Hydroethanolic extract of Cirsium setidens ameliorates doxorubicin-induced cardiotoxicity by AMPK-PGC-1α-SOD-mediated mitochondrial protection.

BACKGROUND: Doxorubicin (DOX) is an effective anticancer agent. However, the clinical outcomes of DOX-based therapies are severely hampered by their significant cardiotoxicity. PURPOSE: We investigated the beneficial effects of an ethanol extract of Cirsium setidens (CSE) on DOX-induced cardiomyotoxicity (DICT). METHODS: UPLC-TQ/MS analysis was used to identify CSE metabolite profiles. H9c2 rat cardiomyocytes and MDA-MB-231 human breast cancer cells were used to evaluate the effects of CSE on DICT-induced cell death. To elucidate the mechanism underlying it, AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma co-activator l-alpha (PGC1-α), nuclear respiratory factor 1 (NRF1), NRF2, superoxide dismutase (SOD1), and SOD2 expression was detected using western blot analysis. The oxygen consumption rate (OCR), cellular ROS, and mitochondrial membrane potential were measured. Finally, we confirmed the cardioprotective effect of CSE against DICT in both C57BL/6 mice and human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) by observing various parameters, such as electrophysiological changes, cardiac fibrosis, and cardiac cell death. RESULTS: Chlorogenic acid and nicotiflorin were the major compounds in CSE. Our data demonstrated that CSE blocked DOX-induced cell death of H9c2 cells without hindrance of its apoptotic effects on MDA-MB-231 cells. DOX-induced defects of OCR and mitochondrial membrane potential were recovered in a CSE through upregulation of the AMPK-PGC1-α-NRF1 signaling pathway. CSE accelerated NRF1 translocation to the nucleus, increased SOD activity, and consequently blocked apoptosis in H9c2 cells. In mice treated with 400 mg/kg CSE for 4 weeks, electrocardiogram data, creatine kinase and lactate dehydrogenase levels in the serum, and cardiac fibrosis, were improved. Moreover, various electrophysiological features indicative of cardiac function were significantly enhanced following the CSE treatment of hiPSCCMs. CONCLUSION: Our findings demonstrate CSE that ameliorates DICT by protecting mitochondrial dysfunction via the AMP- PGC1α-NRF1 axis, underscoring the therapeutic potential of CSE and its underlying molecular pathways, setting the stage for future investigations into its clinical applications.

Differentiation of Human-induced Pluripotent Stem Cell-derived Dental Stem Cells through Epithelial-Mesenchymal Interaction.

Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental regeneration. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchymal-epithelial interactions play an essential role in dental development. We reported the establishment of hiPSCs-derived dental epithelial-like cell (EPI-iPSCs), but hiPSCs-derived dental mesenchymal stem cells (MSCs) have not yet been reported. This study was conducted to establish hiPSCs-derived MSCs and to differentiate them into dental cells with EPI-iPSCs. Considering that dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. To differentiate hiPSCs into MSCs through neural crest formation, established hiPSCs were cultured and differentiated with PA6 stromal cells and differentiated hiPSCs formed neurospheres on ultralow-attachment plates. Neurospheres were differentiated into MSCs in serum-supplemented medium. Neural crest-mediated MSCs (NC-MSCs) continuously showed typical MSC morphology and expressed MSC markers. After 8 days of odontogenic induction, the expression levels of odontogenic/mineralization-related genes and dentin sialophosphoprotein (DSPP) proteins were increased in the NC-MSCs alone group in the absence of coculturing with dental epithelial cells. The NC-MSCs and EPI-iPSCs coculture groups showed high expression levels of amelogenesis/odontogenic/mineralization-related genes and DSPP proteins. Furthermore, the NC-MSCs and EPI-iPSCs coculture group yielded calcium deposits earlier than the NC-MSCs alone group. These results indicated that established NC-MSCs from hiPSCs have dental differentiation capacity with dental epithelial cells. In addition, it was confirmed that hiPSCs-derived dental stem cells could be a novel cell source for autologous dental regeneration.

Induced pluripotent stem cells-based disease modeling, drug screening, clinical trials, and reverse translational research for amyotrophic lateral sclerosis.

It has been more than 10 years since the hopes for disease modeling and drug discovery using induced pluripotent stem cell (iPSC) technology boomed. Recently, clinical trials have been conducted with drugs identified using this technology, and some promising results have been reported. For amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, several groups have identified candidate drugs, ezogabine (retigabine), bosutinib, and ropinirole, using iPSCs-based drug discovery, and clinical trials using these drugs have been conducted, yielding interesting results. In our previous study, an iPSCs-based drug repurposing approach was utilized to show the potential of ropinirole hydrochloride (ROPI) in reducing ALS-specific pathological phenotypes. Recently, a phase 1/2a trial was conducted to investigate the effects of ropinirole on ALS further. This double-blind, randomized, placebo-controlled study confirmed the safety and tolerability of and provided evidence of its ability to delay disease progression and prolong the time to respiratory failure in ALS patients. Furthermore, in the reverse translational research, in vitro characterization of patient-derived iPSCs-motor neurons (MNs) mimicked the therapeutic effects of ROPI in vivo, suggesting the potential application of this technology to the precision medicine of ALS. Interestingly, RNA-seq data showed that ROPI treatment suppressed the sterol regulatory element-binding protein 2-dependent cholesterol biosynthesis pathway. Therefore, this pathway may be involved in the therapeutic effect of ROPI on ALS. The possibility that this pathway may be involved in the therapeutic effect of ALS was demonstrated. Finally, new future strategies for ALS using iPSCs technology will be discussed in this paper.

Safety pharmacology 2023 and implementation of the ICH E14/S7B Q&A guidance document.

This editorial prefaces the annual themed issue on safety pharmacology (SP) methods published since 2004 in the Journal of Pharmacological and Toxicological Methods (JPTM). We highlight here the content derived from the recent 2022 Safety Pharmacology Society (SPS) and Canadian Society of Pharmacology and Therapeutics (CSPT) joint meeting held in Montreal, Quebec, Canada. The meeting also generated 179 abstracts (reproduced in the current volume of JPTM). As in previous years the manuscripts reflect various areas of innovation in SP including a comparison of the sensitivity of cross-over and parallel study designs for QTc assessment, use of human-induced pluripotent stem cell (hi-PSC) neuronal cell preparations for use in neuropharmacological safety screening, and hiPSC derived cardiac myocytes in assessing inotropic adversity. With respect to the latter, we anticipate the emergence of a large data set of positive and negative controls that will test whether the imperative to miniaturize, humanize and create a high throughput process is offset by any loss of precision and accuracy.

Generating Human Glomeruli from Pluripotent Stem Cells for Disease Modelling and Drug Screening.

The functional unit of human kidney is the nephron. This structure is composed of a glomerulus, connected to a tubule that drains into a collecting duct. The cells which make up the glomerulus are critically important to the appropriate function of this specialised structure. Damage to glomerular cells, particularly the podocytes, is the primary cause of numerous kidney diseases. However, access to and the subsequent culture of human glomerular cells is limited. As such, the ability to generate human glomerular cell types from induced pluripotent stem cells (iPSCs) at scale has garnered great interest. Here, we describe a method to isolate, culture and study 3D human glomeruli from induced pluripotent stem cell (iPSC)-derived kidney organoids in vitro. These 3D glomeruli retain appropriate transcriptional profiles and can be generated from any individual. As isolated glomeruli, they have applicability for disease modelling and drug discovery.

Differentiation, Transcriptomic Profiling, and Calcium Imaging of Human Hypothalamic Neurons.

Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Human pluripotent stem cells (hPSCs) can be differentiated into many types of hypothalamic neurons, progenitors, and glia. This updated unit includes published studies and protocols with new advances in the differentiation, maturation, and interrogation by transcriptomic profiling and calcium imaging of human hypothalamic cell populations. Specifically, new methods to freeze and thaw hypothalamic progenitors after they have been patterned and before substantial neurogenesis has occurred are provided that will facilitate experimental flexibility and planning. Also included are updated recipes and protocols for neuronal maturation, with details on the equipment and methods for examining their transcriptomic response and cell-autonomous properties in culture in the presence of synaptic blockers. Together, these protocols facilitate the adoption and use of this model system for fundamental biological discovery and therapeutic translation to human diseases such as obesity, diabetes, sleep disorders, infertility, and chronic stress. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hPSC maintenance Basic Protocol 2: Hypothalamic neuron differentiation Support Protocol 1: Cortical neuron (control) differentiation Basic Protocol 3: Neuronal maturation Support Protocol 2: Cryopreservation and thawing of neuronal progenitors Support Protocol 3: Quality control: Confirmation of hypothalamic patterning and neurogenesis Support Protocol 4: Bulk RNA sequencing of hypothalamic cultures Basic Protocol 4: Calcium imaging of hypothalamic neurons using Fura-2 AM Alternate Protocol: Calcium imaging of green fluorescent hypothalamic neurons using Rhod-3 AM.

Nutritional requirements of human induced pluripotent stem cells.

The nutritional requirements for human induced pluripotent stem cell (hiPSC) growth have not been extensively studied. Here, building on our prior work that established the suitable non-basal medium components for hiPSC growth, we develop a simplified basal medium consisting of just 39 components, demonstrating that many ingredients of DMEM/F12 are either not essential or are at suboptimal concentrations. This new basal medium along with the supplement, which we call BMEM, enhances the growth rate of hiPSCs over DMEM/F12-based media, supports derivation of multiple hiPSC lines, and allows differentiation to multiple lineages. hiPSCs cultured in BMEM consistently have enhanced expression of undifferentiated cell markers such as POU5F1 and NANOG, along with increased expression of markers of the primed state and reduced expression of markers of the naive state. This work describes titration of the nutritional requirements of human pluripotent cell culture and identifies that suitable nutrition enhances the pluripotent state.

Generation of hypothalamic neural stem cell-like cells in vitro from human pluripotent stem cells.

When damaged, restoring the function of the hypothalamus is currently impossible. It is unclear whether neural stem cells exist in the hypothalamus. Studies have reported that adult rodent tanycytes around the third ventricle function as hypothalamic neural stem cell-like cells. However, it is currently impossible to collect periventricular cells from humans. We attempted to generate hypothalamic neural stem cell-like cells from human embryonic stem cells (ESCs). We focused on retina and anterior neural fold homeobox (RAX) because its expression is gradually restricted to tanycytes during the late embryonic stage. We differentiated RAX::VENUS knockin human ESCs (hESCs) into hypothalamic organoids and sorted RAX+ cells from mature organoids. The isolated RAX+ cells formed neurospheres and exhibited self-renewal and multipotency. Neurogenesis was observed when neurospheres were transplanted into the mouse hypothalamus. We isolated RAX+ hypothalamic neural stem cell-like cells from wild-type human ES organoids. This is the first study to differentiate human hypothalamic neural stem cell-like cells from pluripotent stem cells.

Immortalized hippocampal astrocytes from 3xTg-AD mice, a new model to study disease-related astrocytic dysfunction: a comparative review.

Alzheimer's disease (AD) is characterized by complex etiology, long-lasting pathogenesis, and cell-type-specific alterations. Currently, there is no cure for AD, emphasizing the urgent need for a comprehensive understanding of cell-specific pathology. Astrocytes, principal homeostatic cells of the central nervous system, are key players in the pathogenesis of neurodegenerative diseases, including AD. Cellular models greatly facilitate the investigation of cell-specific pathological alterations and the dissection of molecular mechanisms and pathways. Tumor-derived and immortalized astrocytic cell lines, alongside the emerging technology of adult induced pluripotent stem cells, are widely used to study cellular dysfunction in AD. Surprisingly, no stable cell lines were available from genetic mouse AD models. Recently, we established immortalized hippocampal astroglial cell lines from amyloid-ß precursor protein/presenilin-1/Tau triple-transgenic (3xTg)-AD mice (denominated as wild type (WT)- and 3Tg-iAstro cells) using retrovirus-mediated transduction of simian virus 40 large T-antigen and propagation without clonal selection, thereby maintaining natural heterogeneity of primary cultures. Several groups have successfully used 3Tg-iAstro cells for single-cell and omics approaches to study astrocytic AD-related alterations of calcium signaling, mitochondrial dysfunctions, disproteostasis, altered homeostatic and signaling support to neurons, and blood-brain barrier models. Here we provide a comparative overview of the most used models to study astrocytes in vitro, such as primary culture, tumor-derived cell lines, immortalized astroglial cell lines, and induced pluripotent stem cell-derived astrocytes. We conclude that immortalized WT- and 3Tg-iAstro cells provide a non-competitive but complementary, low-cost, easy-to-handle, and versatile cellular model for dissection of astrocyte-specific AD-related alterations and preclinical drug discovery.

Electroacupuncture and human iPSC-derived small extracellular vesicles regulate the gut microbiota in ischemic stroke via the brain-gut axis.

Electroacupuncture (EA) and induced pluripotent stem cell (iPSC)-derived small extracellular vesicles (iPSC-EVs) have substantial beneficial effects on ischemic stroke. However, the detailed mechanisms remain unclear. Here, we explored the mechanisms underlying the regulation of EA and iPSC-EVs in the microbiome-gut-brain axis (MGBA) after ischemic stroke. Ischemic stroke mice (C57BL/6) were subjected to middle cerebral artery occlusion (MCAO) or Sham surgery. EA and iPSC-EVs treatments significantly improved neurological function and neuronal and intestinal tract injury, downregulated the levels of IL-17 expression and upregulated IL-10 levels in brain and colon tissue after cerebral ischemia-reperfusion. EA and iPSC-EVs treatments also modulated the microbiota composition and diversity as well as the differential distribution of species in the intestines of the mice after cerebral ischemia-reperfusion. Our results demonstrated that EA and iPSC-EVs treatments regulated intestinal immunity through MGBA regulation of intestinal microbes, reducing brain and colon damage following cerebral ischemia and positively impacting the outcomes of ischemic stroke. Our findings provide new insights into the application of EA combined with iPSC-EVs as a treatment for ischemic stroke.

Investigating the effect of Shenmai injection on cardiac electrophysiology and calcium signaling using human-induced pluripotent stem cell-derived cardiomyocytes.

Traditional Chinese medicine injection (TCMI) refers to the use of modern technology to make Chinese patent medicines in injectable forms, which shorten the onset time of the traditional Chinese medicine (TCM). Although there have been clinical cases in which Shenmai injection (SMI) was used to treat cardiovascular diseases (CVDs), there are no pharmacological experiments that investigate the efficacy of the drug in vitro or the underlying mechanisms. Aim of the study: We aimed to systemically evaluate the efficacy and investigate the mechanisms of SMI in modulating electrophysiology and calcium (Ca2+) signaling using a microelectrode array (MEA) and a genetically encoded Ca2+ indicator, GCaMP6s, respectively, in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Materials and methods: A MEA system was employed to record field potentials (FPs) in hiPSC-CMs. The QT interval is corrected by the RR interval, the reciprocal of the beating rate. GCaMP6s was used to measure Ca2+ signaling in hiPSC-CMs. Meanwhile, the transcriptome changes in hiPSC-CMs treated with 2% SMI were examined using RNAseq. In addition, the ingredients of SMI were investigated using liquid chromatography-mass spectrometry (LC-MS). Results: It was found that 0.5%, 1%, and 2% (v/v) SMIs could increase corrected QT (QTc) but did not change other FP parameters. GCaMP6s was successfully applied to measure the chronic function of SMI. The full width at half maximum (FWHM), rise time, and decay time significantly decreased after treatment with SMI for 1 h and 24 h, whereas an increased Ca2+ transient frequency was observed. Conclusions: We first used the Ca2+ indicator to measure the chronic effects of TCM. We found that SMI treatment can modulate electrophysiology and calcium signaling and regulate oxidative phosphorylation, cardiac muscle contraction, and the cell cycle pathway in hiPSC-CMs.

Metabolism-based cardiomyocytes production for regenerative therapy.

Human pluripotent stem cells (hPSCs) are currently used in clinical applications such as cardiac regenerative therapy, studying disease models, and drug screening for heart failure. Transplantation of hPSC-derived cardiomyocytes (hPSC-CMs) can be used as an alternative therapy for heart transplantation. In contrast to differentiated somatic cells, hPSCs possess unique metabolic programs to maintain pluripotency, and understanding their metabolic features can contribute to the development of technologies that can be useful for their clinical applications. The production of hPSC-CMs requires stepwise specification during embryonic development and metabolic regulation is crucial for proper embryonic development. These metabolic features have been applied to hPSC-CM production methods, such as mesoderm induction, specifications for cardiac progenitors, and their maturation. This review describes the metabolic programs in hPSCs and the metabolic regulation in hPSC-CM production for cardiac regenerative therapy.

In Vitro Drug Screening Using iPSC-Derived Cardiomyocytes of a Long QT-Syndrome Patient Carrying KCNQ1 & TRPM4 Dual Mutation: An Experimental Personalized Treatment.

Congenital long QT syndrome is a type of inherited cardiovascular disorder characterized by prolonged QT interval. Patient often suffer from syncopal episodes, electrocardiographic abnormalities and life-threatening arrhythmia. Given the complexity of the root cause of the disease, a combination of clinical diagnosis and drug screening using patient-derived cardiomyocytes represents a more effective way to identify potential cures. We identified a long QT syndrome patient carrying a heterozygous KCNQ1 c.656G>A mutation and a heterozygous TRPM4 c.479C>T mutation. Implantation of implantable cardioverter defibrillator in combination with conventional medication demonstrated limited success in ameliorating long-QT-syndrome-related symptoms. Frequent defibrillator discharge also caused deterioration of patient quality of life. Aiming to identify better therapeutic agents and treatment strategy, we established a patient-specific iPSC line carrying the dual mutations and differentiated these patient-specific iPSCs into cardiomyocytes. We discovered that both verapamil and lidocaine substantially shortened the QT interval of the long QT syndrome patient-specific cardiomyocytes. Verapamil treatment was successful in reducing defibrillator discharge frequency of the KCNQ1/TRPM4 dual mutation patient. These results suggested that verapamil and lidocaine could be alternative therapeutic agents for long QT syndrome patients that do not respond well to conventional treatments. In conclusion, our approach indicated the usefulness of the in vitro disease model based on patient-specific iPSCs in identifying pharmacological mechanisms and drug screening. The long QT patient-specific iPSC line carrying KCNQ1/TRPM4 dual mutations also represents a tool for further understanding long QT syndrome pathogenesis.

Human stem cell-derived neurons and neural circuitry therapeutics: Next frontier in spinal cord injury repair.

Spinal cord injury (SCI) remains a life-altering event that devastates those injured and the families that support them. Numerous laboratories are engaged in preclinical and clinical trials to repair the injured spinal cord with stem cell-derived therapeutics. A new developmental paradigm reveals early bifurcation of brain or trunk neurons in mammals via neuromesodermal progenitors (NMPs) relevant to therapies requiring homotypic spinal cord neural populations. Human-induced pluripotent stem cell (hiPSC) NMP-derived spinal motor neurons generated ex vivo following this natural developmental route demonstrate robust survival in vivo when delivered as suspension grafts or as in vitro preformed encapsulated neuronal circuitry when transplanted into a rat C4-C5 hemicontusion injury site. Use of in vitro matured neurons avoids in vivo differentiation challenges of using pluripotent hiPSC or multipotent neural stem cell (NSC) or mesenchymal stem cell therapeutics. In this review, we provide an injury to therapeutics overview focusing on how stem cell and developmental fields are merging to generate exquisitely matched spinal motor neurons for SCI therapeutic studies. The complexity of the SCI microenvironment generated by trauma to neurons and vasculature, along with infiltrating inflammatory cells and scarring, underlies the challenging cytokine microenvironment that therapeutic cells encounter. An overview of evolving but limited stem cell-based SCI therapies that have progressed from preclinical to clinical trials illustrates the challenges and need for additional stem cell-based therapeutic approaches. The focus here on neurons describes how NMP-based neurotechnologies are advancing parallel strategies such as transplantation of preformed neuronal circuitry as well as human in vitro gastruloid multicellular models of trunk central and peripheral nervous system integration with organs. NMP-derived neurons are expected to be powerful drivers of the next generation of SCI therapeutics and integrate well with combination therapies that may utilize alternate biomimetic scaffolds for bridging injuries or flexible biodegradable electronics for electrostimulation.

Therapeutic Potential of a Combination of Electroacupuncture and Human iPSC-Derived Small Extracellular Vesicles for Ischemic Stroke.

This paper aimed to explore the roles of the combination of electroacupuncture (EA) and induced pluripotent stem cell-derived small extracellular vesicles (iPSC-EVs) on mice with ischemic stroke and the underlying mechanisms. A focal cerebral ischemia model was established in C57BL/6 mice through middle cerebral artery occlusion (MCAO). After 3 days, neurological impairment and motor function were examined by performing behavioral tests. The infarct volume and neuronal apoptosis were examined using TTC staining and TUNEL assays. Flow cytometry was performed to assess the proliferation of T lymphocytes. The changes in the interleukin (IL)-33/ST2 axis were evaluated by immunofluorescence and Western blotting. The combination of EA and iPSC-EVs treatment ameliorated neurological impairments and reduced the infarct volume and neuronal apoptosis in MCAO mice. EA plus iPSC-EVs suppressed T helper (Th1) and Th17 responses and promoted the regulatory T cell (Treg) response. In addition, EA plus iPSC-EVs exerted neuroprotective effects by regulating the IL-33/ST2 axis and inhibiting the microglia and astrocyte activation. Taken together, the study shows that EA and iPSC-EVs exerted a synergistic neuroprotective effect in MCAO mice, and this treatment may represent a novel potent therapy for ischemic stroke and damage to other tissues.

Evaluation of the Effects of Exposure to Power-Frequency Magnetic Fields on the Differentiation of Hematopoietic Stem/Progenitor Cells Using Human-Induced Pluripotent Stem Cells.

The causal association between exposure to power-frequency magnetic fields (MFs) and childhood leukemia has been under discussion. Although evidence from experimental studies is required for a conclusion to be reached, only a few studies have focused on the effects of MF exposure on the human hematopoietic system directly related to leukemogenesis. Here, we established an in vitro protocol to simulate the differentiation of human mesodermal cells to hematopoietic stem progenitor cells (HSPCs) using human-induced pluripotent stem cells. Furthermore, we introduced MF in the protocol to study the effects of exposure. After a continuous exposure to 0-300 mT of 50-Hz MFs during the differentiation process, the efficiency of differentiation of mesodermal cells into HSPCs was analyzed in a single-blinded manner. The percentage of emerged HSPCs from mesodermal cells in groups exposed to 50-Hz MFs indicated a lack of significant changes compared with those in the sham-exposed group. These results suggest that exposure to 50-Hz MFs up to 300 mT does not affect the differentiation of human mesodermal cells to HSPCs, which may be involved in the initial process of leukemogenesis. © 2022 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.

Perspectives of future lung toxicology studies using human pluripotent stem cells.

The absence of in vitro platforms for human pulmonary toxicology studies is becoming an increasingly serious concern. The respiratory system has a dynamic mechanical structure that extends from the airways to the alveolar region. In addition, the epithelial, endothelial, stromal, and immune cells are highly organized in each region and interact with each other to function synergistically. These cells of varied lineage, particularly epithelial cells, have been difficult to use for long-term culture in vitro, thus limiting the development of useful experimental tools. This limitation has set a large distance between the bench and the bedside for analyzing the pathogenic mechanisms, the efficacy of candidate therapeutic agents, and the toxicity of compounds. Several researchers have proposed solutions to these problems by reporting on methods for generating human lung epithelial cells derived from pluripotent stem cells (PSCs). Moreover, the use of organoid culture, organ-on-a-chip, and material-based techniques have enabled the maintenance of functional PSC-derived lung epithelial cells as well as primary cells. The aforementioned technological advances have facilitated the in vitro recapitulation of genetic lung diseases and the detection of ameliorating or worsening effects of genetic and chemical interventions, thus indicating the future possibility of more sophisticated preclinical compound assessments in vitro. In this review, we will update the recent advances in lung cell culture methods, principally focusing on human PSC-derived lung epithelial organoid culture systems with the hope of their future application in toxicology studies.

The Use of Induced Pluripotent Stem Cells to Study the Effects of Adenosine Deaminase Deficiency on Human Neutrophil Development.

Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.

Abnormalities of mitochondrial dynamics and bioenergetics in neuronal cells from CDKL5 deficiency disorder.

CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental disorder caused by pathogenic variants in the Cyclin-dependent kinase-like 5 (CDKL5) gene, resulting in dysfunctional CDKL5 protein. It predominantly affects females and causes seizures in the first few months of life, ultimately resulting in severe intellectual disability. In the absence of targeted therapies, treatment is currently only symptomatic. CDKL5 is a serine/threonine kinase that is highly expressed in the brain, with a critical role in neuronal development. Evidence of mitochondrial dysfunction in CDD is gathering, but has not been studied extensively. We used human patient-derived induced pluripotent stem cells with a pathogenic truncating mutation (p.Arg59*) and CRISPR/Cas9 gene-corrected isogenic controls, differentiated into neurons, to investigate the impact of CDKL5 mutation on cellular function. Quantitative proteomics indicated mitochondrial defects in CDKL5 p.Arg59* neurons, and mitochondrial bioenergetics analysis confirmed decreased activity of mitochondrial respiratory chain complexes. Additionally, mitochondrial trafficking velocity was significantly impaired, and there was a higher percentage of stationary mitochondria. We propose mitochondrial dysfunction is contributing to CDD pathology, and should be a focus for development of targeted treatments for CDD.