording neural signals from 3D models.

Distinct calcium sources regulate temporal profiles of NMDAR and mGluR-mediated protein synthesis.

Calcium signaling is integral for neuronal activity and synaptic plasticity. We demonstrate that the calcium response generated by different sources modulates neuronal activity-mediated protein synthesis, another process essential for synaptic plasticity. Stimulation of NMDARs generates a protein synthesis response involving three phases-increased translation inhibition, followed by a decrease in translation inhibition, and increased translation activation. We show that these phases are linked to NMDAR-mediated calcium response. Calcium influx through NMDARs elicits increased translation inhibition, which is necessary for the successive phases. Calcium through L-VGCCs acts as a switch from translation inhibition to the activation phase. NMDAR-mediated translation activation requires the contribution of L-VGCCs, RyRs, and SOCE. Furthermore, we show that IP3-mediated calcium release and SOCE are essential for mGluR-mediated translation up-regulation. Finally, we signify the relevance of our findings in the context of Alzheimer's disease. Using neurons derived from human fAD iPSCs and transgenic AD mice, we demonstrate the dysregulation of NMDAR-mediated calcium and translation response. Our study highlights the complex interplay between calcium signaling and protein synthesis, and its implications in neurodegeneration.

Progresses in overcoming the limitations of in vitro erythropoiesis using human induced pluripotent stem cells.

Researchers have attempted to generate transfusable oxygen carriers to mitigate RBC supply shortages. In vitro generation of RBCs using stem cells such as hematopoietic stem and progenitor cells (HSPCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) has shown promise. Specifically, the limited supplies of HSPCs and ethical issues with ESCs make iPSCs the most promising candidate for in vitro RBC generation. However, researchers have encountered some major challenges when using iPSCs to produce transfusable RBC products, such as enucleation and RBC maturation. In addition, it has proven difficult to manufacture these products on a large scale. In this review, we provide a brief overview of erythropoiesis and examine endeavors to recapitulate erythropoiesis in vitro using various cell sources. Furthermore, we explore the current obstacles and potential solutions aimed at enabling the large-scale production of transfusable RBCs in vitro.

Cardiac differentiation of chimpanzee induced pluripotent stem cell lines with different subspecies backgrounds.

The comparative analysis between humans and non-human primates is an instrumental approach for elucidating the evolutional traits and disease propensity of humans. However, in primates, cross-species analyses of their developmental events have encountered constraints because of the ethical and technical limitations in available sample collection, sequential monitoring, and manipulations. In an endeavor to surmount these challenges, in recent years, induced pluripotent stem cells (iPSCs) have garnered escalating interest as an in vitro tool for cross-species analyses between humans and non-human primates. Meanwhile, compared to humans, there is less information on in vitro differentiation of non-human primate iPSCs, and their genetic diversity including subspecies may cause different eligibility to in vitro differentiation methods. Therefore, antecedent to embarking on a comparative analysis to humans, it is a prerequisite to develop the efficacious methodologies for in vitro differentiation regardless of the intraspecies genetic background in non-human primates. In this study, we executed the in vitro differentiation of cardiomyocytes from four chimpanzee iPSC lines with different subspecies and individual backgrounds. To induce cardiomyocytes from chimpanzee iPSCs, we evaluated our methodology for in vitro cardiac differentiation of human iPSCs. Eventually, with minor alterations, our cardiac differentiation method was applicable to all chimpanzee iPSC lines tested as assessed by the expression of cardiac marker genes and the beating ability. Hence, our in vitro differentiation method will advance iPSC-based research of chimpanzee cardiac development and also hold possible utility to cross-species analyses among primate species.

Knockdown H19 Accelerated iPSCs Reprogramming through Epigenetic Modifications and Mesenchymal-to-Epithelial Transition.

H19 is an essential imprinted gene that is expressed to govern normal embryonic development. During reprogramming, the parental pronuclei have asymmetric reprogramming capacities and the critical reprogramming factors predominantly reside in the male pronucleus. After inhibiting the expression of H19 and Gtl2, androgenetic haploid ESCs (AG-haESCs) can efficiently and stably support the generation of healthy SC pups at a rate of ~20%, and double-knockout parthenogenetic haESCs can also produce efficiently. Induced pluripotent stem (iPS) cell reprogramming is thought to have a characteristic epigenetic pattern that is the reverse of its developmental potential; however, it is unclear how H19 participates in iPS cell reprogramming. Here, we showed that the expression of H19 was transiently increased during iPSC reprogramming. H19 knockdown resulted in greater reprogramming efficiency. The genes associated with pluripotency showed enhanced expression during the early reprogramming process, and the Oct4 promoter was demethylated by bisulfite genomic sequencing analysis. Moreover, expression analysis revealed that the mesenchymal master regulators associated with epithelial-to-mesenchymal transition (EMT) were downregulated during reprogramming in H19 knockdown. These findings provide functional insight into the role of H19 as a barrier to the early reprogramming process.

A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors.

Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein-Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors.

Altered Expression of PDE4 Genes in Schizophrenia: Insights from a Brain and Blood Sample Meta-Analysis and iPSC-Derived Neurons.

Schizophrenia symptomatology includes negative symptoms and cognitive impairment. Several studies have linked schizophrenia with the PDE4 family of enzymes due to their genetic association and function in cognitive processes such as long-term potentiation. We conducted a systematic gene expression meta-analysis of four PDE4 genes (PDE4A-D) in 10 brain sample datasets (437 samples) and three blood sample datasets (300 samples). Subsequently, we measured mRNA levels in iPSC-derived hippocampal dentate gyrus neurons generated from fibroblasts of three groups: healthy controls, healthy monozygotic twins (MZ), and their MZ siblings with schizophrenia. We found downregulation of PDE4B in brain tissues, further validated by independent data of the CommonMind consortium (515 samples). Interestingly, the downregulation signal was present in a subgroup of the patients, while the others showed no differential expression or even upregulation. Notably, PDE4A, PDE4B, and PDE4D exhibited upregulation in iPSC-derived neurons compared to healthy controls, whereas in blood samples, PDE4B was found to be upregulated while PDE4A was downregulated. While the precise mechanism and direction of altered PDE4 expression necessitate further investigation, the observed multilevel differential expression across the brain, blood, and iPSC-derived neurons compellingly suggests the involvement of PDE4 genes in the pathophysiology of schizophrenia.