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Several lines of evidence have established that proliferation and differentiation of neural stem cells into neurons within the sub-granular zone of the dentate gyrus, a process named adult hippocampal neurogenesis, contribute to maintaining healthy cognitive functions throughout life. The rate of adult hippocampal neurogenesis decreases with aging and a premature impairment of adult hippocampal neurogenesis has been observed both in animal models of Alzheimer’s disease and human post-mortem tissues. The causal relationship between adult hippocampal neurogenesis and the development of Alzheimer’s disease pathology has, however, not been established. This is partly due to the limitation of recapitulating the development of Alzheimer’s disease pathology in rodent models and the lack of translatable biomarkers to identify tractable targets in humans. While it is tempting to postulate that adult hippocampal neurogenesis could be leveraged to improve cognitive deficits in Alzheimer’s disease, consensual results have yet to be reached to fully explore this hypothesis. In this review, we discuss how the recent progress in identifying molecular pathways in adult hippocampal neurogenesis provides a good framework to initiate strategies for drug-based intervention in neurodegenerative diseases, especially in Alzheimer’s disease. We outline how discrepancies in pre-clinical disease models and experimental methodology have resulted in contradictory findings and propose a shift towards using more translatable approaches to model neurogenesis in Alzheimer’s disease. In particular, we review how exploring novel experimental paradigms including the use of human induced pluripotent stem cells and more complex cell culture systems, as well as standardizing protocols used to investigate evidence of neurogenesis in human tissues, could deliver deeper mechanistic insights that would kick-start innovative drug discovery efforts to promote healthy aging and cellular rejuvenation.

Objective: Lifestyle choices such as tobacco and e-cigarette use are a risk factor for peripheral arterial disease (PAD) and may influence therapeutic outcomes. The effect of chronic nicotine exposure on the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) was assessed in a murine model of PAD. Methods: Mice were exposed to nicotine or phosphate-buffered saline (PBS) for 28 days, followed by induction of limb ischemia and iPSC-EC transplantation. Cells were injected into the ischemic limb immediately after induction of hindlimb ischemia and again 7 days later. Limb perfusion was assessed by laser Doppler spectroscopy, and transplant cell survival was monitored for 14 days afterward using bioluminescence imaging, followed by histological analysis of angiogenesis. Results: Transplant cell retention progressively decreased over time after implantation based on bioluminescence imaging, and there were no significant differences in cell survival between mice with chronic exposure to nicotine or PBS. However, compared with mice without nicotine exposure, mice with prior nicotine exposure had had an impaired therapeutic response to iPSC-EC therapy based on decreased vascular perfusion recovery. Mice with nicotine exposure, followed by cell transplantation, had significantly lower mean perfusion ratio after 14 days (0.47 ± 0.07) compared with mice undergoing cell transplantation without prior nicotine exposure (0.79 ± 0.11). This finding was further supported by histological analysis of capillary density, in which animals with prior nicotine exposure had a lower capillary density (45.9 ± 4.7 per mm2) compared with mice without nicotine exposure (66.5 ± 8.1 per mm2). Importantly, the ischemic limbs mice exposed to nicotine without cell therapy also showed significant impairment in perfusion recovery after 14 days, compared with mice that received PBS + iPSC-EC treatment. This result suggested that mice without chronic nicotine exposure could respond to iPSC-EC implantation into the ischemic limb by inducing perfusion recovery, whereas mice with chronic nicotine exposure did not respond to iPSC-EC therapy. Conclusions: Together, these findings show that chronic nicotine exposure adversely affects the ability of iPSC-EC therapy to promote vascular perfusion recovery and angiogenesis in a murine PAD model. : Clinical Relevance: Cell therapy is a promising approach to induce revascularization in ischemic tissues associated with PAD. However, the role of lifestyle choices in modulating the efficacy of cell therapy is largely unknown. Using a murine model of PAD, this work demonstrates that chronic nicotine exposure can impair the ability of cell therapy to exert a therapeutic benefit. These results have important implications in the design of treatment options for patients with PAD with a history of tobacco or e-cigarette use.

Introduction: With the increase in aging populations around the world, the development of in vitro human cell models to study neurodegenerative disease is crucial. A major limitation in using induced pluripotent stem cell (hiPSC) technology to model diseases of aging is that reprogramming fibroblasts to a pluripotent stem cell state erases age-associated features. The resulting cells show behaviors of an embryonic stage exhibiting longer telomeres, reduced oxidative stress, and mitochondrial rejuvenation, as well as epigenetic modifications, loss of abnormal nuclear morphologies, and age-associated features.Methods: We have developed a protocol utilizing stable, non-immunogenic chemically modified mRNA (cmRNA) to convert adult human dermal fibroblasts (HDFs) to human induced dorsal forebrain precursor (hiDFP) cells, which can subsequently be differentiated into cortical neurons. Analyzing an array of aging biomarkers, we demonstrate for the first time the effect of direct-to-hiDFP reprogramming on cellular age.Results: We confirm direct-to-hiDFP reprogramming does not affect telomere length or the expression of key aging markers. However, while direct-to-hiDFP reprogramming does not affect senescence-associated β-galactosidase activity, it enhances the level of mitochondrial reactive oxygen species and the amount of DNA methylation compared to HDFs. Interestingly, following neuronal differentiation of hiDFPs we observed an increase in cell soma size as well as neurite number, length, and branching with increasing donor age suggesting that neuronal morphology is altered with age.Discussion: We propose direct-to-hiDFP reprogramming provides a strategy for modeling age-associated neurodegenerative diseases allowing the persistence of age-associated signatures not seen in hiPSC-derived cultures, thereby facilitating our understanding of neurodegenerative disease and identification of therapeutic targets.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in adults, causing progressive degeneration of motor neurons, which results in muscle atrophy, respiratory failure and ultimately death of the patients. The pathogenesis of ALS is complex, and extensive efforts have focused on unravelling the underlying molecular mechanisms with a large emphasis on the dying motor neurons. However, a recent shift in focus towards the supporting glial population has revealed a large contribution and influence in ALS, which stresses the need to explore this area in more detail. Especially studies into astrocytes, the residential homeostatic supporter cells of neurons, have revealed a remarkable astrocytic dysfunction in ALS, and therefore could present a target for new and promising therapeutic entry points. In this review, we provide an overview of general astrocyte function and summarize the current literature on the role of astrocytes in ALS by categorizing the potentially underlying molecular mechanisms. We discuss the current efforts in astrocyte-targeted therapy, and highlight the potential and shortcomings of available models.

Parkinson's disease (PD) is the second most common neurodegenerative disease and its pathogenic mechanisms are poorly understood. The majority of PD cases are sporadic but a number of genes are associated with familial PD. Sporadic and familial PD have many molecular and cellular features in common, suggesting some shared pathogenic mechanisms. Induced pluripotent stem cells (iPSCs) have been derived from patients harboring a range of different mutations of PD-associated genes. PD patient-derived iPSCs have been differentiated into relevant cell types, in particular dopaminergic neurons and used as a model to study PD. In this review, we describe how iPSCs have been used to improve our understanding of the pathogenesis of PD. We describe what cellular and molecular phenotypes have been observed in neurons derived from iPSCs harboring known PD-associated mutations and what common pathways may be involved.

Here, we report that MYC rescues early human cells undergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). We identified ESRG as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC.

Stem cells are precursor cells capable of self-renew and of generating numerous mature cell types. As the field of human embryonic stem cells harvesting has been put under questionable ethic issues, other sources are under investigation and present tremendous potential: tissue specific progenitor stem cells, mesenchymal stem cells, umbilical cord cells, bone marrow stem cells, and induced pluripotent stem cells. Stem cells interest different departments of regenerative medicine as well as conservative wildlife. Stem cells might be a viable option for the treatment of pathologies such as spinal injuries, cardiovascular disease, diabetes, liver injuries or even osteoarthritis. Scientists are looking forward to developing molecules that can activate tissue specific stem cells, promote stem cells to migrate to the side of tissue injury, and promote their differentiation to tissue specific cells, so that many health issues could have an alternative and efficient treatment and or even be cured.

Objectives: To establish a protocol to prepare and transplant clinical-grade human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) and to evaluate the therapeutic potential in an animal myocardial infarction (MI) model. Methods: We simultaneously differentiated clinical-grade hiPSCs into cardiovascular cell lineages with or without the administration of canonical Wnt inhibitors, generated 5- layer cell sheets with insertion of gelatin hydrogel microspheres (GHMs) (HiCTs), and transplanted them onto an athymic rat MI model. Cardiac function was evaluated by echocardiography and cardiac magnetic resonance imaging and compared with that in animals with sham and transplantation of 5-layer cell sheets without GHMs. Graft survival, ventricular remodeling, and neovascularization were evaluated histopathologically. Results: The administration of Wnt inhibitors significantly promoted cardiomyocyte (CM) (P

Pompe disease (PD) is a rare, autosomal recessive, inherited, and progressive metabolic disorder caused by α-glucosidase defect in lysosomes, resulting in abnormal glycogen accumulation. Patients with PD characteristically have multisystem pathological disorders, particularly hypertrophic cardiomyopathy, muscle weakness, and hepatomegaly. Although the pathogenesis and clinical outcomes of PD are well-established, disease-modeling ability, mechanism elucidation, and drug development targeting PD have been substantially limited by the unavailable PD-relevant cell models. This obstacle has been overcome with the help of induced pluripotent stem cell (iPSC) reprogramming technology, thus providing a powerful tool for cell replacement therapy, disease modeling, drug screening, and drug toxicity assessment. This review focused on the exciting achievement of PD disease modeling and mechanism exploration using iPSC.

Successful recovery from vascular diseases has typically relied on the surgical repair of damaged blood vessels (BVs), with the majority of current approaches involving the implantation of autologous BVs, which is plagued by donor site tissue damage. Researchers have attempted to develop artificial vessels as an alternative solution to traditional approaches to BV repair. However, the manufacturing of small-diameter (< 6 mm) BVs is still considered one of the biggest challenges due to its difficulty in the precise fabrication and the replication of biomimetic architectures. In this study, we successfully developed 3D printed flexible small-diameter BVs that consist of smooth muscle cells and a vascularized endothelium. In the developed artificial BV, a rubber-like elastomer was printed as the outermost layer of the vessel, which demonstrated enhanced mechanical properties, while and human induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iSMCs) and endothelial cells (iECs) embedded fibrinogen solutions were coaxially extruded with thrombin solution to form cell-laden fibrin gel inner layers. Our results showed that the 3D BVs possessed proper mechanical properties, and the cells in the fibrin layers substantially proliferated over time to form a stable BV construct. Our study demonstrated that the 3D printed flexible small-diameter BV using iPSCs could be a promising platform for the treatment of vascular diseases.

Itch (pruritus) is a sensation that drives a desire to scratch, a behavior observed in many animals. Although generally short-lasting and not causing harm, there are several pathological conditions where chronic itch is a hallmark symptom and in which prolonged scratching can induce damage. Finding medications to counteract the sensation of chronic itch has proven difficult due to the molecular complexity that involves a multitude of triggers, receptors and signaling pathways between skin, immune and nerve cells. While much has been learned about pruritus from in vivo animal models, they have limitations that corroborate the necessity for a transition to more human disease-like models. Also, reducing animal use should be encouraged in research. However, conducting human in vivo experiments can also be ethically challenging. Thus, there is a clear need for surrogate models to be used in pre-clinical investigation of the mechanisms of itch. Most in vitro models used for itch research focus on the use of known pruritogens. For this, sensory neurons and different types of skin and/or immune cells are stimulated in 2D or 3D co-culture, and factors such as neurotransmitter or cytokine release can be measured. There are however limitations of such simplistic in vitro models. For example, not all naturally occurring cell types are present and there is also no connection to the itch-sensing organ, the central nervous system (CNS). Nevertheless, in vitro models offer a chance to investigate otherwise inaccessible specific cell–cell interactions and molecular pathways. In recent years, stem cell-based approaches and human primary cells have emerged as viable alternatives to standard cell lines or animal tissue. As in vitro models have increased in their complexity, further opportunities for more elaborated means of investigating itch have been developed. In this review, we introduce the latest concepts of itch and discuss the advantages and limitations of current in vitro models, which provide valuable contributions to pruritus research and might help to meet the unmet clinical need for more refined anti-pruritic substances. [ABSTRACT FROM AUTHOR]

Background: The most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), which refers to the lack of all spermatogenic cells in the testes. The genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells. Methods: Whole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS, and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human-induced pluripotent stem cells (hiPSCs) into germ cell-like cells. Results: The homozygous loss-of-function (LoF) variant p.His244ArgfsTer31 (c.731_732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking all functional domains, and no PIWIL2 expression was detected in the patient's testes. The patient and PIWIL2−/− hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses suggested the inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2−/− group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs. Conclusions: Our study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments. [ABSTRACT FROM AUTHOR]

The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used 1H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that 1H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Potentials of immortal proliferation and unlimited differentiation into all the three germ layers and germ cells in induced pluripotent stem cells (iPSCs) render them important bioresources for in vitro reconstitution and modeling of intravital tissues and organs in various animal models, thus contributing to the elucidation of pathomechanisms, drug discovery and stem cell‐based regenerative medicine. We previously reported promising approaches for deriving transgene‐free iPSCs from somatic fibroblasts of multiple mammalian species by episomal vector or RNA transfection, although the respective step‐by‐step protocols and the combinatorial usage of these methods, which achieved high induction efficiency, have not been described in the literature so far. Here, we provide a detailed step‐by‐step description of these methods with critical tips and slight modifications (improvements) to previously reported methods. We also report a novel method for the establishment of iPSCs from the Syrian hamster (also known as golden hamster; Mesocricetus auratus), a unique animal model of hibernation. We anticipate this methodology will contribute to stem cell biology and regenerative medicine research. [ABSTRACT FROM AUTHOR]

Tropomyosins coat actin filaments to impact actin-related signaling and cell morphogenesis. Genome-wide association studies have linked Tropomyosin 1 (TPM1) with human blood trait variation. TPM1 has been shown to regulate blood cell formation in vitro, but it remains unclear how or when TPM1 affects hematopoiesis. Using gene-edited induced pluripotent stem cell (iPSC) model systems, we found that TPM1 knockout augmented developmental cell state transitions and key signaling pathways, including tumor necrosis factor alpha (TNF-α) signaling, to promote hemogenic endothelial (HE) cell specification and hematopoietic progenitor cell (HPC) production. Single-cell analyses revealed decreased TPM1 expression during human HE specification, suggesting that TPM1 regulated in vivo hematopoiesis via similar mechanisms. Analyses of a TPM1 gene trap mouse model showed that TPM1 deficiency enhanced HE formation during embryogenesis, without increasing the number of hematopoietic stem cells. These findings illuminate novel effects of TPM1 on developmental hematopoiesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Neural crest cells (NCCs) are cell populations that originate during the formation of neural crest in developmental stages. They are characterized by their multipotency, self-renewal and migration potential. Given their ability to differentiate into various types of cells such as neurons and Schwann cells, NCCs hold promise for cell therapy applications. The conventional method for obtaining NCCs involves inducing them from stem cells like induced pluripotent stem cells (iPSCs), followed by a long-term passage or purification using fluorescence-activated cell sorting (FACS). Although FACS allows high purity induced neural crest cells (iNCCs) to be obtained quickly, it is complex and costly. Therefore, there is a need for a simpler, cost-effective and less time-consuming method for cell therapy application., Methods: To select differentiated iNCCs from heterogeneous cell populations quickly without using FACS, we adopted the use of scaffold material full-length laminin 211 (LN211), a recombinant, xeno-free protein suitable for cell therapy. After fist passage on LN211, iNCCs characterization was performed using polymerase chain reaction and flow cytometry. Additionally, proliferation and multipotency to various cells were evaluated., Result: The iNCCs obtained using our new method expressed cranial NCC- related genes and exhibited stable proliferation ability for at least 57 days, while maintaining high expression level of the NCCs marker CD271. They demonstrated differentiation ability into several cell types: neurons, astrocytes, melanocytes, smooth muscle cells, osteoblasts, adipocytes and chondrocytes. Furthermore, they could be induced to differentiate into induced mesenchymal stem cells (iMSCs) which retain the essential functions of somatic MSCs., Conclusion: In this study, we have developed novel method for obtaining high purity iNCCs differentiated from iPSCs in a short time using LN211 under xeno-free condition. Compared with traditional methods, like FACS or long-term passage, this approach enables the acquisition of a large amount of cells at a lower cost and labor, and it is expected to contribute to stable supply of large scale iNCCs for future cell therapy applications., Competing Interests: We hereby declare that there are no conflicts of interest to report. We confirm that there have been no financial or personal relationships with other people or organizations that could inappropriately influence or bias our work., (© 2024 The Author(s).)

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease of the central nervous system (CNS), resulting in neurological disability that worsens over time. While progress has been made in defining the immune system's role in MS pathophysiology, the contribution of intrinsic CNS cell dysfunction remains unclear. Here, we generated a collection of induced pluripotent stem cell (iPSC) lines from people with MS spanning diverse clinical subtypes and differentiated them into glia-enriched cultures. Using single-cell transcriptomic profiling and orthogonal analyses, we observed several distinguishing characteristics of MS cultures pointing to glia-intrinsic disease mechanisms. We found that primary progressive MS-derived cultures contained fewer oligodendrocytes. Moreover, MS-derived oligodendrocyte lineage cells and astrocytes showed increased expression of immune and inflammatory genes, matching those of glia from MS postmortem brains. Thus, iPSC-derived MS models provide a unique platform for dissecting glial contributions to disease phenotypes independent of the peripheral immune system and identify potential glia-specific targets for therapeutic intervention., Competing Interests: Declaration of interests P.J.T. and B.L.L.C. are listed as inventors on issued and pending patent claims covering compositions and methods of enhancing glial cell function. P.J.T. is a co-founder and consultant for Convelo Therapeutics, which has licensed patents from Case Western Reserve University (CWRU). P.J.T. and CWRU retain equity in Convelo Therapeutics. V.F. and L.B. are listed as inventors on issued and pending patent claims covering glial cell generation methods., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Abstract Background NRXN1 deletions are identified as one of major rare risk factors for autism spectrum disorder (ASD) and other neurodevelopmental disorders. ASD has 30% co-morbidity with epilepsy, and the latter is associated with excessive neuronal firing. NRXN1 encodes hundreds of presynaptic neuro-adhesion proteins categorized as NRXN1α/β/γ. Previous studies on cultured cells show that the short NRXN1β primarily exerts excitation effect, whereas the long NRXN1α which is more commonly deleted in patients involves in both excitation and inhibition. However, patient-derived models are essential for understanding functional consequences of NRXN1α deletions in human neurons. We recently derived induced pluripotent stem cells (iPSCs) from five controls and three ASD patients carrying NRXN1α+/- and showed increased calcium transients in patient neurons. Methods In this study we investigated the electrophysiological properties of iPSC-derived cortical neurons in control and ASD patients carrying NRXN1α+/- using patch clamping. Whole genome RNA sequencing was carried out to further understand the potential underlying molecular mechanism. Results NRXN1α + / - cortical neurons were shown to display larger sodium currents, higher AP amplitude and accelerated depolarization time. RNASeq analyses revealed transcriptomic changes with significant upregulation glutamatergic synapse and ion channels/transporter activity including voltage-gated potassium channels (GRIN1, GRIN3B, SLC17A6, CACNG3, CACNA1A, SHANK1), which are likely to couple with the increased excitability in NRXN1α + / - cortical neurons. Conclusions Together with recent evidence of increased calcium transients, our results showed that human NRXN1α + / - isoform deletions altered neuronal excitability and non-synaptic function, and NRXN1α + / - patient iPSCs may be used as an ASD model for therapeutic development with calcium transients and excitability as readouts.

Itch (pruritus) is a sensation that drives a desire to scratch, a behavior observed in many animals. Although generally short-lasting and not causing harm, there are several pathological conditions where chronic itch is a hallmark symptom and in which prolonged scratching can induce damage. Finding medications to counteract the sensation of chronic itch has proven difficult due to the molecular complexity that involves a multitude of triggers, receptors and signaling pathways between skin, immune and nerve cells. While much has been learned about pruritus from in vivo animal models, they have limitations that corroborate the necessity for a transition to more human disease-like models. Also, reducing animal use should be encouraged in research. However, conducting human in vivo experiments can also be ethically challenging. Thus, there is a clear need for surrogate models to be used in pre-clinical investigation of the mechanisms of itch. Most in vitro models used for itch research focus on the use of known pruritogens. For this, sensory neurons and different types of skin and/or immune cells are stimulated in 2D or 3D co-culture, and factors such as neurotransmitter or cytokine release can be measured. There are however limitations of such simplistic in vitro models. For example, not all naturally occurring cell types are present and there is also no connection to the itch-sensing organ, the central nervous system (CNS). Nevertheless, in vitro models offer a chance to investigate otherwise inaccessible specific cell–cell interactions and molecular pathways. In recent years, stem cell-based approaches and human primary cells have emerged as viable alternatives to standard cell lines or animal tissue. As in vitro models have increased in their complexity, further opportunities for more elaborated means of investigating itch have been developed. In this review, we introduce the latest concepts of itch and discuss the advantages and limitations of current in vitro models, which provide valuable contributions to pruritus research and might help to meet the unmet clinical need for more refined anti-pruritic substances.

Although pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have been proved to be a new platform for heart regeneration, the lack of maturity significantly hinders the clinic application. Recent researches indicate that the function of stem cell is associated with the nanoscale geometry/topography of the extracellular matrix (ECM). However, the effects of 3D nanofibrous scaffolds in maturation of iPSC-CMs still remain unclear. Thus, we explored the effects of restructuring iPSC-CMs in 3D nano-scaffolds on cell morphology, cardiac-specific structural protein, gap junction and calcium transient kinetics. Using the electrospinning technology, poly-(ε-caprolactone) (PCL) nanofibrous scaffold were constructed and iPSC-CMs were seeded into these forms. As expected, strong sarcolemmal remodeling processes and myofilament reorientation were observed in 3D nano-scaffolds culture, as well as more expression of cardiac mature proteins, such as β-MHC and MLC2v. The mature morphology of 3D-shaped iPSC-CMs leaded to enhanced calcium transient kinetics, with increased calcium peak transient amplitude and the maximum upstroke velocity (Vmax). The results revealed that the maturation of iPSC-CMs was enhanced by the electrospun 3D PCL nanofibrous scaffolds treatment. These findings also proposed a feasible strategy to improve the myocardium bioengineering by combining stem cells with scaffolds.

Background: The African spiny mouse (Acomys) is an emerging mammalian model for scar-free regeneration, and further study of Acomys could advance the field of regenerative medicine. Isolation of pluripotent stem cells from Acomys would allow for development of transgenic or chimeric animals and in vitro study of regeneration; however, the reproductive biology of Acomys is not well characterized, complicating efforts to derive embryonic stem cells. Thus, we sought to generate Acomys induced pluripotent stem cells (iPSCs) by reprogramming somatic cells back to pluripotency. Methods: To generate Acomys iPSCs, we attempted to adapt established protocols developed in Mus. We utilized a PiggyBac transposon system to genetically modify Acomys fibroblasts to overexpress the Yamanaka reprogramming factors as well as mOrange fluorescent protein under the control of a doxycycline-inducible TetON operon system. Results: Reprogramming factor overexpression caused Acomys fibroblasts to undergo apoptosis or senescence. When SV40 Large T antigen (SV40 LT) was added to the reprogramming cocktail, Acomys cells were able to dedifferentiate into pre-iPSCs. Although use of 2iL culture conditions induced formation of colonies resembling Mus PSCs, these Acomys iPS-like cells lacked pluripotency marker expression and failed to form embryoid bodies. An EOS-GiP system was unsuccessful in selecting for bona fide Acomys iPSCs; however, inclusion of Nanog in the reprogramming cocktail along with 5-azacytidine in the culture medium allowed for generation of Acomys iPSC-like cells with increased expression of several naïve pluripotency markers. Conclusions: There are significant roadblocks to reprogramming Acomys cells, necessitating future studies to determine Acomys-specific reprogramming factor and/or culture condition requirements. The requirement for SV40 LT during Acomys dedifferentiation may suggest that tumor suppressor pathways play an important role in Acomys regeneration and that Acomys may possess unreported cancer resistance.

Natural killer (NK) cells are a part of innate immunity that can be activated rapidly in response to malignant transformed cells without prior sensitization. Engineering NK cells to express chimeric antigen receptors (CARs) allows them to be directed against corresponding target tumor antigens. CAR-NK cells are regarded as a promising candidate for cellular immunotherapy alternatives to conventional CAR-T cells, due to the relatively low risk of graft-versus-host disease and safer clinical profile. Human induced pluripotent stem cells (iPSCs) are a promising renewable cell source of clinical NK cells. In the present study, we successfully introduced a third-generation CAR targeting CD19, which was validated to have effective signaling domains suitable for NK cells, into umbilical cord blood NK-derived iPSCs, followed by a single-cell clone selection and thorough iPSC characterization. The established single-cell clone of CAR19-NK/iPSCs, which is highly desirable for clinical application, can be differentiated using serum- and feeder-free protocols into functional CAR19-iNK-like cells with improved anti-tumor activity against CD19-positive hematologic cancer cells when compared with wild-type (WT)-iNK-like cells. With the feasibility of being an alternative source for off-the-shelf CAR-NK cells, a library of single-cell clones of CAR-engineered NK/iPSCs targeting different tumor antigens may be created for future clinical application.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting the upper and lower motor neurons, causing patients to lose control over voluntary movement, and leading to gradual paralysis and death. There is no cure for ALS, and the development of viable therapeutics has proved challenging, demonstrated by a lack of positive results from clinical trials. One strategy to address this is to improve the tool kit available for pre-clinical research. Here, we describe the creation of an open-access ALS iPSC biobank generated from patients carrying mutations in the TARDBP, FUS, ANXA11, ARPP21, and C9ORF72 genes, alongside healthy controls. To demonstrate the utilisation of these lines for ALS disease modelling, a subset of FUS-ALS iPSCs were differentiated into functionally active motor neurons. Further characterisation revealed an increase in cytoplasmic FUS protein and reduced neurite outgrowth in FUS-ALS motor neurons compared to the control. This proof-of-principle study demonstrates that these novel patient-derived iPSC lines can recapitulate specific and early disease-related ALS phenotypes. This biobank provides a disease-relevant platform for discovery of ALS-associated cellular phenotypes to aid the development of novel treatment strategies.

The arginine‐glycine‐aspartic acid (RGD) motif is a cell adhesion sequence that binds to integrins. Some RGD‐containing peptides promote adhesion of both embryonic stem cells and induced pluripotent stem cells (iPSCs); however, not all such RGD‐containing peptides are active. In this study, we elucidated the role of RGD‐neighboring sequences on iPSC adhesion using diverse synthetic peptides and recombinant proteins. Our results indicate that iPSC adhesion requires RGDX1X2 sequences, such as RGDVF and RGDNY, and that the X1X2 residues are essential for the adhesion via integrin αvβ5 but not αvβ3. iPSCs express integrin αvβ5 but not αvβ3; therefore, iPSC adhesion requires the RGDX1X2‐containing sequences. The importance of the X1X2 residues was confirmed with both HeLa and A549 cells, which express integrin αvβ5 but not αvβ3. Analysis of RGD‐neighboring sequences provides important insights into ligand‐binding specificity of integrins. Identification of integrin αvβ5‐binding motifs is potentially useful in drug development, drug delivery, cell culture, and tissue engineering. [ABSTRACT FROM AUTHOR]

Background: Reagent red blood cells (RBCs) are prepared from donated whole blood, resulting in various combinations of blood group antigens. This inconsistency can be resolved by producing RBCs with uniform antigen expression. Induced pluripotent stem cells (iPSCs) generated directly from mature cells constitute an unlimited source for RBC production. We aimed to produce erythroid cells from iPSCs for diagnostic purposes. We hypothesized that cultured erythroid cells express surface antigens that can be recognized by blood group antibodies. Methods: iPSCs were co-cultured with OP9 stromal cells to stimulate differentiation into the erythroid lineage. Cell differentiation was examined using microscopy and flow cytometry. Hemoglobin electrophoresis and oxygen-binding capacity testing were performed to verify that the cultured erythroid cells functioned normally. The agglutination reactions of the cultured erythroid cells to antibodies were investigated to confirm that the cells expressed blood group antigens. Results: The generated iPSCs showed stemness characteristics and could differentiate into the erythroid lineage. As differentiation progressed, the proportion of nucleated RBCs increased. Hemoglobin electrophoresis revealed a sharp peak in the hemoglobin F region. The oxygen-binding capacity test results were similar between normal RBCs and cultured nucleated RBCs. ABO and Rh-Hr blood grouping confirmed similar antigen expression between the donor RBCs and cultured nucleated RBCs. Conclusions: We generated blood group antigen-expressing nucleated RBCs from iPSCs co-cultured with OP9 cells that can be used for diagnostic purposes. iPSCs from rare blood group donors could serve as an unlimited source for reagent production. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) are terminally differentiated somatic cells that differentiate into various cell types. iPSCs are expected to be used for disease modeling and for developing novel treatments because differentiated cells from iPSCs can recapitulate the cellular pathology of patients with genetic mutations. However, a barrier to using iPSCs for comprehensive drug screening is the difficulty of evaluating their pathophysiology. Recently, the accuracy of image analysis has dramatically improved with the development of artificial intelligence (AI) technology. In the field of cell biology, it has become possible to estimate cell types and states by examining cellular morphology obtained from simple microscopic images. AI can evaluate disease-specific phenotypes of iPS-derived cells from label-free microscopic images; thus, AI can be utilized for disease-specific drug screening using iPSCs. In addition to image analysis, various AI-based methods can be applied to drug development, including phenotype prediction by analyzing genomic data and virtual screening by analyzing structural formulas and protein–protein interactions of compounds. In the future, combining AI methods may rapidly accelerate drug discovery using iPSCs. In this review, we explain the details of AI technology and the application of AI for iPSC-based drug screening. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem (iPS) cells have been widely studied in regenerative medicine, pathology modeling, and drug screening. Stable mass culture of iPS cells is essential for these applications. iPS cells can spontaneously differentiate into other cells during culture, and removal of these differentiated cells is necessary. Herein, a cost-effective culture method suitable for mass culture and a detailed analysis of the selective detachment of iPS cells are presented. A simple method for coating the water-insoluble thermoresponsive polymer poly (butyl methacrylate- block - N -isopropylacrylamide) on commercially available polystyrene dishes was employed. Analysis of the effects of the polymer composition, coating thickness, and surface structure on iPS cell culture/detachment showed that a coating thickness of approximately 10–40 nm using a polymer with a high poly (N -isopropylacrylamide) content was suitable for iPS cell detachment. Moreover, an interesting change in surface morphology was observed following temperature variation, thereby affecting laminin adsorption. Second, selective detachment in cocultures of iPS cells and differentiated cells enabled collection of iPS cells with more than 98% purity. Finally, long-term iPS cell culture was conducted using temperature-responsive cell detachment. Overall, long-term maintenance-free culture of iPS cells was possible without manual removal of differentiated cells. [Display omitted] • Water-insoluble thermoresponsive polymers were used in iPS cell culture. • Surface structure of coated polymer affected laminin adsorption and iPS cell culture. • iPS cells were cocultured with mesoderm cells and selectively detached via cooling. • Long-term maintenance-free culture of iPS cells was possible. [ABSTRACT FROM AUTHOR]

The tumorigenicity and toxicity of induced pluripotent stem cells (iPSCs) and their derivatives are major safety concerns in their clinical application. Recently, we developed granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing proliferating myeloid cells (GM-pMCs) from mouse iPSCs as a source of unlimited antigen-presenting cells for use in cancer immunotherapy. As GM-pMCs are generated by introducing c-Myc and Csf2 into iPSC-derived MCs and are dependent on self-produced GM-CSF for proliferation, methods to control their proliferation after administration should be introduced to improve safety. In this study, we compared the efficacy of two promising suicide gene systems, herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and inducible caspase-9 (iCasp9)/AP1903, for safeguarding GM-pMCs in cancer immunotherapy. The expression of HSV-TK or iCasp9 did not impair the fundamental properties of GM-pMCs. Both of these suicide gene-expressing cells selectively underwent apoptosis after treatment with the corresponding apoptosis-inducing drug, and they were promptly eliminated in vivo. iCasp9/AP1903 induced apoptosis more efficiently than HSV-TK/GCV. Furthermore, high concentrations of GCV were toxic to cells not expressing HSV-TK, whereas AP1903 was bioinert. These results suggest that iCasp9/AP1903 is superior to HSV-TK/GCV in terms of both safety and efficacy when controlling the fate of GM-pMCs after priming antitumor immunity.

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) could potentially provide unlimited HSCs for clinical transplantation, a curative treatment for numerous blood diseases. However, to date, bona fide HSC generation has been largely unsuccessful in vitro. We have previously described proof of concept for in vivo HSC generation from PSCs via teratoma formation. However, our first-generation system was complex and the output low. Here, we further optimize this technology and demonstrate the following: (1) simplified HSC generation using transcription factor overexpression; (2) improved HSC output using c-Kit-deficient host mice, and (3) that teratomas can be transplanted and cryopreserved. We demonstrate that overexpression of Gfi1b, c-Fos, and Gata2, previously reported to transdifferentiate fibroblasts into hematopoietic progenitors in vitro, can induce long-term HSC formation in vivo. Our in vivo system provides a useful platform to investigate new strategies and re-evaluate existing strategies to generate HSCs and study HSC development.

Brain microvascular endothelial cells (BMECs) are an essential component of the blood-brain barrier (BBB) that shields the brain against toxins and immune cells. While BBB dysfunction exists in neurological disorders, including Huntington's disease (HD), it is not known if BMECs themselves are functionally compromised to promote BBB dysfunction. Further, the underlying mechanisms of BBB dysfunction remain elusive given limitations with mouse models and post-mortem tissue to identify primary deficits. We undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived BMECs (iBMEC) from HD patients or unaffected controls. We demonstrate that HD iBMECs have intrinsic abnormalities in angiogenesis and barrier properties, as well as in signaling pathways governing these processes. Thus, our findings provide an iPSC-derived BBB model for a neurodegenerative disease and demonstrate autonomous neurovascular deficits that may underlie HD pathology with implications for therapeutics and drug delivery.

Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport, the trafficking of macromolecules between the nucleus and cytoplasm, is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore, we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD, including mouse and fly models, neurons transfected with mHTT, HD iPSC-derived neurons, and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally, overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity, providing future novel therapy targets.

Advances in induced pluripotent stem cell (iPSC) techniques have opened up new perspectives in research on developmental biology. Compared with other sources of human cellular models, iPSCs present a great advantage in hosting the unique genotype background of donors without ethical concerns. A wide spectrum of cellular and organoid models can be generated from iPSCs under appropriate in vitro conditions. The pluripotency of iPSCs is orchestrated by external signalling and regulated at the epigenetic, transcriptional and posttranscriptional levels. Recent decades have witnessed the progress of studying tissue-specific expressions and functions of microRNAs (miRNAs) using iPSC-derived models. MiRNAs are a class of short non-coding RNAs with regulatory functions in various biological processes during development, including cell migration, proliferation and apoptosis. MiRNAs are key modulators of gene expression and promising candidates for biomarker in development; hence, research on the regulation of human development by miRNAs is expanding. In this review, we summarize the current progress in the application of iPSC-derived models to studies of the regulatory roles of miRNAs in developmental processes.

Introduction: Sickle cell disease (SCD) is a monogenic disease and it is estimated that 300,000 infants are born annually with it. Most treatments available are only palliative, whereas the allogeneic hematopoietic stem cell transplantation offers the only potential cure for SCD. Objective: Generation of human autologous cells, when coupled with induced pluripotent stem cell (iPSC) technology, is a promising approach for developing study models. In this study, we provide a simple and efficient model for generating hematopoietic cells using iPSCs derived from a sickle cell anemia patient and an inexpensive in-house-prepared medium. Method: This study used iPSCs previously generated from peripheral blood mononuclear cells (PBMCs) from a patient with sickle cell anemia (iPSC_scd). Hematopoietic and erythroid differentiation was performed in two steps. Firstly, with the induction of hematopoietic differentiation through embryoid body formation, we evaluated the efficiency of two serum-free media; and secondly, the induction of hematopoietic stem/progenitor cells to erythroid progenitor cells was performed. Results: The patient-specific cell line generated CD34+/CD45+ and CD45+/CD43+ hematopoietic stem/progenitor cells and erythroid progenitors, comprising CD36+, CD71+ and CD235a+ populations, as well as the formation of hematopoietic colonies, including erythroid colonies, in culture in a semi-solid medium. Conclusion: In conjunction, our results described a simple serum-free platform to differentiate human the iPSCs into hematopoietic progenitor cells. This platform is an emerging application of iPSCs in vitro disease modeling, which can significantly improve the search for new pharmacological drugs for sickle cell disease.

Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.

Abstract Background Spinal cord injury (SCI) is a common disease that results in motor and sensory disorders and even lifelong paralysis. The transplantation of stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), or subsequently generated stem/progenitor cells, is predicted to be a promising treatment for SCI. In this study, we aimed to investigate effect of human iPSC-derived neural stem cells (hiPSC-NSCs) and umbilical cord-derived MSCs (huMSCs) in a mouse model of acute SCI. Methods Acute SCI mice model were established and were randomly treated as phosphate-buffered saline (PBS) (control group), repaired with 1 × 105 hiPSC-NSCs (NSC group), and 1 × 105 huMSCs (MSC group), respectively, in a total of 54 mice (n = 18 each). Hind limb motor function was evaluated in open-field tests using the Basso Mouse Scale (BMS) at days post-operation (dpo) 1, 3, 5, and 7 after spinal cord injury, and weekly thereafter. Spinal cord and serum samples were harvested at dpo 7, 14, and 21. Haematoxylin-eosin (H&E) staining and Masson staining were used to evaluate the morphological changes and fibrosis area. The differentiation of the transplanted cells in vivo was evaluated with immunohistochemical staining. Results The hiPSC-NSC-treated group presented a significantly smaller glial fibrillary acidic protein (GFAP) positive area than MSC-treated mice at all time points. Additionally, MSC-transplanted mice had a similar GFAP+ area to mice receiving PBS. At dpo 14, the immunostained hiPSC-NSCs were positive for SRY-related high-mobility-group (HMG)-box protein-2 (SOX2). Furthermore, the transplanted hiPSC-NSCs differentiated into GFAP-positive astrocytes and beta-III tubulin-positive neurons, whereas the transplanted huMSCs differentiated into GFAP-positive astrocytes. In addition, hiPSC-NSC transplantation reduced fibrosis formation and the inflammation level. Compared with the control or huMSC transplanted group, the group with transplantation of hiPSC-NSCs exhibited significantly improved behaviours, particularly limb coordination. Conclusions HiPSC-NSCs promote functional recovery in mice with acute SCI by replacing missing neurons and attenuating fibrosis, glial scar formation, and inflammation. Graphical abstract

Extracellular vesicles (EVs) contain various cargo molecules, including RNAs and proteins. EVs, which include exosomes, are predicted to be suitable surrogates of their source cells for liquid biopsy to measure biomarkers. Several studies have performed qualitative comparisons of cargo molecule repertoires between source cells and their EVs. However, quantitative comparisons have not been reported so far. Furthermore, many studies analyzed microRNAs or proteins in EVs, but not mRNAs. In this study, we analyzed mRNAs in motor neurons and their EVs. Normal human‐induced pluripotent stem cells were differentiated into motor neurons, and comprehensive analysis of mRNAs in the cells and their EVs was performed by RNA sequencing. Differential analysis between cellular and EV mRNAs was performed by edgeR after normalization of read count. The results suggest that signatures in the abundance of EV mRNAs were different from those of cellular mRNAs. Comparison of intracellular vesicle and EV mRNA abundance showed negatively and positively biased genes in the EVs. Gene Ontology analysis revealed that the genes showing negatively biased abundance in the EVs were enriched in many functions regarding neuronal development. In contrast, the positively biased genes were enriched in functions regarding cellular metabolism and protein synthesis. These results suggest that mRNAs in motor neurons are loaded into EVs to regulate certain mechanisms, which are yet to be elucidated.

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids are valuable tools for researching developmental biology and personalized therapies, but their closed topology and relative immature state limit applications. Here, we use organ-on-chip technology to develop a hiPSC-derived intestinal barrier with apical and basolateral access in a more physiological in vitro microenvironment. To replicate growth factor gradients along the crypt-villus axis, we locally expose the cells to expansion and differentiation media. In these conditions, intestinal epithelial cells self-organize into villus-like folds with physiological barrier integrity, and myofibroblasts and neurons emerge and form a subepithelial tissue in the bottom channel. The growth factor gradients efficiently balance dividing and mature cell types and induce an intestinal epithelial composition, including absorptive and secretory lineages, resembling the composition of the human small intestine. This well-characterized hiPSC-derived intestine-on-chip system can facilitate personalized studies on physiological processes and therapy development in the human small intestine. [Display omitted] • Intestinal epithelial cells self-organize into villus-like folds in the top channel • Myofibroblasts and neurons co-develop and migrate to the bottom channel • Growth factor gradients induce epithelial compositions resembling the human small intestine • The intestine-on-chip displays physiological barrier integrity and easy access to both sides Moerkens et al. developed a hiPSC-derived intestine-on-chip that emulates the small intestinal barrier. They demonstrate that hiPSC-derived intestinal cells in a two-channel microfluidic system and exposed to a growth factor gradient form a self-organizing tissue containing dividing, absorptive, and secretory epithelial cells; myofibroblasts; and neurons. [ABSTRACT FROM AUTHOR]