The V-ATPase/ATG16L1 axis is controlled by the V1H subunit.

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.

Research progress of brain organoids in the field of diabetes.

Human embryonic stem cells and human induced pluripotent stem cells may be used to create 3D tissues called brain organoids. They duplicate the physiological and pathological characteristics of human brain tissue more faithfully in terms of both structure and function, and they more precisely resemble the morphology and cellular structure of the human embryonic brain. This makes them valuable models for both drug screening and in vitro studies on the development of the human brain and associated disorders. The technical breakthroughs enabled by brain organoids have a significant impact on the research of different brain regions, brain development and sickness, the connections between the brain and other tissues and organs, and brain evolution. This article discusses the development of brain organoids, their use in diabetes research, and their progress.

The interferon γ pathway enhances pluripotency and X-chromosome reactivation in iPSC reprogramming.

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here, we reveal important pathways for pluripotency acquisition and X-reactivation using a genome-wide CRISPR screen during neural precursor to iPSC reprogramming. In particular, we discover that activation of the interferon γ (IFNγ) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFNγ stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFNγ in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes.

iPSC-derived lung and lung cancer organoid model to evaluate cisplatin encapsulated autologous iPSC-derived mesenchymal stromal cell-isolated extracellular vesicles.

BACKGROUND: Lung cancer remains a leading cause of cancer-related mortality globally. Although recent therapeutic advancements have provided targeted treatment approaches, the development of resistance and systemic toxicity remain primary concerns. Extracellular vesicles (EVs), especially those derived from mesenchymal stromal cells (MSC), have gained attention as promising drug delivery systems, offering biocompatibility and minimal immune responses. Recognizing the limitations of conventional 2D cell culture systems in mimicking the tumor microenvironment, this study aims to describe a proof-of-principle approach for using patient-specific organoid models for both lung cancer and normal lung tissue and the feasibility of employing autologous EVs derived from induced pluripotent stem cell (iPSC)-MSC in personalized medicine approaches. METHODS: First, we reprogrammed healthy fibroblasts into iPSC. Next, we differentiated patient-derived iPSC into branching lung organoids (BLO) and generated patient-matched lung cancer organoids (LCO) from patient-derived tumor tissue. We show a streamlined process of MSC differentiation from iPSC and EV isolation from iPSC-MSC, encapsulated with 0.07 µg/mL of cytotoxic agent cisplatin and applied to both organoid models. Cytotoxicity of cisplatin and cisplatin-loaded EVs was recorded with LDH and CCK8 tests. RESULTS: Fibroblast-derived iPSC showed a normal karyotype, pluripotency staining, and trilineage differentiation. iPSC-derived BLO showed expression of lung markers, like TMPRSS2 and MUC5A while patient-matched LCO showed expression of Napsin and CK5. Next, we compared the effects of iPSC-MSC derived EVs loaded with cisplatin against empty EVs and cisplatin alone in lung cancer organoid and healthy lung organoid models. As expected, we found a cytotoxic effect when LCO were treated with 20 µg/mL cisplatin. Treatment of LCO and BLO with empty EVs resulted in a cytotoxic effect after 24 h. However, EVs loaded with 0.07 µg/mL cisplatin failed to induce any cytotoxic effect in both organoid models. CONCLUSION: We report on a proof-of-principle pipeline towards using autologous or allogeneic iPSC-MSC EVs as drug delivery tests for lung cancer in future. However, due to the time and labor-intensive processes, we conclude that this pipeline might not be feasible for personalized approaches at the moment.

Human stem cell-derived neurons and astrocytes to detect novel auto-reactive IgG response in immune-mediated neurological diseases.

Background and objectives: Up to 46% of patients with presumed autoimmune limbic encephalitis are seronegative for all currently known central nervous system (CNS) antigens. We developed a cell-based assay (CBA) to screen for novel neural antibodies in serum and cerebrospinal fluid (CSF) using neurons and astrocytes derived from human-induced pluripotent stem cells (hiPSCs). Methods: Human iPSC-derived astrocytes or neurons were incubated with serum/CSF from 99 patients [42 with inflammatory neurological diseases (IND) and 57 with non-IND (NIND)]. The IND group included 11 patients with previously established neural antibodies, six with seronegative neuromyelitis optica spectrum disorder (NMOSD), 12 with suspected autoimmune encephalitis/paraneoplastic syndrome (AIE/PNS), and 13 with other IND (OIND). IgG binding to fixed CNS cells was detected using fluorescently-labeled antibodies and analyzed through automated fluorescence measures. IgG neuronal/astrocyte reactivity was further analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were used as CNS-irrelevant control target cells. Reactivity profile was defined as positive using a Robust regression and Outlier removal test with a false discovery rate at 10% following each individual readout. Results: Using our CBA, we detected antibodies recognizing hiPSC-derived neural cells in 19/99 subjects. Antibodies bound specifically to astrocytes in nine cases, to neurons in eight cases, and to both cell types in two cases, as confirmed by microscopy single-cell analyses. Highlighting the significance of our comprehensive 96-well CBA assay, neural-specific antibody binding was more frequent in IND (15 of 42) than in NIND patients (4 of 57) (Fisher's exact test, p = 0.0005). Two of four AQP4+ NMO and four of seven definite AIE/PNS with intracellular-reactive antibodies [1 GFAP astrocytopathy, 2 Hu+, 1 Ri+ AIE/PNS)], as identified in diagnostic laboratories, were also positive with our CBA. Most interestingly, we showed antibody-reactivity in two of six seronegative NMOSD, six of 12 probable AIE/PNS, and one of 13 OIND. Flow cytometry using hiPSC-derived CNS cells or PBMC-detected antibody binding in 13 versus zero patients, respectively, establishing the specificity of the detected antibodies for neural tissue. Conclusion: Our unique hiPSC-based CBA allows for the testing of novel neuron-/astrocyte-reactive antibodies in patients with suspected immune-mediated neurological syndromes, and negative testing in established routine laboratories, opening new perspectives in establishing a diagnosis of such complex diseases.

Poly-GR repeats associated with ALS/FTD gene C9ORF72 impair translation elongation and induce a ribotoxic stress response in neurons.

Hexanucleotide repeat expansion in the C9ORF72 gene is the most frequent inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion results in multiple dipeptide repeat proteins, among which arginine-rich poly-GR proteins are highly toxic to neurons and decrease the rate of protein synthesis. We investigated whether the effect on protein synthesis contributes to neuronal dysfunction and degeneration. We found that the expression of poly-GR proteins inhibited global translation by perturbing translation elongation. In iPSC-differentiated neurons, the translation of transcripts with relatively slow elongation rates was further slowed, and stalled, by poly-GR. Elongation stalling increased ribosome collisions and induced a ribotoxic stress response (RSR) mediated by ZAKα that increased the phosphorylation of the kinase p38 and promoted cell death. Knockdown of ZAKα or pharmacological inhibition of p38 ameliorated poly-GR-induced toxicity and improved the survival of iPSC-derived neurons from patients with C9ORF72-ALS/FTD. Our findings suggest that targeting the RSR may be neuroprotective in patients with ALS/FTD caused by repeat expansion in C9ORF72.

Rapid retinoic acid-induced trophoblast cell model from human induced pluripotent stem cells.

A limited number of accessible and representative models of human trophoblast cells currently exist for the study of placentation. Current stem cell models involve either a transition through a naïve stem cell state or precise dynamic control of multiple growth factors and small-molecule cues. Here, we demonstrated that a simple five-day treatment of human induced pluripotent stem cells with two small molecules, retinoic acid (RA) and Wnt agonist CHIR 99021 (CHIR), resulted in rapid, synergistic upregulation of CDX2. Transcriptomic analysis of RA + CHIR-treated cells showed high similarity to primary trophectoderm cells. Multipotency was verified via further differentiation towards cells with syncytiotrophoblast or extravillous trophoblast features. RA + CHIR-treated cells were also assessed for the established criteria defining a trophoblast cell model, and they possess all the features necessary to be considered valid. Collectively, our data demonstrate a facile, scalable method for generating functional trophoblast-like cells in vitro to better understand the placenta.

iPSC-derived NK cells expressing high-affinity IgG Fc receptor fusion CD64/16A to mediate flexible, multi-tumor antigen targeting for lymphoma.

Introduction: NK cells can mediate tumor cell killing by natural cytotoxicity and by antibody-dependent cell-mediated cytotoxicity (ADCC), an anti-tumor mechanism mediated through the IgG Fc receptor CD16A (FcγRIIIA). CD16A polymorphisms conferring increased affinity for IgG positively correlate with clinical outcomes during monoclonal antibody therapy for lymphoma, linking increased binding affinity with increased therapeutic potential via ADCC. We have previously reported on the FcγR fusion CD64/16A consisting of the extracellular region of CD64 (FcγRI), a high-affinity Fc receptor normally expressed by myeloid cells, and the transmembrane/cytoplasmic regions of CD16A, to create a highly potent and novel activating fusion receptor. Here, we evaluate the therapeutic potential of engineered induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as an "off-the-shelf", antibody-armed cellular therapy product with multi-antigen targeting potential. Methods: iNK cells were generated from iPSCs engineered to express CD64/16A and an interleukin (IL)-15/IL-15Rα fusion (IL-15RF) protein for cytokine independence. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells to examine in in vitro and in vivo assays using the Raji lymphoma cell line. ADCC was evaluated in real-time by IncuCyte assays and using a xenograft mouse model with high circulating levels of human IgG. Results: Our data show that CD64/16A expressing iNK cells can mediate potent anti-tumor activity against human B cell lymphoma. In particular, (i) under suboptimal conditions, including low antibody concentrations and low effector-to-target ratios, iNK-CD64/16A cells mediate ADCC, (ii) iNK-CD64/16A cells can be pre-loaded with tumor-targeting antibodies (arming) to elicit ADCC, (iii) armed iNK-CD64/16A cells can be repurposed with additional antibodies to target new tumor antigens, and (iv) cryopreserved, armed iNK-CD64/16A are capable of sustained ADCC in a tumor xenograft model under saturating levels of human IgG. Discussion: iNK-CD64/16A cells allow for a flexible use of antibodies (antibody arming and antibody targeting), and an "off-the-shelf" platform for multi-antigen recognition to overcome limitations of adoptive cell therapies expressing fixed antigen receptors leading to cancer relapse due to antigen escape variants.

Cholangiocyte Organoids in Liver Transplantation; a Comprehensive Review.

Liver transplantation is the only curative option for many liver diseases that end up in liver failure, and cholangiopathy remains a challenging complication post-liver transplant, associated with significant morbidity and potential graft loss. The low availability of organs and high demand for transplantation motivate scientists to find novel interventions. Organoids, as three-dimensional cell cultures derived from adult cells or induced pluripotent cells, may help to address this problem. Different types of organoids have been described, from which cholangiocyte organoids offer a high level of versatility and plasticity for a deeper study of liver disease mechanisms. Cholangiocytes can be obtained from different segments of the biliary tree and have shown a remarkable capacity to adapt to new environments, presenting an effective system for studying cholangiopathies. Studies using cholangiocyte organoids show promising results for disease modeling, where organoids offer fundamental features to recapitulate the complexities of tissues in vitro and uncover fundamental pathological pathways to potentially reveal therapeutic strategies for personalized medicine. Organoids could hold the potential for regeneration of injured livers, representing tools of clinical impact in regenerative medicine when tissue damage is already present.

Human heart-on-a-chip microphysiological system comprising endothelial cells, fibroblasts, and iPSC-derived cardiomyocytes.

In recent years, research on organ-on-a-chip technology has been flourishing, particularly for drug screening and disease model development. Fibroblasts and vascular endothelial cells engage in crosstalk through paracrine signaling and direct cell-cell contact, which is essential for the normal development and function of the heart. Therefore, to faithfully recapitulate cardiac function, it is imperative to incorporate fibroblasts and vascular endothelial cells into a heart-on-a-chip model. Here, we report the development of a human heart-on-a-chip composed of induced pluripotent stem cell (iPSC)-derived cardiomyocytes, fibroblasts, and vascular endothelial cells. Vascular endothelial cells cultured on microfluidic channels responded to the flow of culture medium mimicking blood flow by orienting themselves parallel to the flow direction, akin to in vivo vascular alignment in response to blood flow. Furthermore, the flow of culture medium promoted integrity among vascular endothelial cells, as evidenced by CD31 staining and lower apparent permeability. The tri-culture condition of iPSC-derived cardiomyocytes, fibroblasts, and vascular endothelial cells resulted in higher expression of the ventricular cardiomyocyte marker IRX4 and increased contractility compared to the bi-culture condition with iPSC-derived cardiomyocytes and fibroblasts alone. Such tri-culture-derived cardiac tissues exhibited cardiac responses similar to in vivo hearts, including an increase in heart rate upon noradrenaline administration. In summary, we have achieved the development of a heart-on-a-chip composed of cardiomyocytes, fibroblasts, and vascular endothelial cells that mimics in vivo cardiac behavior.

mTOR activation induces endolysosomal remodeling and nonclassical secretion of IL-32 via exosomes in inflammatory reactive astrocytes.

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo-IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states and was upregulated in astrocytes in multiple sclerosis lesions. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

Neonicotinoid Pesticides Affect Developing Neurons in Experimental Mouse Models and in Human Induced Pluripotent Stem Cell (iPSC)-Derived Neural Cultures and Organoids.

Neonicotinoids are synthetic, nicotine-derived insecticides used worldwide to protect crops and domestic animals from pest insects. The reported evidence shows that they are also able to interact with mammalian nicotine receptors (nAChRs), triggering detrimental responses in cultured neurons. Exposure to high neonicotinoid levels during the fetal period induces neurotoxicity in animal models. Considering the persistent exposure to these insecticides and the key role of nAChRs in brain development, their potential neurotoxicity on mammal central nervous system (CNS) needs further investigations. We studied here the neurodevelopmental effects of different generations of neonicotinoids on CNS cells in mouse fetal brain and primary cultures and in neuronal cells and organoids obtained from human induced pluripotent stem cells (iPSC). Neonicotinoids significantly affect neuron viability, with imidacloprid (IMI) inducing relevant alterations in synaptic protein expression, neurofilament structures, and microglia activation in vitro, and in the brain of prenatally exposed mouse fetuses. IMI induces neurotoxic effects also on developing human iPSC-derived neurons and cortical organoids. Collectively, the current findings show that neonicotinoids might induce impairment during neuro/immune-development in mouse and human CNS cells and provide new insights in the characterization of risk for the exposure to this class of pesticides.

Insight into the role of TXNRD2 in steroidogenesis through a novel homozygous TXNRD2 splice variant.

OBJECTIVE: Adrenal cortisol production occurs through a biosynthetic pathway which depend on NADH and NADPH for energy supply. The mitochondrial respiratory chain and the reactive oxygen species (ROS) detoxification system are therefore important for steroidogenesis. Mitochondrial dysfunction leading to oxidative stress has been implicated in the pathogenesis of several adrenal conditions. Nonetheless, only very few patients with variants in one gene of the ROS detoxification system, Thioredoxin Reductase 2 (TXNRD2), have been described with variable phenotypes. DESIGN: Clinical, genetic, structural, and functional characterization of a novel, biallelic TXNRD2 splice variant. METHODS: On human biomaterial, we performed whole exome sequencing to identify and RNA analysis to characterize the specific TXNRD2 splice variant. Amino acid conservation analysis and protein structure modeling were performed in silico. Using patient's fibroblast-derived human induced pluripotent stem cells, we generated adrenal-like cells (iALC) to study the impact of wild-type (WT) and mutant TXNRD2 on adrenal steroidogenesis and ROS production. RESULTS: The patient had a complex phenotype of primary adrenal insufficiency (PAI), combined with genital, ophthalmological, and neurological features. He carried a homozygous splice variant c.1348-1G > T in TXNRD2 which leads to a shorter protein lacking the C-terminus and thereby affecting homodimerization and flavin adenine dinucleotide binding. Patient-derived iALC showed a loss of cortisol production with overall diminished adrenal steroidogenesis, while ROS production was significantly increased. CONCLUSION: Lack of TXNRD2 activity for mitochondrial ROS detoxification affects adrenal steroidogenesis and predominantly cortisol production.

Zika virus vertical transmission induces neuroinflammation and synapse impairment in brain cells derived from children born with Congenital Zika Syndrome.

Zika virus (ZIKV) infection was first reported in 2015 in Brazil as causing microcephaly and other developmental abnormalities in newborns, leading to the identification of Congenital Zika Syndrome (CZS). Viral infections have been considered an environmental risk factor for neurodevelopmental disorders outcome, such as Autism Spectrum Disorder (ASD). Moreover, not only the infection per se, but maternal immune system activation during pregnancy, has been linked to fetal neurodevelopmental disorders. To understand the impact of ZIKV vertical infection on brain development, we derived induced pluripotent stem cells (iPSC) from Brazilian children born with CZS, some of the patients also being diagnosed with ASD. Comparing iPSC-derived neurons from CZS with a control group, we found lower levels of pre- and postsynaptic proteins and reduced functional synapses by puncta co-localization. Furthermore, neurons and astrocytes derived from the CZS group showed decreased glutamate levels. Additionally, the CZS group exhibited elevated levels of cytokine production, one of which being IL-6, already associated with the ASD phenotype. These preliminary findings suggest that ZIKV vertical infection may cause long-lasting disruptions in brain development during fetal stages, even in the absence of the virus after birth. These disruptions could contribute to neurodevelopmental disorders manifestations such as ASD. Our study contributes with novel knowledge of the CZS outcomes and paves the way for clinical validation and the development of potential interventions to mitigate the impact of ZIKV vertical infection on neurodevelopment.

Generation of Human-Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells of C9ORF72-Associated Amyotrophic Lateral Sclerosis Patients.

Disease modeling of neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), is hindered by limited accessibility of affected cells. This problem can be overcome by generation of human induced pluripotent stem cells (hiPSC), which can be then differentiated into required cells. Here, we describe the detailed protocol of hiPSC establishment from peripheral blood mononuclear cells (PBMC) of two ALS patients with detected expansion of G4C2 (GGGGCC) repeats in the first intron of C9ORF72 gene, known to be linked with the most common form of familial ALS.Successful PBMC reprogramming with non-integrating Sendai vectors was confirmed by expression of pluripotency markers: OCT4, NANOG, SSEA4, and TRA-1-60 in obtained hiPSC and their ability to differentiate into cells of three germ layers.The generated ALS-patient-specific hiPSC create a possibility for deciphering molecular basis of this devastating neuromuscular disease.

The Efficiency of In Vitro Differentiation of Primate iPSCs into Cardiomyocytes Depending on Their Cell Seeding Density and Cell Line Specificity.

A thorough characterization of induced pluripotent stem cells (iPSCs) used with in vitro models or therapeutics is essential. Even iPSCs derived from a single donor can exhibit variability within and between cell lines, which can lead to heterogeneity in results and hinder the promising future of cell replacement therapies. In this study, the cell seeding density of human and rhesus monkey iPSCs was tested to maximize the cell line-specific yield of the generated cardiomyocytes. We found that, despite using the same iPSC generation and differentiation protocols, the cell seeding density for the cell line-specific best differentiation efficiency could differ by a factor of four for the four cell lines used here. In addition, the cell lines showed differences in the range of cell seeding densities that they could tolerate without the severe loss of differentiation efficiency. Overall, our data show that the cell seeding density is a critical parameter for the differentiation inefficiency of primate iPSCs to cardiomyocytes and that iPSCs generated with the same episomal approach still exhibit considerable heterogeneity. Therefore, individual characterization of iPSC lines is required, and functional comparability with in vivo processes must be ensured to warrant the translatability of in vitro research with iPSCs.

Exosomal Preconditioning of Human iPSC-Derived Cardiomyocytes Beneficially Alters Cardiac Electrophysiology and Micro RNA Expression.

Experimental evidence, both in vitro and in vivo, has indicated cardioprotective effects of extracellular vesicles (EVs) derived from various cell types, including induced pluripotent stem cell-derived cardiomyocytes. The biological effects of EV secretion, particularly in the context of ischemia and cardiac electrophysiology, remain to be fully explored. Therefore, the goal of this study was to unveil the effects of exosome (EXO)-mediated cell-cell signaling during hypoxia by employing a simulated preconditioning approach on human-induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs). Electrophysiological activity of hIPSC-CMs was measured using a multielectrode array (MEA) system. A total of 16 h of hypoxic stress drastically increased the beat period. Moreover, hIPSC-CMs preconditioned with EXOs displayed significantly longer beat periods compared with non-treated cells after 16 h of hypoxia (+15.7%, p < 0.05). Furthermore, preconditioning with hypoxic EXOs resulted in faster excitation-contraction (EC) coupling compared with non-treated hIPSC-CMs after 16 h of hypoxia (-25.3%, p < 0.05). Additionally, microRNA (miR) sequencing and gene target prediction analysis of the non-treated and pre-conditioned hIPSC-CMs identified 10 differentially regulated miRs and 44 gene targets. These results shed light on the intricate involvement of miRs, emphasizing gene targets associated with cell survival, contraction, apoptosis, reactive oxygen species (ROS) regulation, and ion channel modulation. Overall, this study demonstrates that EXOs secreted by hIPSC-CM during hypoxia beneficially alter electrophysiological properties in recipient cells exposed to hypoxic stress, which could play a crucial role in the development of targeted interventions to improve outcomes in ischemic heart conditions.

Engineered vascular grafts lend unique insight to pathophysiology of aortic aneurysms.

Yang et al.1 generate tissue engineered blood vessels from hiPSC-derived smooth muscle cells harboring a mutation found in Loeys-Dietz syndrome. In vitro and in vivo data from these vessels provide new insight into the molecular physiology of aortic aneurysms and may create a paradigm for understanding a suite of vascular diseases.

A Three-Step Protocol to Differentiate iPSC into Colon Organoids.

Organoids, three-dimensional, stem cell-based structures that mimic the cellular and functional architecture of tissues, have emerged as an innovative in vitro tool. They offer highly efficient models for studying both embryonic development and disease progression processes. Colon organoids can also be generated from biopsies obtained during a colonoscopy. However, the invasive nature of biopsy collection poses practical challenges and introduces biases when studying patients who are already afflicted. Therefore, the use of iPSC-derived colon organoids can be considered a more practical approach for researchers and patients alike. Numerous protocols have been published for generating colon organoids from iPSCs. While most of these protocols share a common developmental process, some are labor-intensive or require additional equipment. Taking these considerations into account, we present a cost-effective and straightforward yet functionally robust colon organoid protocol: (1) definitive endoderm differentiation, (2) hindgut endoderm differentiation, and (3) maturation of colon spheroids into mature organoids.

Safe and Noninvasive Method for Generating Induced Mesenchymal Stem Cells from Urinary Epithelial Cells.

Mesenchymal stem cells (MSCs) exhibit remarkable versatility and hold immense potential for tissue regeneration. They are actively investigated in clinical trials for various diseases and injuries, showcasing their therapeutic promise. However, traditional sources of MSCs have limitations in terms of scalability and storage. To address these challenges, this study aims to provide a method of creating an alternative source of induced pluripotent stem cells (iPSCs)-derived MSCs (iMSCs) from urinary epithelial cells (UECs) through a noninvasive procedure. This distinct subset of UECs found in urine samples offers an invaluable resource for generating autologous UE-iPSCs. iPSCs have distinct advantages over embryonic stem cells, as they can be generated from somatic cells, eliminating the need for human embryos and associated ethical concerns. Advancements in iPSC technology enable the differentiation of iMSCs, allowing researchers to create disease models, gain insights into disease mechanisms, and develop targeted therapies. This straightforward and noninvasive method aims to enhance the production of high-quality, autologous iMSCs with significant replicative and differentiation potential, making them suitable for regenerative therapy.